PresequenceProtease (PreP), a novel Peptidasome in Mitochondria and Chloroplasts:

PresequenceProtease (PreP), a novel Peptidasome in Mitochondria and Chloroplasts:
Localization, Function, Structure and Mechanism of Proteolysis
Shashi Bhushan
Department of Biochemistry and Biophysics
Arrhenius Laboratories for Natural Sciences
Stockholm University
S-10691 Stockholm
Sweden
Stockholm, 2007
 Shashi Bhushan, Stockholm 2007
ISBN: 978-91-7155-435-2
Typesetting: Intellecta Docusys
Printed in Sweden by Intellecta Docusys, Stockholm 2007
Distributor: Stockholm University Library
Cover picture: Dual targeting of the PreP1:GFP fusion protein to mitochondria and chloroplasts
(Paper I) and a proposed mechanism for the PreP peptidasome substrate binding, proteolysis and
release (Paper III).
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- To my family
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CONTENTS
Abstract
List of publications
Abbreviations
The mitochondria and chloroplasts
The origin and evolution
Structure and function
Genome
Proteome
Organellar import machineries
Mitochondrial protein import machinery
Mitochondrial targeting peptides - the presequences
Cytosolic factors
Translocase of the outer membrane (TOM)
Import receptors
The general import pore
Translocase of the inner membrane (TIM)
TIM23 complex
TIM22 complex
Protein Import to the intermembrane space
Processing peptidases
Chloroplastic protein import machinery
Chloroplastic targeting peptides - the transit peptides
Cytosolic factors
Translocase of the outer envelope membrane (TOC)
Translocase of the inner envelope membrane (TIC)
Stromal processing peptidase
Dual targeting to mitochondria and chloroplasts
Studying dual targeting
Mechanisms of dual targeting
Proteolytic system in mitochondria
ATP-dependent proteases
The FtsH (AAA) protease
Lon-like protease
Clp-like protease
ATP-independent proteases
Oligopeptidases
OMA protease
Rhomboid protease
Proteolytic system in chloroplasts
ATP-dependent proteases
The FtsH (AAA) protease
Clp-like protease
Lon-like protease
ATP-independent proteases
DegP-like protease
SppA-like protease
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The PresequenceProtease, PreP
Dual targeting of PreP in mitochondria and chloroplasts
Function of PreP in mitochondria and chloroplasts
Expression of the AtPreP1 and AtPreP2 in A. thaliana plants
Crystal structure of AtPreP1, a Peptidasome
Mechanism of proteolysis by PreP Peptidasome
The role of the PreP peptidasome in the degradation of the amyloid βpeptide: A possible link to Alzheimer’s disease
Alzheimer’s disease (AD) and amyloid β-peptide (Aβ)
Amyloid β-peptide in mitochondria
PresequenceProtease in human - The hPreP
A novel function of hPreP in mitochondria: Aβ degradation
Future perspectives
Acknowledgements
References
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Abstract
Mitochondria and chloroplasts contain several thousand different proteins, almost all of which
are synthesized in the cytosol as precursor proteins and imported into the correct organelle.
The information for the organellar targeting and import generally resides in the N-terminal
part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the
organellar processing peptidases after import of a precursor protein. Free targeting peptides
generated inside the organelle after import are rapidly degraded by proteolysis as their
accumulation can have severe effects on the functional and structural integrity of the organelle
since they can penetrate membranes, induce channel formation in membranes, dissipate
membrane potential and uncouple respiration. The aim of this thesis has been a thorough
investigation of the newly identified targeting peptide degrading protease, the
PresequenceProtease (PreP).
We have shown that the two isoforms of Arabidopsis thaliana PresequenceProteases
(AtPreP1 and AtPreP1) are dually targeted and localized to both mitochondria and
chloroplasts. Dual targeting of the AtPreP1 is due to an ambiguous targeting peptide with a
domain organization for mitochondrial and chloroplastic targeting. Both the AtPreP1 and
AtPreP2 are expressed in A. thaliana plants in an organ specific manner and they have distinct
but overlapping substrate specificity for efficient degradation of a wide variety of peptides.
The crystal structure of the recombinant AtPreP1 E80Q was solved at 2.1 Å resolution. The
structure represents the first substrate bound, closed conformation of a protease from the
pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a
huge cavity of more than 10 000 Å3 in which the active site resides. Cysteine mutants of
AtPreP1 designed for locking the PreP in a closed conformation showed no proteolytic
activity when disulfide bonds were allowed to form, while activity was normal in absence of
disulfide bonds. A novel mechanism for proteolysis is proposed involving hinge-bending
motions that cause the PreP protease to open and close in response to substrate binding.
PreP is localized to the mitochondrial matrix in human mitochondria where, beside
degradation of targeting peptides, it has a novel function: degradation of amyloid β-peptide
(Aβ). Immunoinactivation of PreP in human brain mitochondria resulted in complete loss of
the proteolytic activity against Aβ-peptide, showing that under circumstances when Aβ is
present in mitochondria, human PreP is the protease responsible for degradation of this toxic
peptide. These findings contribute to studies of the mitochondrial component in Alzheimer’s
disease.
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List of publications included in the thesis
I. Bhushan, S., Lefebvre, B., Ståhl, A., Wright, S.J., Bruce, B.D., Boutry, M. and Glaser,
E. (2003) Dual targeting and function of a protease in mitochondria and chloroplasts.
EMBO Rep, 11, 1073-1078.
II. Bhushan, S., Ståhl, A., Nilsson, S., Lefebvre, B., Seki, M., Roth, C., McWilliam, D.,
Wright, S.J., Liberles, D.A., Shinozaki, K., Bruce, B.D., Boutry, M. and Glaser, E.
(2005) Catalysis, subcellular localization, expression and evolution of the targeting
peptides degrading protease, AtPreP2. Plant Cell Phys, 46, 985-996.
III. Johnson, K.A.*, Bhushan, S.*, Ståhl, A., Hällberg, B.M., Frohn, A., Glaser, E. and
Eneqvist, T. (2006) The closed structure of PresequenceProtease PreP forms a unique
10.000Å3 chamber for proteolysis. EMBO J, 25, 1977-1986.
*
Both authors have contributed equally to this work.
IV. Bhushan, S., Johnson, K.A., Eneqvist, T. and Glaser, E. (2006) Proteolytic mechanism
of a novel mitochondrial and chloroplastic PreP peptidasome. Biol Chem, 387, 10871090.
V. Falkevall, A., Alikhani, N., Bhushan, S., Pavlov, P.F., Busch, K., Johnson, K.A.,
Eneqvist, T., Tjernberg, L., Ankarcrona, M. and Glaser, E. (2006) Degradation of the
amyloid β-protein by the novel mitochondrial peptidasome, PreP. J Biol Chem, 281,
29096-29104.
Additional publications
VI. Moberg, P., Ståhl, A., Bhushan, S., Wright, S.J., Bruce, B.D. and Glaser, E. (2003)
Characterization of a novel metalloprotease involved in degrading targeting peptides
in mitochondria and chloroplasts. Plant J, 36, 616-628.
VII. Ståhl, A., Nilsson, S., Lundberg, P., Bhushan, S., Biverståhl, H., Moberg, P., Morisset,
M., Vener, A., Mäler, L., Langel, U. and Glaser, E. (2005) Two Novel Targeting
Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and
Still Different. J Mol Biol, 349, 847-860.
VIII. Pesaresi, P., Masiero, S., Eubel, H., Bhushan, S., Glaser, E., Braun, H.P., Dietzmann,
A., Rosso, M., Salamini, F. and Leister, D. (2006) Mutational analysis of Arabidopsis
ProRSI, encoding an essential mitochondrial and chloroplastic targeted prolyl-tRNA
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synthetase, reveals mitochondrial-dependent down regulation of photosynthesis. Plant
Cell, 18, 970-991.
IX. Bhushan, S., Kuhn, C., Berglund, A.K., Roth, C. and Glaser, E. (2006) The role of the
N-terminal domain of chloroplast targeting peptides in organellar protein import and
miss-sorting. FEBS Lett, 580, 3966-3972.
X. Glaser, E., Nilsson, S. and Bhushan, S. (2006) Two novel mitochondrial and
chloroplastic targeting peptide degrading peptidasomes in A. thaliana, AtPreP1 and
AtPreP2. Biol Chem, 387, 1441-1447.
XI. Pavlov, P., Rudhe, C., Bhushan, S. and Glaser, E. (2007) In vitro and in vivo protein
import into plant mitochondria. Methods Mol Biol - Mitochondria, Leister, D. ed., in
press.
XII. Bhushan, S., Pavlov, P., Rudhe, C. and Glaser, E. (2007) Plant mitochondrial protein
import. Methods Mol Biol - Protein targeting, van der Giezen, M. ed., in press.
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Abbreviations
AAA
AAC
Aβ
ABAD
ABC
AD
AIP
APP
Clp
GIP
GFP
GST
Hsp70
IDE
IM
IMP
IMS
LC
MIP
MOP
MPP
MS
MSF
NEM
OM
Oma
PBF
PiC
Pim1
PreP
RISP
SPP
TIC
TIM
TM
TOC
TOM
Yme1
Yta
Zn-MP
∆ψ
∆pH
ATPases associated with a number of cellular activities
ADP/ATP translocator
Amyloid β-peptide
Amyloid β-binding alcohol dehydrogenase
ATP-binding cassette
Alzheimer’s disease
Aryl hydrocarbon receptor interacting protein
Amyloid precursor protein
Caseinolytic protease
General import pore
Green fluorescent protein
Glutathione-S-transferase
Heat shock protein 70
Insulin degrading enzyme
Inner membrane
Inner membrane peptidase
Intermembrane space
Liquid chromatography
Mitochondrial intermediate peptidase
Mitochondrial oligopeptidase
Mitochondrial processing peptidase
Mass spectrometry
Mitochondrial import stimulating factor
N-ethylmaleimide
Outer membrane
Overlapping activity with m-AAA proteases
Presequence binding factor
Phosphate carrier
Protease in mitochondria 1
PresequenceProtease
Rieske Fe-S protein of the cytochrome bf complex
Stromal processing peptidase
Translocase of the inner envelope membrane of chloroplast
Translocase of the inner membrane of mitochondria
Transmembrane
Translocase of the outer envelope membrane of chloroplast
Translocase of the outer membrane of mitochondria
Yeast mitochondrial escape 1
Yeast tat-binding-like proteins
Zinc metalloprotease
Membrane potential
Proton gradient
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The mitochondria and chloroplast
The name mitochondrion arises from Greek words mitos = thread and chondrion = granule.
Mitochondria were originally identified as the site of oxidative energy metabolism (Kennedy
and Lehninger, 1950). Mitochondria are also the host for enzymes of the Krebs cycle and βoxidation of fatty acids. In today’s world mitochondria are known not only as the “power
station” of the cell, but also for playing a vital role in the transmission of extra- and
intracellular signals that activate reaction cascades leading to cellular senescence and
programmed cell death (PCD) (Wang, 2001). The discovery of a number of human diseases
associated with mitochondrial dysfunctions once again brought mitochondria into the spotlight of biological research.
The name chloroplast is derived from the Greek words chloros = green and plast = form or
entity. Chloroplasts are members of a class of plant cell organelles known as plastids that all
originate from protoplastids. During plant development the protoplastids differentiate to form
three major groups of plastids, the green chloroplasts, the colored chromoplasts and the
colorless leucoplasts. The most abundant and important plastids are the chloroplasts.
Chloroplasts harvest energy from sunlight to split water and fix carbon dioxide to produce
sugars. This process called photosynthesis also converts harvested solar energy into a
conserved form of energy: ATP and NADPH through a complex set of processes.
The origin and evolution
Mitochondria and chloroplasts are not synthesized de novo, but originate from pre-existing
organelles by partition in a fission process. The most accepted theory about the origin of these
two organelles is the endosymbiotic theory proposed by Lynn Margulis in 1970 (Margulis, 1970).
According to this theory mitochondria arose from aerobic prokaryotes that began to live in
symbiosis with a primitive, anaerobic eukaryotic cell. The endosymbiotic theory is supported by
many facts such as e.g. 1) mitochondria and chloroplasts contain their own genome and divides
independently from the cell where they reside, 2) in most organisms mitochondrial and
chloroplastic DNA is circular, just like in bacteria, 3) transcriptional and translational
machineries of these organelles are very similar to those found in bacteria and 4) the
mitochondrial inner membrane (IM) contains certain lipids, such as cardiolipins, only found in
the mitochondrial IM and in the plasma membrane of bacteria. Based on the genome sequences
from a number of different organisms, phylogenetic reconstitutions of mitochondria have been
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made. These analyses show that mitochondrial genome sequences are the descendents of αproteobacterial homologues (Lang et al., 1999). The closest known ancestor to mitochondria is
Rickettsia prowazekii, an obligate intracellular parasite (Andersson et al., 1998, 2003).
Phylogenetic data suggests that the chloroplasts were engulfed after mitochondria in an
endosymbiotic event. In a first primary endosymbiosis event about 1.5 billion years ago an
ancient cyanobacterium was engulfed by a mitochondria containing eukaryote. The plastids
formed in this way are surrounded by two membranes and are found in land plants and in red
and green algae (Gray, 1999). However, a growing body of evidence indicates that the
chloroplasts of some algae have not been derived by engulfing cyanobacteria in a primary
endosymbiosis like those discussed above, but by engulfing photosynthetic eukaryotes. This
is called secondary endosymbiosis and these plastids are called secondary plastids
characterized by the presence of three or four surrounding membranes. Secondary plastids are
found in lineages such as apicomplexa, dinoflagellates and ciliates (as reviewed by Stoebe
and Maier, 2002).
Structure and function
Mitochondria are present in virtually all eukaryotic cells. They are typically 0.5-1.8 µm wide
and 1-2 µm long in size. The number and distribution of mitochondria are dependent on the
metabolic activity of the cell. Mitochondria are distinct organelles surrounded by two
membranes. The two membranes divide the mitochondrion into two distinct compartments,
the intermembrane space (IMS) and the mitochondrial matrix. Electron tomography has
provided the three-dimensional (3D) structure of mitochondria and gives new insight to the
internal organization (Frey and Mannella, 2000). The outer membrane (OM) is smooth and
permeable to ions and molecules smaller than 10 kDa. The inner membrane (IM) is
impermeable and highly convoluted, forming folds called cristae. Cristae are connected to the
IM by narrow tubular segments, called cristae junctions. The narrowness of the cristae
junctions has led to the hypothesis that the IM is further divided into distinct sub
compartments (Frey and Mannella, 2000).
The mitochondrial matrix contains the enzymes of the tricarboxylic acid cycle (TCA) and
fatty acid oxidation. The important function of mitochondria is to produce the ATP required
by the cell. ATP production in mitochondria starts with the oxidative decarboxylation of
pyruvate in the matrix to form NADH and acetyl-CoA. Acetyl-CoA enters the TCA cycle to
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generate NADH and FADH2 from NAD+ and FAD. The electrons from NADH and FADH2
are funnelled to oxygen through a chain of electron carriers in the respiratory chain. The
transfer of electrons between these carriers releases energy that is used to transfer protons
across the IM from matrix to the IMS side. The proton translocation generates an
electrochemical proton motive force consisting of a membrane potential (∆ψ) and a transmembrane proton gradient (∆pH) (Mitchell, 1974). The energy from the proton gradient is
utilized to synthesize ATP from ADP by the ATP synthase in mitochondria. ATP formed in
this way is exported from the mitochondria and provides the energy required for different
purposes in a cell.
Chloroplasts look like as flat discs usually 2 to 10 µm in diameter and 1 µm thick.
Chloroplasts are surrounded by two membranes, the outer envelope (OE) membrane and the
inner envelope (IE) membrane. The OE membrane is permeable to small molecules up to 10
kDa, but impermeable to bigger molecules such as proteins and nucleic acids. The IE
membrane is impermeable to most molecules and those that are permeable can only cross this
membrane through specific translocases. The compartment between these two envelopes is
called the interenvelope space (IES). The soluble material within the chloroplast is called
stroma, corresponding evolutionary to the cytosol of a bacterium, and contains one or more
molecules of small circular DNA. Chloroplasts perform the very important function of
photosynthesis within plant cells and contain the chlorophyll molecules that are essential for
this process. All reactions of photosynthesis occur in this organelle including CO2 fixation.
The chloroplasts use photosynthetic chlorophyll pigment and take in sunlight, water and
carbon dioxide to produce glucose and oxygen. An important structure in the chloroplasts is
the inter-connected, flattened, membranous sacs called thylakoids. There are many thylakoid
stacks in a chloroplast, providing a vast surface area within a compact volume for harvesting
light energy to drive photosynthesis. These structures are the site of the photosynthetic light
reactions.
Genome
Mitochondria and chloroplasts harbor their own genome in form of the circular DNA. The
mitochondrial and chloroplastic DNA exhibit a remarkable variation in terms of structure and
size as well as gene content and expression. Currently known mitochondrial genome sizes
range from 5 966 base pairs in Plasmodium reichenowi (malaria parasite) to 569 630 base
pairs in Zea mays (maize) (Conway et al., 2000; Clifton et al., 2004). Chloroplasts genome
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sizes range from 110 000 base pairs to 160 000 base pairs, depending on the species (Cui et
al., 2006). Mitochondrial DNA encodes a limited number of proteins and RNAs that are
essential for the formation of functional mitochondria. Generally, mitochondrial DNA
encoded proteins are core components of the respiratory chain complexes and the ATP
synthase (Boore, 1999). Land plant chloroplast genomes typically contain around 110-120
unique genes. Some algae have retained a large chloroplast genome with more than 200
genes, while the plastid genomes from non-photosynthetic organisms have retained only a
few dozen genes (Cui et al., 2006).
Over evolutionary time both mitochondria and chloroplasts have lost most of their genes
and transitioned from being free living prokaryotes to organelles with key roles in eukaryotic
cellular function. Two distinct modes of genetic loss are responsible for the reduced genome
we now see in these two modern organelles (Berg and Kurland, 2000; Kurland and
Andersson, 2000). First these organelles reside in a cell so they can import rather than
synthesize a number of biomolecules from the host. Second, many genes were transferred to,
and are now expressed, in the nuclear genome. The low gene content of mitochondrial DNA
implies a rapid and extensive loss or transfer of genetic material during early stages of
mitochondrial evolution (Grey at al., 1999). In plants, mitochondrial gene transfer can still be
traced (Adams et al., 2000; Daley et al., 2002). The subunit 2 of cytochrome oxidase (CoxII)
gene from legumes (Nugent and Palmer, 1991; Adams et al., 1999), the Rps12 gene from
Oenothera (Grohmann et al., 1992), Rps10 and Rps19 genes from A. thaliana (Wischmann
and Schuster, 1995; Sanchez et al., 1996) and the Rps11 gene from Oryza sativa (Kadowaki et
al., 1996) have all been identified as recent gene transfers from the mitochondrial genome to
the nuclear genome by comparing gene content in nuclear and mitochondrial DNA.
What are the evolutionary advantages driving gene transfer from mitochondria and
chloroplasts to nucleus? One popular hypothesis is Muller’s rachet which suggests that the
asexual reproduction of mitochondria and chloroplasts can lead to a faster accumulation of
deleterious mutations (Kurland, 1992; Berg and Kurland, 2000). High concentrations of free
radicals can be produced in mitochondria and chloroplasts because of the high redox activity
of these organelles (Martin and Palumbi, 1992; Allen and Raven, 1996). Increased levels of
oxygen free radicals have been shown to cause an increase in DNA mutation rates and
therefore it would be advantageous to relocate genetic material from organelles to the nucleus.
However, the genome of plant mitochondria tends to be less mutation prone than the nuclear
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genome (Martin and Herrmann, 1998). Therefore, free radicals that lead to mutations in the
organellar genome might not be the sole driving factor for the organellar gene transfer in
plants.
Why are some genes still maintained in these organelles when most of them have been
transferred to the nucleus? There is no straight answer to this question. A hydrophobicity
hypothesis was proposed by von Heijne, who suggested that genes remaining in these
organelles encode proteins that are too hydrophobic to be imported across membranes back
into the organelles (von Heijne, 1986a). However, there are some very hydrophobic proteins
that have been moved from these organellar genomes to the nucleus and are able to be
imported back to these organelles (Gray et al., 1999). A second theory, CORR (Co-location
for Redox Regulation) was proposed by Allen in 1992. According to this theory co-location
of chloroplast and mitochondrial genes with their gene products is required for rapid and
direct regulatory coupling. Redox control of gene expression is suggested as the common
feature of those mitochondrial and chloroplastic proteins that are encoded in their own
genome (Allen, 1992).
Proteome
Mitochondria contain about 800 to 2500 different proteins (Emanuelsson et al., 2000; Zhang
et al., 2001). The availability of genome databases and recent advances in proteomics have
enabled us to gain a better insight of the mitochondrial proteome. In recent years several
proteomic studies of isolated mitochondria have been reported. The aim of these studies is to
gain a better understanding of the role of mitochondria and its function. Taylor et al. (2003)
have been able to identify 615 proteins of human heart mitochondria using proteomic
approaches and identification by mass spectrometric (MS) analysis. 81% of the identified
proteins were classified amongst protein families with identified functions, while the function
of the remaining 19% proteins remains to be explored. In a separate study Da Cruz et al.
(2003) have been able to identify 183 mitochondrial IM proteins from rat mitochondria using
liquid chromatography (LC) directly coupled to MS analysis. In another study on isolated
Saccharomyces cerevisiae mitochondria, Sickmann et al. (2003) were able to identify 750
proteins which they suggested comprise about 90% of the total mitochondrial proteome.
Plant mitochondria resemble mammalian and yeast mitochondria in many ways but still
have some additional functions, such as an uncoupled bypass of the electron transport chain
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by the alternative oxidase (AOX) and the synthesis of lipids and vitamins (Rebeille et al.,
1997; Bartoli et al., 2000; Gueguen et al., 2000). Heazlewood et al. (2004) have been able to
identify 416 mitochondrial proteins from A. thaliana using a systematic LC-MS/MS
approach. 407 proteins out of those 416 are nuclear encoded and the remaining 9 are encoded
in the mitochondrial genome.
Chloroplasts contain about 3500 different proteins as estimated in silico using different
prediction programs such as TargetP and ChloroP (Emanuelsson et al., 1999, 2000).
Kleffmann et al. (2004) have used different fractionation techniques, followed by LC-ESIMS/MS to identify 687 proteins in isolated A. thaliana chloroplasts. 70% of these identified
proteins could be assigned to one or more known metabolic pathways, whereas the remaining
30% of the proteins were of unknown function. Surprisingly, 48% of the identified nuclear
encoded proteins did not have a predicted targeting peptide when analysed using TargetP. In a
separate study Friso et al. (2004) have been able to identify 198 proteins associated with
thylakoid membranes of A. thaliana.
Organellar import machineries
Mitochondrial protein import machinery
The mitochondrial protein import machinery has been extensively studied in S. cerevisiae and
Neurospora crassa using both biochemical and genetic approaches (as reviewed by Neupert
and Herrmann, 2007). The plant mitochondrial import process has been studied in Solanum
tuberosum, Spinacia oleracea, Pisum sativum and A. thaliana (as reviewed by Glaser and
Whelan, 2007). The plant and yeast mitochondrial import machinery share several aspects,
but differs in some respects.
Most of the mitochondrial proteins are nuclear encoded and synthesized in the cytosol as
precursor proteins. Mitochondrial protein import generally requires a signal for import,
cytosolic factors/chaperones, import receptors on the mitochondrial surface, an import pore,
chaperones inside the mitochondria and processing peptidases for maturation (Figure 1). The
functioning of the general mitochondrial import pathway and machinery can be briefly
described as follows:
-Synthesis of the nuclear encoded precursor proteins in the cytosol.
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-Interaction of the newly synthesized precursor proteins with cytosolic factors/chaperones
that keep them in an unfolded and import competent conformation.
-Recognition of the precursor proteins by import receptors on the mitochondrial surface.
-Translocation of the precursor proteins across the mitochondrial OM via the general
import pore.
(Stefan Nilsson, 2007)
Figure 1. Mitochondrial protein import machinery. General overview of the
protein import pathway into the matrix. TOM and TIM refer to Translocases of the
outer and inner membrane protein complexes in mitochondria. The numbers
represent the molecular masses of the components of the TOM and TIM complexes.
MPP, Mitochondrial processing peptidase; Hsp70, Heat shock protein 70; MGE
and MDJ, mitochondrial co-chaperones, homologs of bacterial DnaJ and GrpE;, αand β-, subunits of MPP; PreP PresequenceProtease.
-Chaperone assisted passage through mitochondrial IMS.
-Interaction of the precursor proteins with import receptors on the mitochondrial IM.
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-ATP and ∆ψ-dependent translocation of the precursor proteins across the mitochondrial
IM.
-Proteolytic maturation of the precursor proteins by removal of the presequence by
processing peptidases.
-Chaperone assisted folding and assembly of the mature proteins.
-Degradation/removal of the free, cleaved targeting peptides/presequences.
Mitochondrial targeting peptides - the presequences
Precursor proteins can be divided into two classes on the basis of their targeting mechanisms
used for import. More than half of the precursor proteins carry a cleavable N-terminal
extension known as the presequence or targeting peptide. Some integral membrane proteins,
such as the metabolic carriers are synthesized without cleavable extensions. These precursor
proteins contain internal targeting signals that are distributed throughout the entire length of
the proteins. The presequence or targeting peptide carries all of the information required for
protein targeting and import into the mitochondria. Most presequences are 20-50 amino acid
residues in length. However, they can vary substantially,,from 13 residues to 136 residues
(Zhang et al., 2001). There is no consensus at the primary structure level among
presequences, only a very loosely conserved motif has been found around the processing site
(von Heijne et al., 1989; Zhang et al., 2001). Presequences are enriched in basic and
hydrophobic residues and are generally deficient in acidic residues (von Heijne et al., 1989).
Plant presequences are about 7-9 amino acid residues longer and contain about 2-5 times
more serine residues than non-plant presequences (Glaser, et al., 1998). Presequences are
known to adopt a positively charged amphiphilic α-helical conformation in membrane
mimicking environments, while they are largely unstructured in aqueous solutions (von
Heijne, 1986b; Moberg et al., 2004).
Cytosolic factors
Mitochondrial protein import is believed to occur in a post-translational manner. This
assumption is based on the fact that in vitro synthesized precursor proteins can be imported
into isolated mitochondria (Hallermayer et al., 1977; Wienhues et al., 1991). However, cotranslational protein import into mitochondria can not be ruled out in vivo (Suissa and Schatz,
1982; Furuya et al., 1991; Verner, 1993). A genome-wide analysis of mRNA encoding
mitochondrial proteins showed that some of the mRNA was closely associated with
mitochondrially bound polyribosomes (Marc et al., 2002). Interestingly, genes producing
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mRNA that are attached to mitochondria were mainly of ancient bacterial origin, while those
producing mRNA that is translated in the cytoplasm were mainly of eukaryotic origin. The
3´UTR of mRNA that were attached to mitochondria carries information required for their
targeting and attachments to mitochondria (Corral-Debrinski et al., 2000; Marc et al., 2002).
A majority of precursor proteins are synthesized in the cytosol at some distance from
mitochondria and must be targeted through the cytosol to mitochondria. Precursor proteins are
also imported into mitochondria in an extended or import competent form, different than their
final native conformation. Because of these requirements precursor proteins in the cytosol are
kept as complexes with chaperones or other factors that are believed to stabilize them, as they
are not in their right conformation and therefore are prone to aggregation and degradation.
Many of these factors have been described such as e.g. the aryl hydrocarbon receptor
interacting protein (AIP), presequence binding factor (PBF), mitochondrial import stimulating
factor (MSF) and cytosolic chaperones Hsp70 and Hsp90 (Mihara and Omura, 1996; Yano et
al., 2003; Young et al., 2003).
Translocase of the outer membrane (TOM)
The first step of recognition and subsequent translocation of precursor proteins across the OM
is accomplished by the multi subunit TOM complex. Virtually all mitochondrial proteins are
translocated across the OM by the TOM complex. The TOM complex was purified from yeast
as a ~ 490 kDa complex and contained TOM70, TOM40, TOM22, TOM20, TOM7, TOM6
and TOM5 (Ahting et al., 1999). The TOM complex acts as a receptor for the recognition of
mitochondrial precursor proteins synthesized in the cytosol and subsequent transfer through
import pores across the OM.
Import Receptors
There are three main receptors in S. cerevisiae mitochondria for precursor protein recognition
on the mitochondrial surface. These are TOM70, TOM20 and TOM22, named according to
their apparent molecular mass (Hines et al., 1990; Kunkele et al., 1998; Brix et al., 1999; Abe
et al., 2000). Each of these receptors is anchored in the OM by a single transmembrane (TM)
segment. TOM20 and TOM70 are anchored to the membrane via their N-terminus, while the
C-terminal domain is exposed to the cytosol. TOM22 has an inverted membrane topology
than TOM20 and TOM70 and also contains an additional small C-terminal domain protruding
into the IMS (Neupert, 1997). Precursor proteins with a presequence are first recognized by
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TOM20 and subsequently transferred to TOM22. TOM22 also plays a critical role for the
general integrity of the TOM complex (van Wilpe et al., 1999). The NMR structure of the
cytosolic domain of rat TOM20 complexed with a peptide derived from the aldehyde
dehydrogenase (ALDH) presequence revealed that the presequence forms an amphiphilic αhelix when bound to TOM20 (Abe et al., 2000) and their interaction is hydrophobic in nature.
Plant TOM20 differs from S. cerevisiae and animal TOM20 in its topology as it is anchored
to the membrane via the C-terminal domain (Macasev et al., 2000). TOM22 in plants is also
different to animal and S. cerevisiae TOM22. The plant TOM22 homologue lacks the
cytosolic acidic domain and is about 9 kDa in size. It has been suggested that the difference is
due to the unique environment in plants where both mitochondria and chloroplasts are present
and it is possible that TOM22 in plants provides targeting specificity (Macasev et al., 2004).
TOM70 is the main receptor for precursor proteins that contain internal targeting signals, such
as the ADP/ATP translocator (AAC) and Phosphate carrier (Pic) of the carrier family.
TOM70 is not only an import receptor but can also act as docking platform for cytosolic
chaperones (Young et al., 2003). Isolated TOM complexes from plants lack TOM70 and no
homologue has been found in the A. thaliana genome. The absence of TOM70 in plants is
puzzling since the carrier import pathway has been demonstrated (Lister et al., 2002).
The general import pore
After recognition and binding to the receptors, the precursor proteins are inserted into the
general import pore. The general import pore of the TOM complex is composed of the
TOM40 and three small TOM proteins TOM5, TOM6 and TOM7, through which all
precursor proteins cross the OM (Ahting et al., 2001). TOM40 is an integral membrane
protein that is believed to form a β-barrel structure. Purified TOM40 forms a cation-selective
channel of about 22 Å when inserted in artificial membranes (Hill, et al., 1998; Kunkele et al.,
1998; Becker et al., 2005). The pore diameter is large enough to accommodate an α-helical
peptide or even a protein loop. TOM40 is the only TOM protein that is essential for cell
viability in yeast under all growth conditions (Baker et al., 1990). The loss of individual small
TOM proteins does not lead to any major effects, but the simultaneous deletion of all three
small TOM is lethal (Sherman et al., 2005). The small TOM6 and TOM7 subunits do not
interact with precursor proteins during protein import, rather they modulate the stability of the
TOM complex (Alconada et al., 1995; Honlinger et al., 1996). TOM6 has been proposed to
support the cooperation between the TOM22 receptor and the general import pore (Alconda et
al., 1995; Dekker et al., 1998; van Wilpe et al., 1999).
20
According to the “acid chain hypothesis” electrostatic interactions are the main driving
forces behind the unidirectional protein translocation across the OM (Komiya et al., 1998).
Precursors with a presequence successively interact with at least five different TOM subunits
(TOM20, TOM22, TOM5, TOM40, and the IMS domain of TOM22) during translocation
across the OM. Most of these TOM subunits contain negatively charged patches and it has
been proposed that the positively charged presequence is recognized by increasing affinity
along the import pathway. However, a report by Muto et al. (2001) showed that the
presequence interacts with TOM20 via hydrophobic patches. This suggests that forces other
than ionic interaction are important for the interaction of the presequence with TOM subunits
and has led to a revised theory named the “binding chain hypothesis”.
Outer mitochondrial membrane proteins are first imported through TOM complex and later
inserted into the outer membrane using SAM (Sorting and Assembly Machinery) complex (as
revived by van der Lann, et al., 2005).
Translocase of the inner membrane (TIM)
After crossing the OM with the TOM complex, precursor proteins interact with either of two
TIM complexes in the IM, the TIM23 complex or the TIM22 complex. Matrix targeted or
presequence carrying precursor proteins are recognized and translocated by TIM23, while
polytopic IM proteins are inserted into the IM by the TIM22 complex. IM also contains the
OXA1 complex that mediates insertion of precursor proteins from the matrix side into the IM
(Hell et al., 1998; Jensen and Dunn, 2002).
TIM23 complex
The TIM23 complex is the main precursor protein translocase in the IM of mitochondria. The
TIM23 complex is responsible for translocating all precursors of matrix proteins, most inner
membrane proteins and many of the IMS proteins. Translocation by TIM23 requires an
electrical membrane potential (∆ψ) across the IM and the hydrolysis of ATP. The TIM23
complex is composed of two parts, the protein conducting channel and the protein import
motor or presequence translocase-associated motor (PAM). The protein conducting channel
consists of TIM50, TIM23, TIM17 and TIM21 (Bauer at al., 1996; Dekker et al., 1997;
Chacinska et al., 2005). The core of the translocase is formed by TIM17 and TIM23 with a
molecular mass of 90 kDa (Dekker et al., 1997). TIM23 and TIM17 are integral membrane
21
proteins with four TM segments. TIM23 and TIM17 are believed to form a pore through
which proteins are translocated into the matrix. TIM23 also exposes an N-terminal
hydrophilic region to the IMS. This part has been proposed to place the TIM23 complex in
the proximity to the OM (Donzeau et al., 2000). TIM50 is anchored into the IM by an Nterminal TM segment. TIM50 interacts with incoming proteins as they come out from the
TOM complex and can pass them to other subunits of the TIM23 complex. In this way TIM50
acts as a receptor for the TIM23 complex. TIM50 has also been proposed to have a role in
regulation of import channel’s permeability (Meinecke et al., 2006). TIM21 was found to be
directly interacting with the IMS domain of TOM22, suggesting a direct interaction between
TIM23 and the TOM complex (Chacinska et al., 2005; Mokranjac et al., 2005).
Most of the presequence carrying precursor proteins are imported into the matrix by the
combined action of the TIM23 protein conducting channel and the protein import motor. The
protein conducting channel can only transfer the presequence part of the precursors, which
requires ∆ψ. After the presequence emerges from the TIM23 pore, PAM has to take over. For
a long time it was thought that the ATP-dependent import motor consists of three proteins, the
peripheral inner membrane protein TIM44, the mitochondrial chaperone Hsp70 and the
nucleotide exchange factor Mge1. Recently two new essential co-chaperones have been
identified, Pam18 and Pam16 (Li et al., 2004; van der Laan et al., 2005). TIM44 recruits
Hsp70 in its ATP bound form, which then immediately can grasp the incoming unfolded
polypeptide as its substrate binding site is open (Schneider et al., 1994). After binding to the
emerging precursor, ATP is hydrolyzed, the substrate binding site closes and Hsp70 is
released. This release of Hsp70 requires the nucleotide exchange factor Mge1. Mge1 removes
the bound nucleotide and allows cycling of the ATP bound Hsp70 to the PAM (Schneider et
al., 1994).
Two mechanisms for the PAM have been proposed and evidence suggested that the
mechanisms co-operate in translocating the precursor proteins across the IM. The Brownian
ratchet mechanism (Neupert and Brunner, 2002) suggests that the precursors are prevented
from sliding back upon binding to the Hsp70 in the matrix. The matrix-bound Hsp70 biases
spontaneous oscillations of the incoming polypeptide chain toward the matrix, and makes new
Hsp70 binding sites accessible. Thus, by successive binding of Hsp70, the precursor protein is
trapped into the matrix. In the pulling mechanism (Matouschek et al., 1997) Hsp70 plays an
active role in the translocation. Upon ATP hydrolysis a conformational change of Hsp70 pulls
22
the polypeptide into the matrix. By using a model protein, Huang et al. (2002) showed that
simple trapping of precursor protein segments by Hsp70 was enough to import loosely folded
precursor proteins, while partially folded precursors also required more efficient Hsp70TIM44 cycling, suggesting that pulling is needed.
TIM22 complex
The TIM22 complex is required for the import and insertion of the carrier proteins and of the
hydrophobic TIM proteins (Kerscher et al., 1997; Kurz et al., 1999). Import via the TIM22
complex only requires membrane potential (∆ψ) and not ATP (Jensen and Dun, 2002). The
TIM22 complex is about 300 kDa in size and consists of the three integral membrane proteins
TIM22, TIM54 and TIM18 (Sirrenberg et al., 1996). TIM22 is a homologue of the TIM23
and TIM17 and is the only essential protein of the TIM22 complex (Kovermann et al., 2002).
TIM22 forms the essential core of the TIM22 complex that can mediate the insertion of
carrier proteins without TIM54 and TIM18 (Kovermann et al., 2002). The precise role of
TIM54 and TIM18 is not known, although both of these TIM proteins are required for the
formation and stability of the TIM22 complex.
A complex of the small TIM subunits comprising of the TIM8, TIM9, TIM10, TIM12 and
TIM13 in the IMS also interacts with the TIM22 complex. These complexes are proposed to
act as chaperones by transporting the hydrophobic IM proteins from the TOM complex to the
TIM22 complex through the aqueous IMS and preventing their aggregation (Koehler et al.,
1998a; Koehler et al., 1998b). TIM54 is believed to act as a binding site for the small TIM
complex because its interaction with TIM22 was destabilized in a TIM54 deletion mutant
(Kovermann et al., 2002). The small TIM complex can also play an important role in substrate
recognition by the TIM22 complex.
Protein Import to the intermembrane space
All of the proteins residing in the intermembrane space (IMS) are nuclear encoded and
imported from the cytosol. Bigger IMS proteins contain bipartite signal sequences consisting
of a matrix-targeting presequences followed by a hydrophobic sorting signal (Hartl et al.,
1987). Bipartite signal sequences direct the proteins to the IM before they are proteolytically
cleaved, thereby releasing the mature part of the protein into the IMS (Glick et al., 1992).
Small IMS proteins with sizes 7-16 kDa (e.g. small TIM) carry a characteristic “twin CX9C
motif”. Import of the Cox17 protein into the IMS was abolished upon mutating one of the
23
cysteine residues present in the CX9C motif (Heaton et al., 2000), indicating critical
importance of cysteine residues in import. Cysteine residues of the twin CX9C motif have
been also shown to be required for stability and folding of the proteins in the IMS (Lu et al.,
2004). Mesecke et al. (2005) have identified a “disulfide relay system” for protein import into
the IMS. It was shown that the newly arrived TIM13 and Cox17 proteins are entrapped in the
IMS by forming disulfide bonds with Mia40, a component of disulfide relay system. It was
also shown that the sulphydryl oxidase Erv1 directly interacts and is required for maintaining
Mia40 in an oxidized state. Depletion of either Erv1 or Mia40 in S. cerevisiae resulted in no
import of Cox17 and TIM13 into the IMS (Mesecke et al., 2005).
Processing peptidases
Once a precursor has been imported into the mitochondrial matrix, the presequence has
fulfilled its function and is no longer needed. The presequence may actually interfere with
further sorting and protein folding or assembly. There are three types of processing peptidases
in mitochondria that mediate removal of the presequence from the precursors. These are
Mitochondrial Processing Peptidase (MPP), Mitochondrial Intermediate Peptidase (MIP) and
Inner Membrane Peptidase (IMP) (as reviewed by Gakh et al., 2002).
MPP is an essential protein in S. cerevisiae and processes precursors that are fully
translocated to the matrix as well as precursors in transit to the IM or the IMS. MPP has been
purified and characterized from different sources including fungi, mammals and plants
(Glaser and Dessi, 1999; Gakh et al., 2002). MPP is an integral part of the cytochrome bc1
complex in plant mitochondria, while it is a soluble protein in fungal and mammalian
mitochondria (Braun et al., 1993; Eriksson et al., 1994). MPP is a heterodimeric protein
composed of α- and β-subunits of about 50 kDa each. The catalytic site is present in the β
subunit with a characteristic inverted zinc binding motif (HXXEH), while substrate
recognition and binding is mediated by the α-subunit (Luciano et al., 1997). MPP is classified
as a member of the pitrilysin family of proteases on the basis of the zinc binding motif
(Kitada et al., 1995). The crystal structure of the recombinant S. cerevisiae MPP in complex
with a synthetic presequence peptide has been determined (Taylor et al., 2001). The crystal
structure showed that the presequence peptide was bound in an extended conformation at the
active site present in a large polar cavity. It was suggested that the presequences adopt
context-dependent conformations through mitochondrial import and processing, helical for
24
recognition by mitochondrial import machinery and extended for cleavage by the main
processing component (Taylor et al., 2001).
A number of mitochondrial precursors destined to the mitochondrial matrix or the IM are
processed in two sequential steps by MPP and MIP. These precursors carry a characteristic RX (F/L/I)-X-X(T/S/G)-X-X-X-X (first arrow indicates cleavage by MPP and the second by
MIP) motif at the C-terminus of the presequence. The first cleavage is made by MPP one
residue downstream from the arginine that yields a processing intermediate with a typical Nterminal octapeptide that is sequentially cleaved by MIP producing a mature size protein
(Gakh et al., 2002). MIPs from different species are soluble monomers of about 70-75 kDa.
MIP is a thiol-dependent metalloprotease and belongs to the thimet (thiol and metal
dependent) oligopeptidase family (Barret et al., 1995). Deletion of MIP in S. cerevisiae causes
loss of respiratory competence, suggesting that MIP is involved in the biogenesis of some of
mitochondrial proteins. A number of substrates for MIP have been identified in S. cerevisiae
including CoxIV, ubiquinol-cytochrome c reductase iron sulphur protein (Fe/S) and malate
dehydrogenase (MDH) (Branda and Isaya, 1995). The biological significance of the
processing by MIP is not certain. It is known that the N-terminal region of MIP processed
precursor proteins is incompatible with cleavage by MPP and octapeptides may have evolved
to overcome this problem (Isaya et al., 1991).
Some of the proteins imported into the IMS carry a bipartite N-terminal targeting signal
consisting of a matrix-targeting signal, (Hartl et al., 1987), that is cleaved by MPP, followed
by a hydrophobic signal that is cleaved by IMP. In S. cerevisiae, IMP exists as a
heterodimeric protein composed of two different subunits: Imp1 and Imp2, both of these
subunits possess catalytic activity (Schneider et al., 1994). Each subunit is bound to the outer
face of the IM through an N-terminal membrane spanning domain and exposes the Cterminus with the catalytic site into the IMS (Daum et al., 1982). The catalytic sites of both
subunits are characterized by a conserved serine/lysine dyad (Chen at al., 1999). Interestingly,
each subunit recognizes different substrates. Imp1 is involved in the maturation of at least
three proteins; CoxII, Cyt b2 and NADH-cytochrome b5 reductase (Mcr1), whereas Imp2
cleaves the targeting signal of cyt c1 (Nunnari et al., 1993). Deletion of the Imp1 or Imp2 gene
in S. cerevisiae leads to no growth on-non fermentable carbon sources, indicating a role of
IMP in mitochondrial biogenesis. Both the Imp1 and Imp2 are homologous to the signal
peptidases of the bacterial and ER membrane (Dalbey et al., 1991). A potential third subunit
25
of the IMP complex, Som1, has been identified using co-immunoprecipitation and cross
linking experiments (Jan et al., 2000). Som1 is required for the processing of two of the three
known substrates of Imp1 (CoxII and Mcr1), but not for the processing of the Cyt b2 (Esser et
al., 1996).
Chloroplastic protein import machinery
The majority of the chloroplastic proteins are nuclear encoded and post translationally
imported into the chloroplasts in a similar way as for mitochondria (Figure 2). Import into the
chloroplast involves transit peptides, cytosolic factors and two translocases present at the
outer and inner envelope of the chloroplasts. Being a recent organelle in the modern
eukaryotic plant cell, the chloroplastic import machinery possesses unique features to ensure
the targeting specificity. Isolated P. sativum chloroplasts have been used as a model system to
identify and characterize the components of the chloroplastic protein import machinery using
a variety of biochemical techniques (Perry and Keegstra, 1994; Schnell et al., 1994).
Sequencing of the A. thaliana and O. sativa genomes has enabled the use of more advanced
genetic techniques in search of the import machinery components.
Chloroplastic targeting peptides – the transit peptides
Chloroplastic targeting peptides called transit peptides do not show any sequence consensus at
the primary structure level and vary greatly in length from 13 to 146 amino acid residues with
an average length of about 60 residues. Generally, transit peptides are longer than
mitochondrial presequences (Zhang and Glaser, 2002; Bhushan et al., 2006). Interestingly,
transit peptides are very similar in overall amino acid composition to the presequences. They
are enriched in hydroxylated and hydrophobic amino acids, have some positively charged
residues and lack negatively charged amino acids. In comparison to presequences, positively
charged amino acids are usually lacking in the very N-terminal part of transit peptides
(Peeters and Small, 2001; Zhang and Glaser, 2002; Bhushan et al., 2006). Transit peptides are
mainly unstructured in an aqueous environment and it has been proposed that transit peptides
have evolved to maximize the potential to form a random coil (Bruce, 2000). Both ferrodoxin
(Fd) and Rubisco activase transit peptides (Lancelin et al., 1994; Krimm et al., 1999) from
Chlamydomonas reinhardtii were shown to contain a helix and a random coil structure as
determined by NMR. NMR structural data available for the higher plant transit peptide from
the Silene Fd shows that addition of micelles to Fd transit peptide induced N- and C-terminal
helical formation in the transit peptide. However, induced helices were short and contained
26
only 3-4 amino acid residues indicating that the major part of the transit peptide remained
unstructured even in the presence of membrane mimicking environment (Wienk et al., 2000).
Figure 2. The chloroplastic protein import machinery. General overview of the
protein import into stroma. TOC and TIC refer to Translocases of the outer and
inner envelope membrane of chloroplasts. The numbers represent the molecular
masses of the components of the TOC and TIC complexes. SPP, Stromal
processing peptidase; Hsp, Heat shock protein; Cpn60, Chaperonin 60; PreP,
PresequenceProtease (modified from Bedard and Jarvis, 2005).
Cytosolic factors
Chloroplastic precursor proteins are synthesized in the cytosol and have to be imported into
the organelle post-translationally and therefore they need to be protected from aggregation
and degradation. During or after translation in the cytosol most of these precursor proteins
associate with cytosolic factors or chaperones. This interaction is believed to be non selective
and due to the unfolded protein exposing hydrophobic amino acids. In vitro import of
27
bacterial overexpressed and urea denatured light-harvesting chlorophyll-binding protein
(LHCP) precursor protein into chloroplasts was greatly stimulated by cytosolic factors
(Waegemann et al., 1990). One of these factors could be replaced by purified Hsp70. Some of
the chloroplastic transit peptides contain a motif that can be phosphorylated on a serine or
threonine residue by a protein kinase. Some of the abundant chloroplastic precursor proteins
such as the precursors of small subunit of ribulose bisphosphate carboxylase/oxygenase
(SSU), LHCP and outer envelop 23 (OE23) have been shown experimentally to become
phosphorylated (Waegemann and Soll, 1996), while many more are predicted to contain the
potential phosphorylation motif. The phosphorylated precursor protein interacts with the 143-3 protein and Hsp70 to form a guidance complex (May and Soll, 2000). 14-3-3 proteins
belong to a ubiquitous protein family of regulatory proteins with their main function being
molecular chaperones mediating protein-protein interaction (Aitken et al., 1992). Binding of
precursor proteins to the guidance complex stimulated the import rate about 4-5 fold into
chloroplasts when compared to the free precursor (May and Soll, 2000). However, removal of
the phosphorylation site does not result in loss of the targeting specificity (Waegemann and
Soll, 1996). Martin et al. (2006) have recently isolated a serine/threonine protein kinase from
A. thaliana that is able to phosphorylate chloroplast targeted precursor proteins.
Translocase of the outer envelope membrane (TOC)
Like the TOM complex of mitochondria the TOC complex is involved in both recognition and
translocation of chloroplastic precursor proteins across the outer envelope membrane of
chloroplasts. Unlike the mitochondrial TOM complex, translocation through the TOC
complex is an energy dependent process (Jarvis and Soll, 2001). The core of the TOC
complex isolated from P. sativum chloroplasts consists of three proteins, TOC34, TOC75 and
TOC159, named according to their molecular masses (Perry and Keegstra, 1994; Waegemann
and Soll, 1995). The molecular stoichiometry of TOC75, TOC34 and TOC159 in the complex
was determined to be 4:4:1 (Schleiff et al., 2003).
TOC34 is anchored to the outer envelope membrane by a C-terminal tail, while a large Nterminal domain is exposed into the cytosol (Seedorf et al., 1995). The N-terminal domain
possesses GTP binding and GTPase activity of TOC34 (Kessler et al., 1994). Becker et al.
(2004) have suggested that TOC34 acts as an initial receptor on the chloroplastic surface.
TOC34 binds precursor proteins with high affinity in its GTP bound form. The precursor
functions as a GTPase activating factor and stimulates the GTP hydrolysis of TOC34 by about
28
40-50 fold (Jelic et al., 2002). TOC34-GDP has a much lower affinity for the precursor,
which continues its path to the next translocon subunit, most likely TOC159. There are two
TOC34 homologues present in A. thaliana, named AtTOC34 and AtTOC33. AtTOC33 is
expressed predominantly in photosynthetic and meristematic tissue, while AtTOC34 is
expressed in all tissues, but at a relatively lower level (Jarvis et al., 1998; Gutensohn et al.,
2000).
TOC159 was the first TOC component to be identified (Waegemann and Soll, 1991; Perry
and Keegstra, 1994; Ma et al., 1996). TOC159 is proposed to be the main receptor for the
chloroplastic import machinery (Kessler et al., 1994; Perry and Keegstra, 1994). TOC159 is
composed of the three domains: an N-terminal A-domain that contains many acidic amino
acid residues, a central G-domain containing a GTP binding domain with sequence homology
to TOC34 (Hirsch et al., 1994, Kessler at al., 1994) and a C-terminal M domain that is
essential for targeting and anchoring to the membrane (Lee at al., 2003). TOC159 is essential
as A. thaliana seedlings with TOC159 knockout die early during development (Bauer et al.,
2000).
TOC75 is the most abundant outer envelope protein. TOC75 forms the pore through which
precursor proteins cross the outer envelope membrane. Overexpressed and purified TOC75
forms a cation-selective channel when inserted into the lipid bilayer (Hinnah et al., 2002).
TOC75 is predicted to be a β-barrel protein with 16 TM β-sheets (Sveshnikova et al., 2000).
Calculation of the pore diameter indicates that the channel is approximately 15-25 Å wide
(Hinnah et al., 2002). This is wide enough to accommodate a polypeptide chain with some
secondary structure (Hinnah et al., 2002). TOC75 has a protein binding site at the cytosolic
face of the channel that can discriminate between the precursor and mature form of the protein
(Ma et al., 1996; Hinnah et al., 1997). There are four TOC75 homologues present in A.
thaliana, however only one isoform is dominantly expressed.
The role of the fourth TOC component, TOC64, is not well defined. TOC64 exposes
tetratricopeptide repeats in the cytosol, like the peroxisomal receptor Pex5 or the
mitochondrial receptor TOM70 (Sohrt and Soll, 2000). On the basis of similarity to TOM70 it
has been proposed that TOC64 has a similar role to that of TOM70 in recognition of polytopic
membrane proteins (Soll and Schleiff, 2004). Becker et al. (2004) have identified a new TOC
component, TOC12. TOC12 carries a J-domain and stimulates the ATPase activity of Hsp70.
29
Translocase of the inner envelope membrane (TIC)
After their translocation through the TOC complex into the IES, the precursor proteins are
transferred to the TIC complex for translocation across the inner envelope membrane. ATP is
required for translocation across the inner envelope membrane (Flugge and Hinz, 1986).
Several TIC components have been identified, however their role in import is less well
defined. The TIC translocase is a multi subunit complex consisting of TIC110, TIC62, TIC55,
TIC40, TIC32, TIC22 and TIC20 (as reviewed by Gutensohn et al., 2006).
TIC110 is an abundant protein in the inner envelope and has one or two TM segments in
its N-terminal region (Kessler and Blobel 1996; Lubeck et al., 1996). TIC110 is believed to
form a pore in the inner envelope and can form a cation-selective channel when inserted in the
lipid bilayer (Heins et al., 2002). The pore diameter was estimated to be between 15-20 Å,
which is the same as for TOC75. TIC40 is an integral membrane protein tightly associated
with TIC110 (Stahl et al., 1999). The exact role of TIC40 is not known, but it shares some
sequence similarity with Hsp70-interacting protein (Hip) in its C-terminal domain (Chou et
al., 2003). Hip is a mammalian co-chaperone that regulates nucleotide exchange by Hsp70
(Hohfeld et al., 1995; Frydman and Hohfeld, 1997) and it may be possible that TIC40 has a
role in chaperone recruitment at the TIC complex during protein import into chloroplasts. A
role for TIC40 as a chaperone recruitment factor is further supported by the demonstration
that Hsp93 and TIC40 can be immunoprecipitated together (Chou et al., 2003). Three of the
subunits of the TIC translocase, TIC62, TIC55 and TIC32 are redox components of the TIC
complex. TIC55 contains a Rieske iron sulphur center and a mononuclear iron binding site,
which indicates the potential for electron transfer (Caliebe et al., 1997). TIC62 contains a
conserved NAD/NADP binding site and a C-terminal motif, which interacts with stromal
ferredoxin-NAD/NADP reductase (Kuchler et al., 2002). Ferredoxin-NADP-reductase
connects photosynthetic electron transfer with metabolically required reducing power. TIC62
might therefore represent a link between the metabolic redox status of the chloroplasts and
TIC translocon (Hirohasi et al., 2001). TIC32 belongs to the family of short chain
dehydrogenases, which also use NAD/NADP as a cofactor. TIC22 is localized to the IES and
has been proposed to be a link between TOC and TIC complexes or in the transfer of proteins
across the IES (Ma et al., 1996; Kouranov and Schnell, 1997). TIC20 is another integral
subunit of the TIC complex with homology to bacterial amino acid transporters and TIM17 of
mitochondria (Kouranov et al., 1998; Rassow et al., 1999), and has been suggested to take
30
part in the channel formation. There is need of more biochemical work on these individual
TIC subunits to define their exact role in chloroplastic protein import.
Stromal processing peptidase
Stromal Processing Peptidase (SPP) is the protease that cleaves off the transit peptides from
precursor proteins after their import into the stroma. SPP is responsible for cleaving off the
transit peptide from a number of different precursor proteins involved in different biosynthetic
pathways and destined for different locations in the chloroplasts (Richter and Lamppa, 1999).
SPP was initially purified from P. sativum chloroplasts as a soluble metalloprotease of about
100 kDa (Oblong and Lamppa 1992). SPP contains an inverted zinc binding motif (HXXEH)
characteristic of members of the metallopeptidase family of pitrilysin proteases such as
pitrilysin, insulin degrading enzyme (IDE) and the catalytic β-subunit of mitochondrial MPP
(Rawlings et al., 2006). Down regulation of SPP in A. thaliana yielded many lines that were
seedling lethal. Import of a model precursor protein was defective in surviving plants,
indicating a critical function for SPP in the chloroplast protein import pathway (Zhong et al.,
2003). SPP initially recognizes a precursor by binding to the transit peptide and then cuts it
off in a single proteolytic event. The mature form of the protein is then released, while SPP
remains bound to the transit peptide. Before the release from SPP, transit peptides are further
cleaved into sub fragments by a second proteolytic event (Richter and Lampaa, 2000, 2003).
Dual targeting to mitochondria and chloroplasts
Plant cells contain both mitochondria and chloroplasts and therefore require more efficient
sorting mechanism than non plant cells. The existence of a higher order protein sorting is
evident from in vivo studies where protein import into these two organelles was shown to be
highly specific (Boutry et al., 1987; Schmitz and Lonsdale 1989; Silva-Filho et al., 1997).
There are a number of proteins present in these two organelles with similar functions that are
encoded by a distinct gene for each organelle. However, there are some proteins encoded by a
single gene but targeted to both mitochondria and chloroplasts, referred to as dual targeted
proteins (Peeters and Small, 2001). Since the first report of dual targeting of P. sativum
glutathione reductase (GR) by Creissen et al. (1995), 33 dually targeted proteins have been
identified and it is expected that there will be many more (Silva-Filho, 2003). In silico
analysis of the A. thaliana genome predicted that as many as 160 proteins may be dually
targeted to both mitochondria and chloroplasts (Small I, personal communication).
31
The mystery of dual targeting lies in the targeting peptide by which the precursors are
targeted and imported into both mitochondria and chloroplasts. Analysis of the dual targeting
peptides has revealed that they are intermediate in length and have an overall amino acids
composition similar to that of mitochondrial and chloroplastic targeting peptides. However,
they contain fewer alanines and a greater abundance of phenylalanine and leucine, suggesting
that dual targeting peptides are more hydrophobic (Peeters and Small, 2001). This implies that
they have potential to be targeted and imported simultaneously to both of these organelles.
Studying dual targeting
Targeting of proteins to mitochondria and chloroplasts has been studied using a number of
different experimental approaches including both in vivo and in vitro methods. When it comes
to studying the targeting of a dual targeted protein, none of these methods alone is ideal. The
most commonly used method to study subcellular localization of dually targeted proteins is an
in vivo method expressing a chimeric construct consisting of a reporter protein such as green
fluorescence protein (GFP) fused to the full length precursor or targeting peptide (Peeters and
Small, 2001). In vivo methods use an intact cellular system and are the best system to study
the in vivo targeting capacity of a targeting peptide. However, there are some limitations of
this system: 1) fusion construct often use a small part of the protein coupled to a reporter
protein and therefore the role of the mature protein is ignored, 2) fusion proteins are usually
under a strong promoter and overexpressed at very high level which can affect targeting and
3) it is not possible to study the kinetics and efficiency of protein recognition and import.
Another method is to import in vitro synthesized radiolabelled precursor proteins into
isolated organelles. This in vitro method can be useful sometimes but has other disadvantages:
1) isolated organelles lack an intact cellular system and other factors required for protein
sorting, 2) protein can be miss-targeted to an incorrect organelle and 3) there is no
competition between organelles. Rudhe et al. (2002a) established an in vitro dual import
system enabling the simultaneous import of radiolabelled precursor proteins into both
mitochondria and chloroplasts minimizing the miss-targeting associated with the classical
single organellar in vitro import system. A combination of different complementary methods
should be applied in order to study the targeting of a dual targeted protein.
32
Mechanisms of dual targeting
The majority of reports on single gene products that are targeted to more than one subcellular
location in a cell are related to mitochondria and chloroplasts. Dual targeting to these
organelles can be achieved in two ways whereby a single gene product can be targeted to both
mitochondria and chloroplasts: either through an ambiguous targeting signal or via a twin
targeting signal (Peeters and Small, 2001). The precursor proteins with an ambiguous
targeting signal are synthesized as a single polypeptide, but can be recognized and transported
by the import machinery of both mitochondria and chloroplasts (Small et al., 1998). The
majority of the known dually targeted proteins carry an ambiguous targeting signal. Most of
these proteins are involved in gene expression e.g. most of the aminoacyl-tRNA synthetases
and RNA polymerases. Other dually targeted proteins are involved in various processes such
as: biosynthetic pathways, phosporibosyl aminoimidazole synthase (Smith et al., 1998) and
phosphatidylglycerophosphate synthase I (Babiychuk et al., 2003); protein modification
function such as methionine amino-peptidase (Giglione et al., 2000) and; anti-oxidant
activities such as GR. It has been suggested by Chew et al. (2003) that the enzymes involved
in entire enzymatic cycles may be dually targeted. They have shown that main components of
the ascorbate glutathione cycle in A. thaliana, ascorbate peroxidase, monodehydroascorbate
reductase (MDAR) and GR were dually targeted to mitochondria and chloroplasts both in
vitro and in vivo (Chew et al., 2003). See also page 46 in this thesis and papers I and II.
Twin targeting signals have two separate targeting signals for mitochondria and
chloroplasts in tandem and at a given time only one targeting signal is present in the precursor
protein. Twin targeting signals may arise by either alternative transcription or translational
initiation, alternative splicing or via post translational modifications resulting in the formation
of two different precursors with distinct targeting specificity. Twin targeting signals seem not
to be common among dual targeting proteins. Protox, a protein involved in the biosynthesis of
chlorophyll and heme, and THI1 involved in thiamine biosynthesis are dually targeted using
alternative translational initiation, with the longer form of protein targeted to chloroplasts and
the shorter to the mitochondria (Chabregas et al., 2003; Watanabe et al., 2001). A. thaliana
MDAR is dually targeted using alternative transcription start sites, producing two forms of
mRNAs, the longer form of mRNA is translated with a mitochondrial targeting signal, while
the shorter one is translated with a chloroplastic targeting signal (Obara et al., 2002). A
domain structure was proposed for the dually targeted P. sativum GR targeting peptide,
33
indicating that targeting information for mitochondria and chloroplasts is located in different
domains (Rudhe et al., 2002b).
Proteolytic system in mitochondria
Proteolysis is important for the biogenesis, morphology and homeostasis of mitochondria.
There are about 40 proteases predicted to be present in mitochondria, but only a very few of
them have been characterized so far (Esser et al., 2002). Non-selective degradation of
mitochondrial proteins occurs in the lysosome after autophagy of the whole organelle,
whereas selective degradation is mediated by proteases within the mitochondrion (as
reviewed by Kaser and Langer 2000). Mitochondrial proteases can be classified into two
classes based on their requirements for ATP: ATP-dependent and ATP-independent.
ATP-dependent proteases
ATP-dependent proteases are involved in the assembly of mature proteins by regulation of the
stoichiometric amount of polypeptides in protein complexes and are also required for the
removal of miss-folded and damaged proteins. These proteases catalyze the first step of
degradation by cleaving the substrate polypeptide into peptides that are later cleaved to free
amino acids by ATP-independent proteases. ATP-dependent proteases do not require ATP for
hydrolysis, but rather for unfolding of target polypeptides and to regulate their proteolytic
activity. Mitochondria contain a few ATP-dependent proteases including the membrane
bound FtsH protease and the soluble Lon and ClpP proteases (Kaser and Langer, 2000; Adam
and Clarke, 2002; Urantowka et al., 2005).
The FtsH (AAA) protease
The FtsH proteases, also called AAA-proteases, are membrane bound, ATP-dependent
metalloproteases. FtsH proteases are required for the assembly of the newly imported proteins
into their native protein complexes by degrading superfluous subunits (Langer, 2000). These
proteases are present in eubacteria and in mitochondria and chloroplasts. S. cerevisiae
mitochondria contain two classes of AAA-proteases with different topologies, named the mAAA and i-AAA proteases (Leonhard et al., 1996, 2000; Klanner et al., 2001). The m-AAA
protease catalytic site faces the matrix side, forms a mega complex of 1 Mega Dalton (MDa),
and is composed of Yta10 and Yta12 subunits (Yeast Tat binding like proteins). The i-AAA
protease catalytic site is exposed to the IMS and also forms a 1 MDa complex (Langer, 2000).
Whereas there is a single gene of FtsH protease in bacteria, there are three homologues
34
present in S. cerevisiae and humans (Arnold and Langer, 2002). It has been suggested that
AAA-proteases function in the degradation of the non-assembled membrane proteins. A non
assembled CoxII protein and protein inhibitors of the ATP synthase have been identified as
native substrates of the i-AAA type of FtsH protease (Pearce and Sherman, 1995; Kominsky,
et al., 2002). The cellular function of mitochondrial FtsH protease has been studied in detail in
S. cerevisiae, where severe phenotypes were shown to be associated with mutations in these
proteases (Leonhard et al., 1996; Weber et al., 1996). The deletion of either Yta10 or Yta12
subunits resulted in inhibition of respiration and impaired the degradation of none assembled
IM proteins and assembly of the respiratory chain complexes and ATP synthase (Arnold and
Langer, 2002). It has been also shown that the mutations in m-AAA proteases affected
splicing of CoxI and Cob, both of which are encoded by genes in the mitochondrial genome
(Arlt et al., 1998). Mitochondrial dysfunction such as impaired respiration and changes in
morphology were shown in S. cerevisiae lacking the i-AAA protease subunit Yme1 (Yeast
mitochondrial escape) (Thorsness et al., 1993; Campbell et al., 1994). Mutations in the human
homologue of the AAA-protease, paraplegin, cause Hereditary Spastic Paraplegia (HSP), a
neurodegenerative disorder (Casari et al., 1998).
In plants, the A. thaliana genome harbors a total of 12 FtsH like genes (Sokolenko et al.,
2002) with four suggested to be localized to mitochondria and rest eight to chloroplasts
(Adam and Clarke, 2002). Our knowledge of FtsH proteases in plants is limited. A plant mAAA protease (PsFtsH) has been identified and studied from P. sativum (Kolodziejczak et al.,
2002). PsFtsH was shown to complement respiratory defects of S. cerevisiae lacking the mAAA protease, indicating a conserved function of AAA-protease from fungi to plants. On the
basis of Blue-native gel electrophoresis, it has been suggested that plants, in contrast to yeast,
have more than one i-AAA protease complex (Urantowka et al., 2005).
Lon-like Protease
Lon Like proteases belong to a conserved protein family with members present in eubacteria,
archaebacteria and eukaryotes (Van Dyck and Langer, 1999). These are ATP-dependent,
serine proteases present in both the mitochondrial matrix and the chloroplastic stroma (Suzuki
et al., 1994). Functional conservation between various members of the Lon protease family
has been demonstrated by complementation studies in S. cerevisiae (Barakat et al., 1998;
Teichmann et al., 1996). Mitochondria in S. cerevisiae contain the Lon homologue, Pim1
(Protease in mitochondria), which is a homo-oligomer composed of 7 subunits and is about
35
800 kDa in molecular mass (Stahlberg et al., 1999). Pim1 consists of two catalytic domains,
an ATPase domain and a protease domain. Pim1 in S. cerevisiae mitochondria cleaves several
non assembled proteins such as the β-subunit of MPP, and the α-, β- and γ-subunits of ATP
synthase (Kaser and Langer, 2000). Inactivation of Pim1 in S. cerevisiae causes severe
phenotypes in mitochondria indicating specific regulatory functions of Pim1. Cells lacking
Pim1 are unable to maintain mitochondrial DNA and are respiratory deficient. Pim1 mutants
are defective in splicing of CoxI and Cob mRNAs (Van Dyck et al., 1994). This role of Pim1
in splicing is similar to that of m-AAA proteases and indicates that Pim1 can also be required
for the processing of enzymes required in mRNA maturation. Down-regulation of human Lon
protease resulted in disruption of mitochondrial structure and function and eventually cell
death within four days. Cell death in the majority of these cells was a result of caspase 3
activated apoptosis (Bota et al., 2005). It has been suggested that the Lon proteolytic system
plays an important role in the degradation of oxidized proteins in the mitochondrial matrix
and in the maintenance of mitochondrial structure and functional integrity (Bulteau et al.,
2006).
There are four Lon homologues present in the A. thaliana genome. Lon1 is localized to
mitochondria, Lon2 to peroxisomes, the remaining two are predicted to be localized to the
chloroplasts (Adam et al., 2001). A gene encoding Lon protease has been reported from Z.
mays (Barakt et al., 1998). Z. mays Lon1 can complement mitochondrial DNA maintenance in
Pim1 deficient S. cerevisiae. In legume mitochondria, Lon1 has been identified as at least one
of the proteases involved in the degradation of orf239, a cytoplasmic male sterility (CMS)
associated protein in mitochondria (Sarria et al., 1998).
ClpP-like protease
Lon and Clp (Caseinolytic protease) are the two major proteases in E. coli accounting for
about 80% of protein degradation. Homologues of Clp proteases are present in mammalian
and plant mitochondria, but are absent in S. cerevisiae (Corydon et al., 1998; Santagata et al.,
1999; Halperin et al., 2001a). Most of our knowledge about Clp proteases is based on E. coli
Clp protease. Clp-like proteases form hetero-oligomeric complexes with an interior chamber
for proteolysis and consist of proteolytic (ClpP) and regulatory (ClpA or ClpX) subunits. The
catalytic ClpP subunit has an active site similar to serine proteases, a catalytic triad of SerHis-Asp, whereas regulatory subunits have a chaperone like function. The crystal structure of
E. coli Clp protease has been solved at 2.3 Å resolution (Wang et al., 1997). The proteolytic
36
chamber is composed of two central heptameric rings of ClpP that form a hollow chamber of
50 Å, flanked by one or two hexameric rings of ClpA or ClpX. The catalytic chamber has two
narrow openings of about 10 Å at either end, indicating that the substrate needs to be unfolded
before entering into the proteolytic chamber. ClpP shares similar structural features to the S.
cerevisiae 20S proteasome and the E. coli Hs1V (Lowe et al., 1995; Bochtler et al., 2000).
Bacterial Clp protease is involved in degradation of specific regulatory proteins, aggregated
and mis-folded proteins, nascent peptide chains that are stalled on the ribosomes, and proteins
involved in the stress and starvation responses (Gottesman et al., 1998). A cellular quality
control role has been suggested for the Clp proteases (Adam et al., 2006).
Plants have a battery of Clp like proteases. There are at least 30 Clp-related genes in the A.
thaliana genome, 15 of these genes encoding plastid localized proteins, 5 serine type ClpP
proteases containing catalytic triad motifs, 4 ClpP related ClpR proteins lacking catalytic triad
motifs, 3 ClpA homologues (ClpC1, ClpC2 and ClpD), and 3 Clp proteins with unknown
function (ClpS1, ClpS2 and ClpT) (Halperin et al., 2001b; Peltier et al., 2004; Adam et al.,
2006). ClpP2, ClpX1 and ClpX2 are predicted to be located in mitochondria. Plant Clp
proteases are believed to play a housekeeping role since they are constitutively expressed
under different growth and environmental conditions (Halperin et al., 2001b; Adam et al.,
2006).
ATP-Independent proteases
Beside the processing peptidases MPP, MIP and IMP, mitochondria also contain a few ATPindependent
proteases,
which
are
oligopeptidases.
These
include:
mitochondrial
oligopeptidases, MOP and MOP112, in the IMS of yeast (Buchler et al., 1994; Kambacheld et
al., 2005); Oma, a membrane bound metalloprotease (Kaser et al., 2003) with overlapping
functions with the m-AAA protease and; a serine rhomboid membrane protease responsible
for the degradation of intermembrane space proteins (Van der Bliek and Koehler, 2003).
Oligopeptidases
Oligopeptidases are endopeptidases that act on shorter peptides of about 6-18 amino acid
residues (Barrett et al., 1995). In S. cerevisiae, MOP is mainly located in the cytosol, but
some of the activity has also been reported in mitochondria. MOP contains an N-terminal
presequence and is targeted to different subcellular locations by using alternative promoters
(Buchler et al., 1994; Serizawa et al., 1995; Kato et al., 1997). Deletion of MOP in S.
37
cerevisiae does not lead to any major defects, but cells exhibit a decrease in the intracellular
degradation of a collagen like substrate, indicating that MOP plays a role in the late stages of
protein degradation (Serizawa et al., 1995).
OMA protease
OMA (overlapping function with the m-AAA proteases) was identified as a conserved
metallopeptidase, a novel component of the quality control system in the inner membrane of
mitochondria from S. cerevisiae (Kaser et al., 2003). OMA1 was shown to have functions
overlapping with the m-AAA protease and cleaves a mis-folded polytopic membrane protein
(OXA1) in an ATP-independent manner at multiple sites. Proteins homologous to OMA1
comprise a large protein family with members present in higher eukaryotes, including plants,
as well as in eubacteria and archaebacteria. Although different in their domain structure, all of
them are predicted to be integral membrane proteins and contain a metallopeptidase domain
characteristic of the M48 family of proteases, suggesting that OMA1 represents a novel
enzyme class capable of degrading membrane proteins (Kaser et al., 2003). It was proposed
that OMA1 functions under conditions of limited m-AAA proteolytic activity (Kaser et al.,
2003).
Rhomboid protease
Rhomboid protease is an interesting integral membrane protease that cleaves the substrate
within the membrane. Rhomboid protease was initially identified in Drosophila Golgi bodies.
Here it regulates epidermal growth factor receptor signaling by cleaving the transmembrane
domain of Spitz, the principal ligand for the receptor in flies, and promoting its release from
signal-sending cells (Urban et al., 2001). Rhomboid protease is a serine protease with a
characteristic Ser-His-Asn catalytic triad. Rhomboid proteases are present in all three domains
of life, archea, bacteria and eukaryotes (Koonin et al., 2003). Wang et al. (2006) have recently
solved a 2.1 Å resolution crystal structure of the rhomboid core domain. The structure
contained six transmembrane segments. Catalytic site residues, the Ser-His dyad, and several
water molecules were found at the protein interior at a depth below the membrane surface.
These observations indicated that, in intramembrane proteolysis, the scission of peptide bonds
takes place within the hydrophobic environment of the membrane bilayer.
Whereas most prokaryotes have a single gene encoding the rhomboid protease, Drosophila
has seven and A. thaliana has eight. One of the S. cerevisiae homologues of rhomboid, Rbd1
38
has been shown to be located in mitochondria. Rbd1 has been shown to be involved in
processing of the bipartite signal peptides of cytochrome c peroxidase, Ccp1 and a Dynaminlike GTPase, Mgm1 (Esser et al., 2002; Herlan et al., 2003, 2004; Mc Quibban et al., 2003;
van der Blick and Koehler, 2003). It has been suggested that cleavage of Mgm1 by the
rhomboid protease regulates mitochondrial membrane remodeling indicating a role of
proteases in regulating membrane biogenesis (McQuibban et al., 2003). Cipolat et al. (2006)
have also shown that rhomboid protease directly regulates apoptosis by controlling
cytochrome c release from mitochondria. There is currently no information on the role of the
rhomboid protease in plants, but two of them are predicted to be localized to mitochondria in
A. thaliana and possibly have a role in processing the N-terminal extension present on few
carrier proteins (Murcha et al., 2004).
Proteolytic system in chloroplasts
Proteolysis is required for a number of processes during the biogenesis and maintenance of
chloroplasts. Proteolysis should therefore be considered a vital homeostatic factor that
influences metabolic functions such as photosynthesis under both normal and adverse growth
conditions (Adam, 2000, 1996). Chloroplasts contain defined proteases within each
compartment; the ATP-dependent Lon, Clp in the stroma and FtsH in stroma exposed
thylakoid membranes; the ATP-independent DegP protease within the thylakoid lumen and
on both sides of thylakoids membrane and the SppA protease on the stromal side of the
thylakoid. All five of these chloroplastic proteases are homologous to bacterial proteases and
are present in multiple copies in higher plants.
ATP-dependent proteases
The FtsH protease
FtsH proteases in chloroplasts were first identified by immunoblot analysis from S. oleracea
leaves. They were characterized as integral thylakoid membrane proteins expressed in a light
dependent manner (Lindahl et al., 1996). The ~70 kDa protein is bound to the stroma exposed
lamellae, with its metal binding and ATP binding sites facing into the soluble stroma. There
are a total of sixteen FtsH genes present in the A. thaliana genome with four of them having
incomplete metal binding site (Sokolenko et al., 2002). These four FtsH with incomplete
metal binding sites might be involved in chaperone like functions because they retain the
AAA-domain. In chloroplasts FtsH1 is involved in the degradation of unassembled subunits
39
of membrane complexes, such as the Rieske Fe-S protein of the cytochrome bf complex and
the degradation of oxidatively damaged proteins such as the D1 protein of the photosystem II
(PSII) reaction centre (Lindahl et al., 2000; Ostersetzer and Adam, 1997).
Mutations in the FtsH2 homologue in A. thaliana leads to a variegated phenotype (leaves
with green and yellow sections) (Chen et al., 2000). Ultrastructural analysis of the variegated
leaves shows underdeveloped plastids in the yellow sections and normal chloroplasts in the
green sections (Chen et al., 2000; Takechi et al., 2000). This suggests a role of FtsH protease
in chloroplasts development. However, the patchy phenotype implies that the loss of FtsH2
can be compensated for, at least partially within the green leaf sections. Because up to eight
different FtsH proteases are believed to be localized to chloroplasts, some of them can
probably compensate for each other. The role of FtsH proteases in biogenesis is supported by
a report from cyanobacteria where the loss of four FtsH genes resulted in reduced level of
functional PSI by 60%, whereas PSII and phycobilisome levels remained unchanged (Mann,
et al., 2000). Recently, the FtsH11 homologue in A. thaliana has been implicated as playing a
critical role in A. thaliana thermo-tolerance (Chen et al., 2006).
Clp-like protease
Clp is a multi-subunit enzyme complex, in which the catalytic and ATPase domain are
present on different subunits. Clp proteases are believed to be the main proteases responsible
for most of the protein degradation in stroma. There are about two dozen genes in A. thaliana
encoding four Clp subunits (Zheng et al., 2006) with ClpP1 encoded in the chloroplast
genome. Chloroplasts do not contain ClpX homologue; instead ClpC and ClpD are suggested
to function as the regulatory subunits of the chloroplastic Clp protease. The identified core
complex of the Clp protease is about 325-350 kDa in size and consists of five ClpP isomers
(ClpP1, ClpP3, ClpP4, ClpP5 and ClpP6), four proteolytically inactive or regulatory subunits
(ClpR1 to ClpR4) and two plant specific subunits ClpS1 and ClpS2 (Peltier et al., 2001).
Although the function of ClpR remains unknown, it might control access of the substrate to
the catalytic site in a chaperone like manner. In A. thaliana, most of the Clp genes showed an
increase in transcript level under high light conditions, but were less responsive to
temperature shifts (Zheng et al., 2002; Sinvany-Villalobo et al., 2004). These results together
with the proteome analysis imply that all isomers constituting the protease core complex
40
substantially accumulate in all plastid types and that regulation of the proteolytic activity of
the Clp complex may depend on regulatory subunits.
The exact role of Clp proteases is unknown, however they are essential for chloroplast
function. Inactivation of the ClpP1 (encoded in the chloroplasts genome) in Nicotiana and
Chlamydomonas shows that ClpP1 is essential for cell viability (Huang et al., 1994; Kuroda
and Maliga, 2003). Knockdown of ClpP4 in Nicotiana by antisense methods caused severely
reduced growth with chlorotic leaf tissues (Shen et al., 2007). This may mean that each
isomer is required for the formation of the functional complex, supporting the heterooligomeric composition as proposed by Peltier et al. (2004). As for other subunits, visible
phenotypes such as yellow and pale green leaves have been reported in the knockout or
knockdown mutants of ClpR1, ClpR2, ClpR4, ClpC1 and ClpB3, whereas no clear
phenotypes were observed for ClpC2, ClpD and ClpT (Sjögren et al., 2006). Interestingly,
mutations in ClpC2 have been shown to suppress a variegated phenotype caused by loss of an
FtsH (Park and Rodermel, 2004). Although the mechanism of this genetic suppression
remains unclear, it demonstrates the interaction of the Clp regulatory components with other
protease family members.
Lon-like protease
Lon was the first ATP-dependent protease found in E. coli. E. coli cells lacking in Lon shows
accumulation of abnormal proteins and increased sensitivity to DNA damage (Gottesman and
Zipser, 1978). A characteristic property of Lon is its affinity to DNA, although the domain
required for DNA binding is not known (Rotanova et a., 2004). There are four homologues of
the Lon present in the A. thaliana genome and two of them are predicted to be chloroplastic.
A gene encoding Lon is not present in Synechocystis. Thus, whether Lon is present in
chloroplasts remains controversial. Based on transient assay and immunoblot analysis Lon4
has been shown to be present in chloroplasts (Sakamoto, 2006).
ATP-independent proteases
Besides Clp, FtsH and Lon, there are few ATP-independent proteases present in the
chloroplasts. These are SPP, DegP and SppA like protease.
41
DegP-like protease
DegP homologues are found in most organisms including bacteria, human and plants (Spies et
al., 1999). DegP is a serine protease that forms a homotrimeric oligomer in E. coli and
humans (Clausen et al., 2002). Two trimers further dimerize to form a hexamer (Krojer et al.,
2002). There are three Deg proteases in E. coli, which are named DegP, Q and S. Each of
these three protease has one or two characteristic PDZ domains in the C-terminus that are
necessary for protease-protease interaction and that possibly regulate recognition of
substrates. DegP also has a chaperone activity at higher temperature (Spiess et al., 1999;
Ehrmann and Clausen, 2004).
There are 16 DegP homologues present in the A. thaliana genome. Four of the A. thaliana
DegP homologs (Deg 1, 2, 5 and 8) are located in chloroplasts (Haussuhl et al., 2001; Chassin
et al., 2002), where they are peripherally attached to the thylakoid membrane. DegP1, 5 and 8
are located on the luminal side, whereas DegP2 is on the stromal side (Haussuhl et al., 2001;
Itzhaki et al., 1998). There is no report on the effects of knockout mutations in any DegP
genes in A. thaliana. Expression of DegP proteins is increased by abiotic stresses such as salt,
light and temperature. DegP2 has been proposed to perform the primary cleavage of photodamaged D1 protein from the PSII complex, prior to its complete degradation by FtsH2
(Haussuhl et al., 2001). Degradation and removal of damaged D1 protein is crucial for the
integration of a new D1 copy and for restoring a functional PSII.
SppA-like protease
SppA, a homologue of E. coli protease IV has been identified in A. thaliana chloroplasts.
There is only one SppA homologue present in the A. thaliana genome (Lensch et al., 2001).
SppA is an ATP-independent serine protease. SppA is tightly bound to the stromal side of the
thylakoid membrane. SppA is present as a 270 kDa complex suggesting a homotetrameric
structure of the SppA complex. SppA is up-regulated by light and might be involved in the
light dependent degradation of antenna and PSII complexes (Lensch et al., 2001).
42
The PresequenceProtease, PreP
After or during import into mitochondria and chloroplasts, most precursor proteins are
proteolytically processed by MPP in mitochondria and SPP in chloroplasts, resulting in
production of the mature protein and free targeting peptides. In general, targeting peptides do
not seem to be required inside organelles. Subunit 9 of the mammalian cytochrome bc1
complex is the only known example of a targeting peptide integrated as a functional subunit
of an oligomeric protein complex. Subunit 9 corresponds to the targeting peptide of the
Rieske iron sulphur protein (RISP) (Brandt et al., 1993; Iwata, et al., 1998). The RISP
precursor is targeted and assembled into the bc1 complex before its targeting peptide is
cleaved off (Deng et al., 1998).
Targeting peptides are potentially harmful for the integrity of the structure and function of
mitochondria and chloroplasts. They can perturb natural and artificial lipid bilayers. Addition
of presequences to mitochondria results in membrane lysis, uncoupling of respiration and
dissipation of the membrane potential (Roise et al. 1986; Glaser and Cumsky, 1990a; Glaser
and Cumsky, 1990b; Hugosson et al., 1994; Nicolay et al., 1994; van't Hof et al., 1991, 1995).
The mechanism of action of presequences on the mitochondrial membrane is not clear, but it
has been proposed that the presequence peptides induce channel opening (Lu et al., 1997), or
that the peptides themselves form a pore (Matsuzaki et al., 1996). Furthermore, mitochondrial
presequences have been shown to possess antimicrobial activity (Hugosson et al., 1994).
Therefore, free targeting peptides generated inside the mitochondria and chloroplasts have to
be rapidly removed, either by proteolysis or export. Mitochondrial export of peptides has been
reported in S. cerevisiae mitochondria. It was shown that the ABC (ATP-binding cassette)
Mdl1 protein of S. cerevisiae transports small peptide fragments, generated by AAAproteases, across the mitochondrial IM (Young et al., 2001), although the efficiency of the
export was very low. The low export efficiency of targeting peptides to the out-side of
mitochondria and the energy requirement for the export of positively charged targeting
peptides against the membrane potential, suggests that the degradation of targeting peptides
inside mitochondria is the most likely system for their disposal.
In agreement with these observations, it was shown by Ståhl et al. (2000) in our laboratory,
that mitochondrial targeting peptides (presequences) are rapidly degraded by a matrix
43
localized ATP-independent protease after import into mitochondria, while the mature part of
the protein remained stable. PresequenceProtease (PreP), the protease responsible for this
degradation was isolated from potato tuber mitochondrial matrix and identified by mass
spectrometric analysis, ESI MS/MS. (Ståhl et al., 2002). There are two homologues of PreP
present in the A. thaliana genome, an A. thaliana zinc metalloprotease (renamed, AtPreP1)
(AAL90904, on chromosome 3: Zn-MP) and an A. thaliana putative zinc metalloprotease
(renamed, AtPreP2) (AAG13049, on chromosome 1: putZn-MP). Both proteases display high
amino acid sequence similarities, with most differences occurring in their predicted organellar
targeting peptides. Both AtPreP1 and AtPreP2 harbor a characteristic inverted zinc-binding
motif, HILEHX96E, and are classified to the pitrilysin protease family (Ståhl et al., 2002).
Dual Targeting of PreP in Mitochondria and Chloroplasts (Paper I and II)
Subcellular prediction programs did not yield conclusive results for the intracellular
localization of the AtPrep1 (AtZn-MP) and the AtPrep2 (AtputZn-MP) (Predotar:
http://www.inra.fr/predotar/ and TargetP: http://www.cbs.dtu.dk/services/TargetP/). However,
the targeting peptide of AtPrep1 and AtPrep2 was predicted to be 85 amino acid residues, by
both
MitoProt
(http://ihg.gsf.de/ihg/mitoprot.html)
and
ChloroP
(http://www.cbs.dtu.dk/services/ChloroP/). In order to investigate the subcellular localization
of AtPreP1 and AtPreP2, a number of different, but complementary techniques were
employed. In the in vivo import assay, the predicted targeting peptide plus 40 amino acid
residues from the mature part of the protein for AtPreP1, and 70 residues for AtPreP2, were
fused to the GFP reporter protein. Transient expression of the GFP fusion constructs was
carried out in N. tabacum protoplasts and leaves. Protoplasts transformed with the AtPreP1GFP (Zn-MP-GFP) or AtPreP2-GFP (putZn-MP-GFP) construct showed GFP fluorescence in
two distinct locations, with both punctuated and large round shaped structures. Fluorescence
in the punctuated shape structures co-localized with Mitotracker, whereas in the large round
shaped structures, GFP fluorescence co-localized with chlorophyll autofluorescence. This
indicated that the targeting peptide of both PrePs is capable of dually targeting the GFP to
both mitochondria and chloroplasts (Paper I and II). In another complementary assay, where
Agrobacterium tumefaciens mediated transient expression was carried out with intact N.
tabacum leaves, the targeting peptide of AtPreP1 and AtPreP2 again dually targeted GFP to
both organelles (Paper I and II). These results indicated that both the AtPreP1 and the AtPreP2
could be members of a dually targeted protein family. The presence of PreP in both organelles
was also verified by Western blot analysis.
44
Interestingly, there is a second methionine at position 29 in the predicted targeting peptide
of AtPreP1 that can be used as a second translational initiation site in AtPreP1 mRNA,
producing two forms of the protein differing in targeting specificity. We therefore used a
deletion construct starting from the second methionine (∆1-28PreP1-GFP), and a mutant with
the second methionine changed to leucine ([M29L]PreP1-GFP). The truncated targeting
peptide in the ∆1-28PreP1-GFP construct targeted the GFP to chloroplasts only, while the
mutant construct, [M29L]PreP1-GFP targeted GFP to both mitochondria and chloroplasts.
Furthermore, no fluorescence was detected when a mutated version of AtPreP1-GFP construct
that contained a frame shift mutation in between the first and the second ATG of the AtPreP1GFP construct, was introduced into the N. tabacum leaves (Paper I). These results excluded
the dual targeting of the AtPreP1 as a result of alternative translational initiation in vivo and
also suggested the presence of a domain organization in AtPreP1 targeting peptide. In
conclusion, the AtPreP1 harbors an ambiguous targeting signal with a distinct domain present
for mitochondrial and chloroplastic targeting that is recognized and transported by both
mitochondrial and chloroplastic import machineries.
Dual targeting of the AtPreP1 and AtPreP2 was also supported by results from in vitro
import assays using full length radiolabelled precursor proteins of both PreP isoforms. Both
full length PreP precursors were imported and processed into mitochondria and chloroplasts,
while the ∆1-28PreP1 precursor was only imported to isolated chloroplasts, with no import
detected into mitochondria. [M29L]PreP1 precursor was again imported and processed in
both mitochondria and chloroplasts. Incubation of the AtPreP1 or AtPreP2 precursors
simultaneously with isolated mitochondria and chloroplasts in a dual import system, followed
by reisolation of the organelles also resulted in import and processing of the precursor inside
both organelles (Paper I and II).
Function of PreP in Mitochondria and Chloroplasts (Paper I and II)
The predicted mature part of AtPreP1 and AtPreP2 was cloned as a fusion protein with
glutathione-S-transferase (GST), and the GST-PreP1 and GST-PreP2 fusion proteins were
overexpressed in E. coli. Recombinant AtPreP1 and AtPreP2 completely degraded the
mitochondrial and chloroplastic targeting peptides as well as a fluorescent P1 peptide. The
proteolytic activity was not dependent on ATP and was specifically inhibited by ortho-
45
phenanthroline (o-ph). (Paper I, II and VI). Furthermore, immunoinactivation studies on
isolated mitochondrial matrix and chloroplastic stroma resulted in complete inhibition of the
proteolytic activity against a mitochondrial and chloroplastic targeting peptide indicating the
function of PreP in both organelles.
The substrate specificity of the recombinant AtPreP1 was investigated using degradation of
a mitochondrial targeting peptide, a chloroplastic targeting peptide, unstructured peptide
(insulin B chain and galanin (Duckworth et al., 1998) and a folded de novo peptide ala-α3w
(Dai et al., 2002). AtPreP1 completely degraded all peptides except for the tightly folded
peptide ala-α3w. Based on these results, it can be concluded that PreP is not specific for
mitochondrial and chloroplastic targeting peptides and does not recognize amino acid residues
per se, but degrades unstructured peptides and is not active against folded substrates (Paper
VI). What are the advantages of having two proteases with the same function? Is it to enhance
the proteolysis? To answer these questions, cleavage specificity of AtPreP1 and AtPreP2 was
investigated by cleavage of a specific fluorescent peptide P1, the mitochondrial presequence
peptide and the chloroplastic transit peptide. After incubation of the peptides for 30 min at
30oC, an intermediate product was produced by AtPreP2 with both the synthetic fluorescent
peptide and mitochondrial targeting peptide, while no such intermediate product was seen
when these two peptides were incubated with AtPreP1 (Paper II and VII). The intermediate
could not be detected upon degradation with AtPreP1 even when much shorter incubation
times and different concentrations of AtPreP1 were used. Accumulation of intermediate
products of the peptides after incubation with AtPreP2 shows that the AtPreP1 and AtPreP2
have different cleavage specificity. Degradation by AtPreP1 and AtPreP2 was also
investigated with the chloroplastic transit peptide and its mutants (Paper II). Mutants were
designed to study the effect of changing the polypeptide chain flexibility of the transit peptide
on import and processing. AtPreP2 had the capacity to degrade both the transit peptide and all
the mutants, whereas AtPreP1 could not degrade the chloroplastic targeting peptide mutant
(P36A), which has decreased polypeptide chain flexibility as proline has been changed to
alanine. Thorough substrate specificity studies of the AtPreP1 and AtPreP2 proteases using
mass spectrometric analysis of degradation products of mutants of mitochondrial targeting
peptides, as well as a number of other synthetic peptides show differences in amino acid
recognition and the cleavage efficiency (Paper VII). In conclusion, AtPreP1 and AtPreP2 may
have overlapping, but complementary proteolytic specificity, allowing a wide variety of
substrate peptides to be efficiently degraded.
46
Expression of the AtPreP1 and AtPreP2 in A. thaliana plants
Expression levels of AtPreP1 and AtPreP2 transcripts in A. thaliana were studied using semi
quantitative RT-PCR, under carefully optimized conditions for the quantitative measurements
of the transcripts. Both the AtPreP1 and the AtPreP2 transcript were detected in young
seedlings, however in varying amounts. The AtPreP1 transcript was detected in silique and
flower tissues, although the transcript level was much higher in flowers. In contrast to the
AtPreP1 transcript, the AtPreP2 transcript was found to be present in leaf, flower and root
tissues with no transcript detected in shoot and silique tissues (Paper II). These results showed
that both AtPreP1 and AtPreP2 are expressed in an organ-specific manner in A. thaliana
plants (Paper II). It will be interesting to investigate the functional importance of the higher
transcript level of AtPreP1 present during flower development.
Crystal structure of AtPreP1, a Peptidasome (Paper III)
The crystal structure of the inactive mutant, AtPreP1 E80Q, was solved at 2.1Å resolution
(Paper III). The asymmetric unit is composed of two protein molecules, each with a zinc atom
and a six-residue peptide substrate entrapped in the active site. Magnesium ions facilitated
crystallization of AtPreP1 and two hydrated ions were found in each protein molecule
coordinated by acidic residues. The PreP polypeptide folded into four topologically similar
domains that formed two bowl-shaped halves. Comparison of the four domains showed that in
spite of only 6-11% sequence identity, the root-mean-square deviation (rmsd) after
superimposing the main chains is 2 Å or higher, despite a similar topology. A unique hinge
region of 82 residues joins the two enzyme halves. The arrangement of the active site is that
of a typical metalloprotease, but the zinc-binding motif is inverted. The first domain forms the
major part of the active site containing the inverted zinc-binding motif (HXXEH) where
His77 and His81 coordinate the zinc and Glu80 (substituted for Gln in the crystallized
mutant) acts as a base catalyst. The inactive mutants H77L, E80Q and H81L generated in a
separate study (Paper VI) confirm the importance of these residues in proteolysis. However,
the third zinc ligand, distal Glu177 was previously unknown. It is also essential for catalysis
as demonstrated by the inactive E177Q mutant. Despite the fact that the protease was
crystallized in the absence of a substrate, the electron density revealed a peptide of six
residues bound in the active site. This substrate peptide was helpful in order to identify amino
acid residues participating in the substrate binding and catalysis (Paper III).
47
An interesting finding was the presence of C-terminal residues, Arg848 and Tyr854 at the
active site, residues separated by almost 800 residues in sequence from the inverted zinc
binding residues. Arg848 forms a hydrogen bond to the main chain oxygen of P2′, while
Tyr854 binds to the main chain oxygen of the scissile bond. Substitution of Arg848 for Ala or
Lys and Tyr854 to Phe resulted in the abolition of the AtPreP1 activity. The catalytic site is
present inside a large proteolytic chamber surrounded by the two enzyme halves. The
chamber has a volume of more than 10 000 Å3. The chamber appears spacious enough to hold
peptide substrates such as the targeting peptide, but is sufficiently small to exclude larger,
folded proteins. Since the active site includes residues from both the N- and C-terminal part of
the protein, proteolysis can occur only when the chamber is closed. Thus the proteolytic
chamber protects against unwarranted degradation by preventing folded proteins from
entering the active protease and limiting the size of the substrate. This is also supported by
another study showing that AtPreP1 only recognized and cleaved peptides of approximately
10 and 65 amino acids residues in length (Paper VII). The way the substrates are cleaved
inside a chamber is reminiscent of the proteasome and therefore PreP was named a
Peptidasome.
Mechanism of Proteolysis by PreP Peptidasome (Paper III and IV)
The PreP structure presented the first, substrate-bound conformation of an M16 protease,
where proteolysis of the substrate takes place inside a chamber formed by two halves of the
protease connected by a hinge region. There is no hole or cavity present in the structure. How
do substrate peptides as long as 65 amino acid residues access the active site that is situated
inside the proteolytic chamber? An open conformation of the AtPreP1 was modeled using the
known crystal structure of S. cerevisiae MPP (Taylor et al., 2001). The modeled conformation
suggested that the closed and open states may differ by as much as a 38° rotation of the two
halves around the hinge. A hinge bending mechanism is suggested where the unbound state is
open and substrate binding triggers a movement that brings the two halves of the enzyme
together so that Arg848 and Tyr854 can complete the active site and stabilize the transition
state. While the opening and closing of the peptidasome revolves around the hinge, it seems
that electrostatic forces could be driving these movements. The electrostatic surface potential
for AtPreP1 E80Q showed that the protease is very acidic, especially around the active site.
As the two negatively charged halves repel each other, the proteasome stays open. However,
when a substrate containing basic residues is bound, some of the negative charge is
neutralized allowing the chamber to close. It is possible that cations such as magnesium
48
reduce the negative charge and further favour the closed conformation. After proteolysis, the
cleaved products are released from the active site and the interactions with Arg848 and
Tyr854 are broken, signaling adoption of the unbound state to the hinge region. The two
negatively charged inner surfaces then contribute to push the two halves open by repulsion.
The mechanism involving the opening and closing of the enzyme was investigated by
introducing disulfide bonds between the two halves and locking the enzyme in a closed
conformation. Disulfide bonds are expected to form under oxidizing conditions, thus locking
the enzyme in a closed conformation, whereas it would be able to open and close normally
under reducing conditions due to the absence of these disulfide bonds. Consequently, mutants
with appropriately positioned cysteine pairs would be inactive under oxidizing conditions and
fully active under reducing conditions. Four double mutants (C1-C4) of AtPreP1 with a pair
of cysteine residues, predicted to form the disulfide bridges, were created by site directed
mutagenesis. Catalytic activity was tested under both conditions using the mitochondrial
targeting peptide and the P1 peptide as substrates. The proteolytic activity of the wild type
was normal under all of the given conditions. The activity of all four cysteine double mutants
was normal under reducing conditions. Interestingly, under oxidizing conditions three of the
four mutants (C1-C3) were catalytically inactive, whereas the C4 mutant had almost normal
activity. The disulfide bond in the C4 mutant is far away from the active site, in a position
where no significant conformational changes are expected during opening and closing. This
may explain why this mutant form showed no effect on catalytic activity when locked by a
disulfide bond. The remaining mutations are located in regions where larger conformational
changes are expected and the suppression of proteolytic activity by the introduced disulfide
bonds supported this. Under oxidizing conditions, the disulfide bonds restricted the opening
of the protease so that the substrate is unable to reach the active site. Modification of the
cysteine residues by N-ethylmaleimide (NEM) showed that the change in activity is directly
linked to the formation of disulfide bonds. Taken together, these results verify that neither the
cysteine substitutions per se, nor the addition of oxidizing or reducing agents, have a negative
impact on proteolysis. In conclusion, these results supported the proposed mechanism
involving opening and closing of the enzyme in response to substrate binding. The crystal
structure of human IDE has also been recently solved at 2.25 Å resolution (Shen et al., 2006).
The active site of IDE is also located inside a cavity, as in the PreP Peptidasome. The
structure and mechanism of proteolysis of IDE closely resembles that of PreP and a similar
opening and closing mechanism is suggested (Shen et al., 2006).
49
The effect of magnesium or calcium ions was investigated on the proteolytic activity of
AtPreP1, since it was not possible to crystallize AtPreP1 without these ions. In the crystal
structure, two hydrated Mg2+ ions were found in each protein molecule, nicely coordinated by
acidic residues. The position of the Mg2+-binding sites suggested that binding of magnesium
ions might have an effect on the conformation of the fourth domain. The position of this
domain is crucial for proteolytic activity. In order to determine the impact of cations for the
proper function of AtPreP1, the proteolytic activity was studied using various concentrations
of MgCl2 and CaCl2. AtPreP1 was inactive in the absence of MgCl2 or CaCl2. The addition of
MgCl2 or CaCl2 resulted in restoration of the proteolytic activity with full activation at about
10 mM concentrations of MgCl2 or CaCl2 (Paper III). These results suggested the requirement
of additional metal ions beside Zn2+ for the activity of AtPreP1. Substituting residues involved
in Mg2+-binding sites in the AtPreP1 shows severe effects on the proteolytic activity of the
AtPreP1 (Bäckman and Bhushan, unpublished results).
The role of the PreP peptidasome in the degradation of the amyloid βpeptide: A possible link to the Alzheimer’s disease
Alzheimer’s disease (AD) and amyloid β-peptide
Alzheimer’s disease (AD), also known simply as Alzheimer’s is a neurodegenerative disease
characterized by progressive cognitive deterioration together with declining activities of daily
living and neuropsychiatric symptoms or behavioral changes. It is the most common type of
dementia. The most striking early symptom is the loss of short term memory (amnesia), which
usually manifests as minor forgetfulness that becomes steadily more pronounced with illness
progression, with relative preservation of older memories. As the disorder progresses,
cognitive (intellectual) impairment extends to the domains of language (aphasia), skilled
movements (apraxia), recognition (agnosia), and those functions (such as decision-making
and planning) closely related to the frontal and temporal lobes of the brain as they become
disconnected from the limbic system, reflecting extension of the underlying pathological
process. These changes affect essential human qualities, and thus AD is sometimes described
as a disease where the victims suffer the loss of the very qualities that define human existence
(as reviewed by Thomas and Fenech, 2006).
Alzheimer’s disease has been identified as a protein misfolding disease due to the
accumulation of abnormally folded amyloid beta (Aβ) peptide in the brain of AD patients
(Hashimoto, et al., 2003). Amyloid beta is a short peptide that is an abnormal proteolytic
50
byproduct of the transmembrane protein amyloid precursor protein (APP) (Figure 3), whose
function is unclear, but thought to be involved in neuronal development (Kerr and Small,
2005). The γ-secretase complex is involved in APP processing and production of Aβ-peptides
(Cai et al., 2003). Although Aβ monomers are soluble and harmless, they undergo a dramatic
conformational change at sufficiently high concentration to form a beta sheet-rich tertiary
structure that aggregates to form amyloid fibrils (Ohnishi and Takano, 2004) that deposit
outside neurons in dense formations known as senile plaques or neuritic plaques, in less dense
aggregates as diffuse plaques, and sometimes in the walls of small blood vessels in the brain
in a process called amyloid angiopathy or congophilic angiopathy. AD is also considered a
tauopathy due to abnormal aggregation of the tau protein, a microtubule-associated protein
expressed in neurons that normally acts to stabilize microtubules in the cell cytoskeleton. Like
most microtubule-associated proteins, tau is normally regulated by phosphorylation however,
in AD patients, hyperphosphorylated tau accumulates as paired helical filaments (Goedert et
al., 2006) that in turn aggregate into masses inside nerve cell bodies known as neurofibrillary
tangles and as dystrophic neurites associated with amyloid plaques.
Amyloid β-peptide in mitochondria
Extracellular plaque formation of Aβ has been the main focus of molecular studies associated
with AD (Selkoe, 1999). However, there are reports indicating intracellular events including
the mitochondrial role in AD (Glabe, 2001). There are many links between mitochondrial
dysfunctions and AD (Hashimoto et al., 2003; Yan and Stern, 2005). Impairment of
mitochondrial energy metabolism and altered cytochrome c oxidase activity are among the
earliest detectable defects in AD (Anandatheerthavarada et al., 2003; Cardoso et al., 2004). It
has been shown that Alzheimer’s amyloid precursor protein 695 (APP) is not only targeted to
the plasma membrane, but also to mitochondria (Anandatheerthavarada et al., 2003).
Accumulation of APP in the outer mitochondrial membrane caused dysfunctions and
impaired energy metabolism. The active γ-secretase, which cleaves APP to generate Aβ, has
been shown to be present in the mitochondrial membrane (Hansson et al., 2004). Furthermore,
the occurrence of Aβ in mitochondria of AD patients and its direct binding to amyloid βbinding alcohol dehydrogenase (ABAD) induces apoptosis and free radical generation in
neurons (Lustbader et al., 2004). A recent study demonstrated that Aβ is present in the
mitochondrial matrix in AD brains and in brains from transgenic mice overexpressing mutant
human APP that impairs neuronal function and contributes to cellular dysfunction in AD
(Caspersen et al., 2005).
51
(Thomas and Fenech, 2006)
Figure 3. Amyloid β-peptide production. APP is a membrane protein producing a number of
isoforms which range in size from 695–770 amino acids. Proteolysis of the APP protein involves
α-, β- and γ- secretases. APP cleavage by α-secretase releases sAPPα from the membrane leaving
an 83 amino acid APP fragment. Cleavage of the APP protein by β-secretase releases sAPPβ
from the membrane and leaves behind a 99 amino acid fragment which can be further cleaved by
γ-secretase to produce Aβ40/42 fragments extracellularly (Thomas and Fenech, 2006).
PresequenceProtease in human - The hPreP
Interestingly, PreP is an organellar functional analogue of the human insulin degrading
enzyme (IDE) that also belongs to the pitrilysin family. IDE has been implicated in AD as it
cleaves Aβ-peptide before insoluble amyloid fibers are formed (Tanzi et al., 2004). These
findings led us to investigate the degradation of Aβ by human PreP (hPreP). hPreP comprises
1037 amino acid residues with a predicted 29 amino acid residue N-terminal mitochondrial
targeting signal. It has been previously identified and referred to as a metalloprotease, hMP1
(Mzhavia et al., 1999).
52
A novel function of hPreP in mitochondria: Aβ degradation (Paper V)
The predicted mature part of the human PreP (hPreP) was cloned as a fusion protein with
GST, and the GST-hPreP fusion protein was overexpressed in E. coli. Recombinant hPreP
was purified to homogeneity on GSTrap FF column after cleavage with PreScission protease
(Paper V). The recombinant hPreP completely degraded both Aβ-(1-40) and Aβ-(1-42) as well
as Aβ Arctic peptide (1-42 E22G). We compared the proteolytic activity of the recombinant
hPreP to IDE and found that, unlike IDE, hPreP was not able to degrade insulin. This makes
hPreP very interesting and a better candidate over IDE to use for Aβ-peptide degradation in
situ. We show that neither PMSF nor bestatin (i.e. serine or aminopeptidase type protease
inhibitors) affected proteolytic activity of the recombinant hPreP, whereas NEM, a cysteinetype protease inhibitor, showed some inhibitory effect (about 10%) and the metalloprotease
inhibitor ortho-phenanthroline (o-Ph) completely inhibited degradation of Aβ, demonstrating
that hPreP is a thiol-sensitive metalloprotease. Apyrase had no effect on the proteolytic
activity showing that Aβ degradation is independent of ATP. hPreP contains an inverted zincbinding motif, H75ILE78H79. The importance of the metal-binding motif for proteolytic activity
was investigated by studying the recombinant hPreP mutant, hPreP(E78Q), in which the
catalytic base Glu78 was changed to Gln. Overexpressed and purified hPreP(E78Q) did not
degrade the Aβ-peptide, confirming the importance of the inverted zinc-binding site for the
proteolytic activity.
PreP was shown to be localized to the mitochondrial matrix in plants and mammals (Paper
I, II and V). Kambacheld et al. (2005) have shown that PreP homologue in yeast, MOP112 is
localized to the IMS where it functions in degradation of the shorter peptide fragments
generated after ATP-dependent proteolysis. This indicates that PreP has different subcellular
localizations in different organisms; it is present in the mitochondrial matrix in mammals and
plants, while it is present in the IMS in S. cerevisiae. In situ, immunoinactivation of PreP in
human brain mitochondria, using anti-hPreP antibodies, revealed complete inhibition of the
proteolytic activity against Aβ. These results show that under circumstances when Aβ is
present in the mitochondria, hPreP is the protease responsible for degradation of this toxic
protein. In conclusion, PreP is localized to the mitochondrial matrix in mammalian
mitochondria where, beside presequence and other unstructured peptide degradation, it has a
novel function: the degradation of Aβ. These findings contribute to studies of the
mitochondrial component in AD.
53
The structure of AtPreP1 (Paper III) allowed molecular modeling of hPreP and the
identification of important amino acid residues involved in substrate binding and proteolysis
(Paper V). Unexpectedly, hPreP contains two native cysteine residues in close proximity to
each other at position Cys90 in the first domain and position Cys527 at the hinge region.
These cysteine residues are conserved in all known mammalian PreP sequences as well as in
the S. cerevisiae PreP homologue. Interestingly, proteolytic activity of hPreP was almost
abolished against the Aβ-peptide in the presence of oxidizing agents, indicating a disulfide
bridge formation between the Cys90 and Cys527 that locks the enzyme in a closed
conformation and limits substrate access to the active site. This finding was further supported
by the full proteolytic activity of hPreP(C90S) mutant under oxidizing conditions.
Furthermore, the importance of the status of cysteine residues for the proteolytic activity of
hPreP may explain the partial inhibitory effect of NEM on hPreP activity. As NEM is a
cysteine-modifying agent, its substitution may cause steric hindrance during proteolysis. The
importance of the redox status of cysteine residues for proteolytic activity implies a possible
inhibition of the enzyme in mitochondria under conditions of elevated reactive oxygen species
(ROS) production that would additionally increase mitochondrial dysfunction. The
physiological consequences of this finding require further study.
54
Future perspectives
Targeting peptides are essential for directing a protein to its final destination in mitochondria
and chloroplasts. However, once a protein has reached its final destination the targeting
peptide is no longer needed and may also be harmful to the organelle because of its membrane
disrupting properties. PresequenceProtease (PreP) was initially identified as the protease
responsible for clearing the mitochondria from free presequence peptides generated after the
import. PreP was also found to be present in the chloroplasts to take care of free transit
peptides. (Paper I and II). We show that PreP is not specific for targeting peptides, but is a
general protease that degrades shorter and unstructured toxic peptides in these organelles
(Paper VI). The crystal structure of AtPreP1 presented the first substrate-bound, closed
conformation of a protease from the pitrilysin family (Paper III). Based on the structure and
proteolytic activity of cysteine mutants designed to lock the PreP in a closed conformation, a
novel hinge bending opening/closing mechanism of proteolysis is proposed (Paper III and
IV). PreP is localized to the mitochondrial matrix in mammals where, beside degradation of
presequences, it has a novel function i.e. degradation of toxic Aβ-peptides present in
Alzheimer’s patients (Paper V).
We have been able to carry out a thorough investigation of the PreP by showing its dual
localization and function in mitochondria and chloroplasts, the structure and mechanism of
proteolysis and a possible link to Alzheimer’s disease. However, there are still as many or
even more interesting questions that remain to be answered as there were prior to beginning
this work.
How is a dual targeting peptide sorted and imported in both mitochondria and
chloroplasts? It seems that dual targeting peptides have some common features of both the
mitochondrial and chloroplastic targeting peptides, but there is no structural information
available as yet for these targeting peptides. It will be interesting to solve the 3D structure for
a dual targeting peptide alone, and in complex with the mitochondrial and chloroplastic
import receptors, in order to understand the molecular mechanism of dual targeting. A short
dual targeting peptide has been identified with promising results to be used for 3D structural
studies by NMR (Bhushan and Berglund, unpublished results).
What is the significance of higher transcript level of AtPreP1 during flowering? Expression
of the many proteases can be induced during stress conditions. During the late stage of
flowering, the state resembles desiccation, which is also a type of stress and the higher level
of AtPreP1 transcripts may have an important role under these conditions. It will be
55
interesting to study the transcript level of PreP under different stress conditions. Stefan
Nilsson and Hans Bäckman in our laboratory have succeeded in generating a double knock
out mutant of PreP1 and PreP2 in A. thaliana, which will enable to investigate the role of
PreP in detail. How distribution of PreP between mitochondria and chloroplasts is regulated,
is another important issue which should be investigated.
Another interesting issue is the different sub-mitochondrial localization of PreP in yeast
compared to mammals and plants. This can be studied either by shuffling the presequences
between plants, mammals and yeast, or by directing yeast PreP to the matrix. It will be also
interesting to study whether yeast PreP can be imported into the mammalian and plant
mitochondria and vice versa.
One basic question is what are the end products of proteolysis catalysed by PreP. Is it
single amino acids or small peptide fragments? From the mass spectrometric analysis it seems
that the main products of proteolysis are small fragments. It is also possible that these are the
remaining fragments that are resistant to further degradation because of their physical
properties. In order to solve this problem a detailed MS detection of degradation products of a
number of different substrate peptides should be performed.
Next question is how the PreP structure will look in an open conformation. We have
modelled an open conformation of the PreP protease on the basis of the known structure of S.
cerevisiae MPP. However, it is a model and the native PreP structure in an open conformation
is not known. It will be of great interest to crystallize and solve the structure of PreP protease
in a substrate-free, open conformation.
Another interesting question will be to study the possible link of human PreP (hPreP) in
Alzheimer’s disease. hPreP is capable of degrading toxic Aβ-peptides of various lengths as
shown by in vitro and in situ studies (Paper V). However, these are preliminary results and
more in vivo studies are needed before coming to a satisfactory conclusion. One possibility is
to overexpress Aβ and hPreP together in cell culture and test whether PreP has the capability
to degrade the overproduced Aβ in vivo. It will be also interesting to knockdown the PreP in
cell culture using RNAi, and study the physiological consequences of PreP’s absence on
mitochondrial functioning. Since we have been able to solve the structure of AtPreP1 it should
not be difficult to solve the crystal structure of hPreP alone, and in a complex with Aβpeptides. There are plenty of opportunities when it comes to the AD. One can also try to find
the activators of hPreP to be used for stimulating the PreP activity in vivo.
56
Acknowledgements
I would like to express my sincere gratitude to all of you who have directly or indirectly been
involved in the making of my thesis. It has been great to work with all of the people in the
DBB. It is difficult to remember and thank all the people who have helped me during all these
years, but I will try my best.
First of all, I would like to express my gratitude to Prof Elzbieta Glaser, my supervisor, for
letting my PhD dream come true. She is the best supervisor one can have. Thanks for being
such a generous, inspiring and encouraging guide. I have learned much from you and it will
help me a lot in days ahead of me.
Patrick Dessi is one of the nicest persons, you will never forget him. He is always willing
to help everyone. Thanks for introducing me to Elzbieta´s group and going through the thesis
on such a short notice. You are not only a great person, but a saint.
To Prof Mark Boutry, my thanks for accepting me in your lab and helping during my stay
in Belgium. Those experiments turned out to be very important for the thesis. Thanks for your
encouragement and ideas. Thank you to Benoit also, for teaching me GFP system in such a
short time.
To Dr Therese Eneqvist and Dr Ken Johnson my thanks for such a wonderful collaboration
and sharing of ideas. It was not possible to come so far in this work without you guys. Thanks
a lot!
To Prof Stefan Nordlund, Dean for the graduate studies, my thanks for making DBB a nice
place to work. You are always there to help all the PhD students. Thanks for evaluating me so
many times.
My thanks to present and former members of EG group: Stefan for your interesting ideas.
It was fun to play Poker with you and Tatiana in Linköping. To Anna-Karin and Nyosha for
being such cheerful people even when nothing work in the lab. Thanks for making such a nice
environment to work in. To Hans for our discussions of stock markets though we never
agreed on any thing. To Charlotta, for helping with dual import system. Former EG group
members, thanks to Annelie and Per for teaching me how import works. It was so much fun to
collaborate with you and share the ideas. Pavel, though we never collaborate directly, you
were always ready to discuss anything in science. It was nice to have an experienced person
like you around all of these years.
My thanks to Prof Gunnar von Heijne, Prof Åke Wieslander and Prof Mikael Oliveberg for
each spending your time evaluating my work and being my examiners.
57
To Prof Birgitta Norling my thanks for giving me shelter in your house when I did not
have any place to live; to Dr Inger and Dr IngMarie for helping in teaching courses; to Dr JanWillem, for being my co-supervisor though I never discussed the project with you.
Thanks Dan, Joy, Tara, Mikaela, Filippa, Carolina, Mirjam, Yoko, Karl and rest of the
Gunnar´s group for being such nice neighbours. Thank you for letting me borrow so many
reagents.
Bogos, thanks for helping me out in everything including translating some of the Swedish
documents. Thank you for being around. And thanks to the Secretaries, Anki, Ann, Lotta,
Sofia and Peter and Eddie for making sure that everything works in the DBB.
I am indebted to my parents in India. Thank you for all your help and support. It would
have never been possible to come to this point without your support. I would also like to
thank all of my relatives in India for their understanding and support.
My wife Anu, for sharing life with me and giving me the most precious jewel of the world,
our little Dixie. I can not imagine life without the two of you.
58
References
Abe, Y., Shodai, T., Muto, T., Mihara, K., Torii, H., Nishikawa, S., Endo, T. and Kohda, D. (2000) Structural basis
of presequence recognition by the mitochondrial protein import receptor Tom20. Cell, 100, 551-560.
Adam, Z. (1996) Protein stability and degradation in chloroplasts. Plant Mol Biol, 32, 773-783.
Adam, Z. (2000) Chloroplast proteases: possible regulators of gene expression? Biochimie, 82, 647-654.
Adam, Z. and Clarke, A.K. (2002) Cutting edge of chloroplast proteolysis. Trends Plant Sci, 7, 451-456.
Adam, Z., Adamska, I., Nakabayashi, K., Ostersetzer, O., Haussuhl, K., Manuell, A., Zheng, B., Vallon, O.,
Rodermel, S.R., Shinozaki, K. and Clarke, A.K. (2001) Chloroplast and mitochondrial proteases in
Arabidopsis. A proposed nomenclature. Plant Physiol, 125, 1912-1918.
Adam, Z., Rudella, A. and van Wijk, K.J. (2006) Recent advances in the study of Clp, FtsH and other proteases
located in chloroplasts. Curr Opin Plant Biol, 9, 234-240.
Adams, K.L., Daley, D.O., Qiu, Y.L., Whelan, J. and Palmer, J.D. (2000) Repeated, recent and diverse transfers of a
mitochondrial gene to the nucleus in flowering plants. Nature, 408, 354-357.
Adams, K.L., Song, K., Roessler, P.G., Nugent, J.M., Doyle, J.L., Doyle, J.J. and Palmer, J.D. (1999) Intracellular
gene transfer in action: dual transcription and multiple silencings of nuclear and mitochondrial cox2
genes in legumes. Proc Natl Acad Sci U S A, 96, 13863-13868.
Ahting, U., Thieffry, M., Engelhardt, H., Hegerl, R., Neupert, W. and Nussberger, S. (2001) Tom40, the poreforming component of the protein-conducting TOM channel in the outer membrane of mitochondria. J
Cell Biol, 153, 1151-1160.
Ahting, U., Thun, C., Hegerl, R., Typke, D., Nargang, F.E., Neupert, W. and Nussberger, S. (1999) The TOM core
complex: the general protein import pore of the outer membrane of mitochondria. J Cell Biol, 147, 959968.
Aitken, A., Collinge D.B., van Heusden, B.P., Isobe, T., Roseboom, P.H., Rosenfeld, G. and Soll, J. (1992) 14-3-3
proteins: a highly conserved, widespread family of eukaryotic proteins. Trends Biochem Sci, 12, 498-501.
Alconada, A., Kubrich, M., Moczko, M., Honlinger, A. and Pfanner, N. (1995) The mitochondrial receptor complex:
the small subunit Mom8b/Isp6 supports association of receptors with the general insertion pore and
transfer of preproteins. Mol Cell Biol, 15, 6196-6205.
Allen, J.F. (1992) Protein phosphorylation in regulation of photosynthesis. Biochim Biophys Acta, 1098, 275-335.
Allen, J.F. and Raven, J.A. (1996) Free-radical-induced mutation vs redox regulation: costs and benefits of genes in
organelles. J Mol Evol, 42, 482-492.
Anandatheerthavarada, H.K., Biswas, G., Robin, M.A. and Avadhani, N.G. (2003) Mitochondrial targeting and a
novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in
neuronal cells. J Cell Biol, 161, 41-54.
Andersson, S.G., Karlberg, O., Canback, B. and Kurland, C.G. (2003) On the origin of mitochondria: a genomics
perspective. Philos Trans R Soc Lond B Biol Sci, 358, 165-177.
Andersson, S.G., Zomorodipour, A., Andersson, J.O., Sicheritz-Ponten, T., Alsmark, U.C., Podowski, R.M.,
Naslund, A.K., Eriksson, A.S., Winkler, H.H. and Kurland, C.G. (1998) The genome sequence of
Rickettsia prowazekii and the origin of mitochondria. Nature, 396, 133-140.
Arlt, H., Steglich, G., Perryman, R., Guiard, B., Neupert, W. and Langer, T. (1998) The formation of respiratory
chain complexes in mitochondria is under the proteolytic control of the m-AAA protease. EMBO J, 17,
4837-4847.
Arnold, I. and Langer, T. (2002) Membrane protein degradation by AAA proteases in mitochondria. Biochim
Biophys Acta, 1592, 89-96.
Babiychuk, E., Muller, F., Eubel, H., Braun, H.P., Frentzen, M. and Kushnir, S. (2003) Arabidopsis
phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for
mitochondrial function. Plant J, 33, 899-909.
Baker, K.P., Schaniel, A., Vestweber, D. and Schatz, G. (1990) A yeast mitochondrial outer membrane protein
essential for protein import and cell viability. Nature, 348, 605-609.
Barakat, S., Pearce, D.A., Sherman, F. and Rapp, W.D. (1998) Maize contains a Lon protease gene that can partially
complement a yeast pim1-deletion mutant. Plant Mol Biol, 37, 141-154.
Barrett, A.J., Brown, M.A., Dando, P.M., Knight, C.G., McKie, N., Rawlings, N.D. and Serizawa, A. (1995) Thimet
oligopeptidase and oligopeptidase M or neurolysin. Methods Enzymol, 248, 529-556.
59
Bartoli, C.G., Pastori, G.M. and Foyer, C.H. (2000) Ascorbate biosynthesis in mitochondria is linked to the electron
transport chain between complexes III and IV. Plant Physiol, 123, 335-344.
Bauer, J., Chen, K., Hiltbunner, A., Wehrli, E., Eugster, M., Schnell, D. and Kessler, F. (2000) The major protein
import receptor of plastids is essential for chloroplast biogenesis. Nature, 403, 203-207.
Bauer, M.F., Hofmann, S., Neupert, W. and Brunner, M. (2000) Protein translocation into mitochondria: the role of
TIM complexes. Trends Cell Biol, 10, 25-31.
Bauer, M.F., Sirrenberg, C., Neupert, W. and Brunner, M. (1996) Role of Tim23 as voltage sensor and presequence
receptor in protein import into mitochondria. Cell, 87, 33-41.
Becker, L., Bannwarth, M., Meisinger, C., Hill, K., Model, K., Krimmer, T., Casadio, R., Truscott, K.N., Schulz,
G.E., Pfanner, N. and Wagner, R. (2005) Preprotein translocase of the outer mitochondrial membrane:
reconstituted Tom40 forms a characteristic TOM pore. J Mol Biol, 353, 1011-1020.
Becker, T., Jelic, M., Vojta, A., Radunz, A., Soll, J. and Schleiff, E. (2004) Preprotein recognition by the Toc
complex. EMBO J, 23, 520-530.
Bedard, J. and Jarvis, P. (2005) Recognition and envelope translocation of chloroplast preproteins. J Exp Bot, 56,
2287-2320.
Berg, O.G. and Kurland, C.G. (2000) Why mitochondrial genes are most often found in nuclei. Mol Biol Evol, 17,
951-961.
Bhushan, S., Kuhn, C., Berglund, A.K., Roth, C. and Glaser, E. (2006) The role of the N-terminal domain of
chloroplast targeting peptides in organellar protein import and miss-sorting. FEBS Lett, 580, 3966-3972.
Bochtler, M., Hartmann, C., Song, H.K., Bourenkov, G.P., Bartunik, H.D. and Huber, R. (2000) The structures of
HsIU and the ATP-dependent protease HsIU-HsIV. Nature, 403, 800-805.
Boore, J. L. (1999) Animal mitochondrial genomes. Nucleic Acids Res, 27, 1767-1780.
Bota, D.A., Ngo, J.K. and Davies, K.J. (2005) Down regulation of the human Lon protease impairs mitochondrial
structure and function and causes cell death. Free Radic Biol Med, 38, 665-677.
Boutry, M., Nagy, F., Poulsen, C., Aoyagi, K. and Chua, N.H. (1987) Targeting of bacterial chloramphenicol
acetyltransferase to mitochondria in transgenic plants. Nature, 328, 340-342.
Branda, S.S. and Isaya, G. (1995) Prediction and identification of new natural substrates of the yeast mitochondrial
intermediate peptidase. J Biol Chem, 270, 27366-27373.
Brandt, U., Yu, L., Yu, C.A. and Trumpower, B.L. (1993) The mitochondrial targeting presequence of the Rieske
iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in
mammals and retained as a subunit in the complex. J Biol Chem, 268, 8387-8390.
Braun, H.P. and Schmitz, U.K. (1993) Purification and sequencing of cytochrome b from potato reveals methionine
cleavage of a mitochondrially encoded protein. FEBS Lett, 316, 128-132.
Brix, J., Rudiger, S., Bukau, B., Schneider-Mergener, J. and Pfanner, N. (1999) Distribution of binding sequences
for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein
and a non-cleavable preprotein. J Biol Chem, 274, 16522-16530.
Bruce, B.D. (2000) Chloroplast transit peptides: structure, function and evolution. Trends Cell Biol, 10, 440-447.
Buchler, M., Tisljar, U. and Wolf, D.H. (1994) Proteinase yscD (oligopeptidase yscD) Structure, function and
relationship of the yeast enzyme with mammalian thimet oilgopeptidase (metalloendopeptidase, EP
24.15). Eur. J. Biochem, 219, 627-639.
Bulteau, A.L., Szweda, L.I. and Friguet, B. (2006) Mitochondrial protein oxidation and degradation in response to
oxidative stress and aging. Exp Gerontol, 41, 653-657.
Cai, D., Leem, J.Y., Greenfield, J.P., Wang, P., Kim, B.S., Wang, R., Lopes, K.O., Kim, S.H., Zheng, H.,
Greengard, P., Sisodia, S.S., Thinakaran, G. and Xu, H. (2003) Presenilin-1 regulates intracellular
trafficking and cell surface delivery of beta-amyloid precursor protein. J Biol Chem, 278, 3446-3454.
Caliebe, A., Grimm, R., Kaiser, G., Lubeck, J., Soll, J. and Heins, L. (1997) The chloroplastic protein import
machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein. EMBO J,
16, 7342-7350.
Campbell, C.L., Tanaka, N., White, K.H. and Thorsness, P.E. (1994) Mitochondrial morphological and functional
defects in yeast caused by yme1 are suppressed by mutation of a 26S protease subunit homologue. Mol
Biol Cell, 5, 899-905.
Cardoso, S.M., Santana, I., Swerdlow, R.H. and Oliveira, C.R. (2004) Mitochondria dysfunction of Alzheimer's
disease cybrids enhances Abeta toxicity. J Neurochem, 89, 1417-1426.
60
Casari, G., De Fusco, M., Ciarmatori, S., Zeviani, M., Mora, M., Fernandez, P., De Michele, G., Filla, A., Cocozza,
S., Marconi, R., Durr, A., Fontaine, B. and Ballabio, A. (1998) Spastic paraplegia and OXPHOS
impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell,
93, 973-983.
Caspersen, C., Wang, N., Yao, J., Sosunov, A., Chen, X., Lustbader, J.W., Xu, H.W., Stern, D., McKhann, G. and
Yan, S.D. (2005) Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in
Alzheimer's disease. FASEB J, 19, 2040-2051.
Chabregas, S.M., Luche, D.D., Van Sluys, M.A., Menck, C.F. and Silva-Filho, M.C. (2003) Differential usage of
two in-frame translational start codons regulates subcellular localization of Arabidopsis thaliana THI1. J
Cell Sci, 116, 285-291.
Chacinska, A., Lind, M., Frazier, A.E., Dudek. J., Meisinger. C., Geissler, A., Sickmann, A., Meyer, H.E., Truscott,
K.N., Guiard, B., Pfanner, N. and Rehling, P. (2005) Mitochondrial presequence translocase: switching
between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell, 120, 817-829.
Chassin, Y., Kapri-Pardes, E., Sinvany, G., Arad, T. and Adam, Z. (2002) Expression and characterization of the
thylakoid lumen protease DegP1 from Arabidopsis. Plant Physiol, 130, 857-864.
Chen, J., Burke, J.J., Velten, J. and Xin, Z. (2006) FtsH11 protease plays a critical role in Arabidopsis
thermotolerance. Plant J, 48, 73-84.
Chen, M., Choi, Y., Voytas, D.F. and Rodermel, S. (2000) Mutations in the Arabidopsis VAR2 locus cause leaf
variegation due to the loss of a chloroplast FtsH protease. Plant J, 22, 303-313.
Chen, X., Van Valkenburgh, C., Fang, H. and Green, N. (1999) Signal peptides having standard and nonstandard
cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease. J Biol Chem,
274, 37750-37754.
Chew, O., Whelan, J. and Millar, A.H. (2003) Molecular definition of the ascorbate-glutathione cycle in Arabidopsis
mitochondria reveals dual-targeting of antioxidant defenses in plants. J Biol Chem, 278, 46869-46877.
Chou, M.L., Fitzpatrick, L.M., Tu, S.L., Budziszewski, G., Potter-Lewis, S., Akita, M., Levin, J.Z., Keegstra, K. and
Li, H.M. (2003) Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein
translocon. EMBO J, 22, 2970-2980.
Cipolat, S., Rudka, T., Hartmann, D., Costa, V., Serneels, L., Craessaerts, K., Metzger, K., Frezza, C., Annaert, W.,
D'Adamio, L., Derks, C., Dejaegere, T., Pellegrini, L., D'Hooge, R., Scorrano, L. and De Strooper, B.
(2006) Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1dependent cristae remodeling. Cell, 126, 163-175.
Clausen, T., Southan, C. and Ehrmann, M. (2002) The HtrA family of proteases: implications for protein
composition and cell fate. Mol Cell, 10, 443-455.
Clifton, S.W., Minx, P., Fauron, C.M., Gibson, M., Allen, J.O., Sun, H., Thompson, M., Barbazuk, W.B.,
Kanuganti. S., Tayloe, C., Meyer, L., Wilson, R.K. and Newton, K.J. (2004) Sequence and comparative
analysis of the maize NB mitochondrial genome. Plant Physiol, 136, 3486-3503.
Conway, D.J., Fanello, C., Lloyd, J.M., Al-Joubori, B.M., Baloch, A.H., Somanath, S.D., Roper, C., Oduola, A.M.,
Mulder, B., Povoa, M.M., Singh, B. and Thomas A.W. (2000) Origin of Plasmodium falciparum malaria
is traced by mitochondrial DNA. Mol Biochem Parasitol, 111, 163-171.
Corral-Debrinski, M., Blugeon, C. and Jacq, C. (2000) In yeast, the 3' untranslated region or the presequence of
ATM1 is required for the exclusive localization of its mRNA to the vicinity of mitochondria. Mol Cell
Biol, 20, 7881-7892.
Corydon, T.J., Bross, P., Holst, H.U., Neve, S., Kristiansen, K., Gregersen, N. and Bolund, L. (1998) A human
homologue of Escherichia coli ClpP caseinolytic protease: recombinant expression, intracellular
processing and subcellular localization. Biochem J, 331, 309-316.
Creissen, G., Reynolds, H., Xue, Y. and Mullineaux, P. (1995) Simultaneous targeting of pea glutathione reductase
and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco. Plant J, 8, 167175.
Cui, L., Veeraraghavan, N., Richter, A., Wall, K., Jansen, R.K., Leebens-Mack, J., Makalowska, I. and dePamphilis,
C.W. (2006) ChloroplastDB: the chloroplast genome database. Nucleic Acids Research, 34, D692-696.
Da Cruz, S., Xenarios, I., Langridge, J., Vilbois, F., Parone, P.A. and Martinou, J.C. (2003) Proteomic analysis of
the mouse liver mitochondrial inner membrane. J Biol Chem, 278, 41566-41571.
Dai, Q.H., Tommos, C., Fuentes, E.J., Blomberg, M.R., Dutton, P.L. and Wand, A.J. (2002) Structure of a de novo
designed protein model of radical enzymes. J Am Chem Soc, 124, 10952-10953.
Dalbey, R.E. (1991) Leader peptidase. Mol Microbiol, 5, 2855-2860.
61
Daley, D.O., Adams, K.L., Clifton, R., Qualmann, S., Millar, A.H., Palmer, J.D., Pratje, E. and Whelan, J. (2002)
Gene transfer from mitochondrion to nucleus: novel mechanisms for gene activation from Cox2. Plant J,
30, 11-21.
Daum, G., Gasser, S.M. and Schatz, G. (1982) Import of proteins into mitochondria. Energy-dependent, two-step
processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J Biol
Chem, 257, 13075-13080.
Dekker, P.J., Martin, F., Maarse, A.C., Bomer, U., Muller, H., Guiard, B., Meijer, M., Rassow, J. and Pfanner, N.
(1997) The Tim core complex defines the number of mitochondrial translocation contact sites and can
hold arrested preproteins in the absence of matrix Hsp70-Tim44. EMBO J, 16, 5408-5419.
Dekker, P.J., Ryan, M.T., Brix, J., Muller, H., Honlinger, A. and Pfanner, N. (1998) Preprotein translocase of the
outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.
Mol Cell Biol, 18, 6515-6524.
Deng, K., Zhang, L., Kachurin, A.M., Yu, L., Xia, D., Kim, H., Deisenhofer, J. and Yu, C.A. (1998) Activation of a
matrix processing peptidase from the crystalline cytochrome bc1 complex of bovine heart mitochondria. J
Biol Chem, 273, 20752-20757.
Donzeau, M., Kaldi, K., Adam, A., Paschen, S., Wanner, G., Guiard, B., Bauer, M.F., Neupert, W. and Brunner, M.
(2000) Tim23 links the inner and outer mitochondrial membranes. Cell, 101, 401-412.
Duckworth, W.C., Bennett, R.G. and Hamel, F.G. (1998) Insulin degradation: progress and potential. Endocr Rev,
19, 608-624.
Ehrmann, M. and Clausen, T. (2004) Proteolysis as a regulatory mechanism. Annu Rev Genet, 38, 709-724.
Emanuelsson, O., Nielsen, H. and von Heijne, G. (1999) ChloroP, a neural network-based method for predicting
chloroplast transit peptides and their cleavage sites. Protein Sci, 8, 978-984.
Emanuelsson, O., Nielsen, H., Brunak, S. and von Heijne, G. (2000) Predicting subcellular localization of proteins
based on their N-terminal amino acid sequence. J Mol Biol, 300, 1005-1016.
Eriksson, A.C., Sjoling, S. and Glaser, E. (1994) The ubiquinol cytochrome c oxidoreductase complex of spinach
leaf mitochondria is involved in both respiration and protein processing. Biochim Biophys Acta, 1186,
221-231.
Esser, K., Pratje, E. and Michaelis, G. (1996) SOM 1, a small new gene required for mitochondrial inner membrane
peptidase function in Saccharomyces cerevisiae. Mol Gen Genet, 252, 437-445.
Esser, K., Tursun, B., Ingenhoven, M., Michaelis, G. and Pratje, E. (2002) A novel two-step mechanism for removal
of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease
Pcp1. J Mol Biol, 323, 835-843.
Flugge, U.I and Hinz, G. (1986) Energy dependence of protein translocation into chloroplasts. Eur J Biochem, 160,
563-570.
Frey, T.G. and Mannella, C.A. (2000) The internal structure of mitochondria. Trends Biochem Sci, 25, 319-324.
Friso, G., Giacomelli, L., Ytterberg, A.J., Peltier, J.B., Rudella, A., Sun, Q. and Wijk, K.J. (2004) In-depth analysis
of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions,
and a plastid proteome database. Plant Cell, 16, 478-499.
Frydman, J. and Hohfeld, J. (1997) Chaperones get in touch: the Hip-Hop connection. Trends Biochem Sci, 22, 8792.
Furuya, S., Mihara, K., Aimoto, S. and Omura, T. (1991) Cytosolic and mitochondrial surface factor-independent
import of a synthetic peptide into mitochondria. EMBO J, 10, 1759-1766.
Gakh, O., Cavadini, P. and Isaya, G. (2002) Mitochondrial processing peptidases. Biochim Biophys Acta, 1592, 6377.
Giglione, C., Serero, A., Pierre, M., Boisson, B. and Meinnel, T. (2000) Identification of eukaryotic peptide
deformylases reveals universality of N-terminal protein processing mechanisms. EMBO J, 19, 59165929.
Glabe, C. (2001) Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer's disease. J Mol
Neurosci, 17, 137-145.
Glaser, E., Sjoling, S., Tanudji, M. and Whelan, J. (1998) Mitochondrial protein import in plants. Signals, sorting,
targeting, processing and regulation. Plant Mol Biol, 38, 311-338.
Glaser, E. and Dessi, P. (1999) Integration of the mitochondrial-processing peptidase into the cytochrome bc1
complex in plants. J Bioenerg Biomembr, 31, 259-274.
62
Glaser, E. and Whelan, J. (2007) "Plant mitochondrial protein import", in Plant mitochondria, Logan D., ed., invited
review, in press.
Glaser, S.M. and Cumsky, M.G. (1990a) Localization of a synthetic presequence that blocks protein import into
mitochondria. J Biol Chem, 265, 8817-8822.
Glaser, S.M. and Cumsky, M.G. (1990b) A synthetic presequence reversibly inhibits protein import into yeast
mitochondria. J Biol Chem, 265, 8808-8816.
Glick, B.S., Brandt, A., Cunningham, K., Muller, S., Hallberg, R.L. and Schatz, G. (1992) Cytochromes c1 and b2
are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell, 69, 809822.
Goedert, M., Klug, A. and Crowther, R.A. (2006) Tau protein, the paired helical filament and Alzheimer's disease. J
Alzheimers Dis, 9, 195-207.
Gottesman, S. and Zipser, D. (1978) Deg phenotype of Escherichia coli lon mutants. J Bacteriol, 133, 844-851.
Gottesman, S., Roche, E., Zhou, Y. and Sauer, R.T. (1998) The ClpXP and ClpAP proteases degrade proteins with
carboxy-terminal peptide tails added by the SsrA-tagging system. Genes Dev, 12, 1338-1347.
Gray, M.W. (1999) Evolution of organellar genomes. Curr Opin Genet Dev, 9, 678-687.
Gray, M.W., Burger, G. and Lang, B.F. (1999) Mitochondrial evolution. Science, 283, 1476-1481.
Grohmann, L., Brennicke, A. and Schuster, W. (1992) The mitochondrial gene encoding ribosomal protein S12 has
been translocated to the nuclear genome in Oenothera. Nucleic Acids Res, 20, 5641-5646.
Gueguen, V., Macherel, D., Jaquinod, M., Douce, R. and Bourguignon, J. (2000) Fatty acid and lipoic acid
biosynthesis in higher plant mitochondria. J Biol Chem, 275, 5016-5025.
Gutensohn, M., Schulz, B., Nicolay, P. and Flugge, U.I. (2000) Functional analysis of the two Arabidopsis
homologues of Toc34, a component of the chloroplast protein import apparatus. Plant J, 23, 771-783.
Gutensohn, M., Fan, E., Frielingsdorf, S., Hanner, P., Hou, B., Hust, B. and Klosgen, R.B. (2006) Toc, Tic, Tat et
al.: structure and function of protein transport machineries in chloroplasts. J Plant Physiol, 163, 333-347.
Hallermayer, G., Zimmermann, R. and Neupert, W. (1977) Kinetic studies on the transport of cytoplasmically
synthesized proteins into the mitochondria in intact cells of Neurospora crassa. Eur J Biochem, 81, 523532.
Halperin, T., Ostersetzer, O. and Adam, Z. (2001a) ATP-dependent association between subunits of Clp protease in
pea chloroplasts. Planta, 213, 614-619.
Halperin, T., Zheng, B., Itzhaki, H., Clarke, A.K. and Adam, Z. (2001b) Plant mitochondria contain proteolytic and
regulatory subunits of the ATP-dependent Clp protease. Plant Mol Biol, 45, 461-468.
Hansson, C.A., Frykman, S., Farmery, M.R., Tjernberg, L.O., Nilsberth, C., Pursglove, S.E., Ito, A., Winblad, B.,
Cowburn, R.F., Thyberg, J. and Ankarcrona, M. (2004) Nicastrin, presenilin, APH-1, and PEN-2 form
active gamma-secretase complexes in mitochondria. J Biol Chem, 279, 51654-51660.
Hartl, F.U., Ostermann, J., Guiard, B. and Neupert, W. (1987) Successive translocation into and out of the
mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell,
51, 1027-1037.
Hashimoto, M., Rockenstein, E., Crews, L. and Masliah, E. (2003) Role of protein aggregation in mitochondrial
dysfunction and neurodegeneration in Alzheimer's and Parkinson's diseases. Neuromolecular Med, 4, 2136.
Hashimoto, Y., Niikura, T., Chiba, T., Tsukamoto, E., Kadowaki, H., Nishitoh, H., Yamagishi, Y., Ishizaka, M.,
Yamada, M., Nawa, M., Terashita, K., Aiso, S., Ichijo, H. and Nishimoto, I. (2003) The cytoplasmic
domain of Alzheimer's amyloid-beta protein precursor causes sustained apoptosis signal-regulating kinase
1/c-Jun NH2-terminal kinase-mediated neurotoxic signal via dimerization. J Pharmacol Exp Ther, 306,
889-902.
Haussuhl, K., Andersson, B. and Adamska, I. (2001) A chloroplast DegP2 protease performs the primary cleavage
of the photodamaged D1 protein in plant photosystem II. EMBO J, 20, 713-722.
Heaton, D., Nittis, T., Srinivasan, C. and Winge, D.R. (2000) Mutational analysis of the mitochondrial copper
metallochaperone Cox17. J Biol Chem, 275, 37582-37587.
Heazlewood, J.L., Tonti-Filippini, J.S., Gout, A.M., Day, D.A., Whelan, J. and Millar, A.H. (2004) Experimental
analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components,
provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins.
Plant Cell, 16, 241-256.
63
Heins, L., Mehrle, A., Hemmler, R., Wagner, R., Kuchler, M., Hormann, F., Sveshnikov, D. and Soll, J. (2002) The
preprotein conducting channel at the inner envelope membrane of plastids. EMBO J, 21, 2616-2625.
Hell, K., Herrmann, J.M., Pratje, E., Neupert, W. and Stuart, R.A. (1998) Oxa1p, an essential component of the Ntail protein export machinery in mitochondria. Proc Natl Acad Sci U S A, 95, 2250-2255.
Herlan, M., Bornhovd, C., Hell, K., Neupert, W. and Reichert, A.S. (2004) Alternative topogenesis of Mgm1 and
mitochondrial morphology depend on ATP and a functional import motor. J Cell Biol, 165, 167-173.
Herlan, M., Vogel, F., Bornhovd, C., Neupert, W. and Reichert, A.S. (2003) Processing of Mgm1 by the rhomboidtype protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA.
J Biol Chem, 278, 27781-27788.
Hill, K., Model, K., Ryan, M.T., Dietmeier, K., Martin, F., Wagner, R. and Pfanner, N. (1998) Tom40 forms the
hydrophilic channel of the mitochondrial import pore for preproteins. Nature, 395, 516-521.
Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brutsch, H. and Schatz, G. (1990) Protein import into yeast
mitochondria is accelerated by the outer membrane protein MAS70. EMBO J, 9, 3191-3200.
Hinnah, S.C., Hill, K., Wagner, R., Schlicher, T and Soll, J. (1997) Reconstitution of a chloroplast protein import
channel. EMBO J, 16, 7351-7360.
Hinnah, S.C., Wagner, R., Sveshnikova, N., Harrer, R. and Soll, J. (2002) The chloroplast protein import channel
Toc75: pore properties and interaction with transit peptides. Biophys J, 83, 899-911.
Hirohashi, T., Hase, T. and Nakai, M. (2001) Maize non-photosynthetic ferredoxin precursor is mis-sorted to the
intermembrane space of chloroplasts in the presence of light. Plant Physiol, 125, 2154-2163.
Hirsch, S., Muckel, E., Heemeyer, F., von Heijne, G. and Soll, J. (1994) A receptor component of the chloroplast
protein translocation machinery. Science, 266, 1989-1992.
Hohfeld, J., Minami, Y. and Hartl, F.U. (1995) Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40
reaction cycle. Cell, 83, 589-598.
Honlinger, A., Bomer, U., Alconada, A., Eckerskorn, C., Lottspeich, F., Dietmeier, K. and Pfanner, N. (1996) Tom7
modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related
role in protein import. EMBO J, 15, 2125-2137.
Huang, C., Wang, S., Chen, L., Lemieux, C., Otis, C., Turmel, M. and Liu, X.Q. (1994) The Chlamydomonas
chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth. Mol
Gen Genet, 244, 151-159.
Huang, S., Ratliff, K.S. and Matouschek, A. (2002) Protein unfolding by the mitochondrial membrane potential. Nat
Struct Biol, 9, 301-307.
Hugosson, M., Andreu, D., Boman, H.G. and Glaser, E. (1994) Antibacterial peptides and mitochondrial
presequences affect mitochondrial coupling, respiration and protein import. Eur J Biochem, 223, 10271033.
Isaya, G., Kalousek, F., Fenton, W.A. and Rosenberg, L.E. (1991) Cleavage of precursors by the mitochondrial
processing peptidase requires a compatible mature protein or an intermediate octapeptide. J Cell Biol,
113, 65-76.
Itzhaki, H., Naveh, L., Lindahl, M., Cook, M. and Adam, Z. (1998) Identification and characterization of DegP, a
serine protease associated with the luminal side of the thylakoid membrane. J Biol Chem, 273, 70947098.
Iwata, S., Lee, J.W., Okada, K., Lee, J.K., Iwata, M., Rasmussen, B., Link, T.A., Ramaswamy, S. and Jap, B.K.
(1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex. Science,
281, 64-71.
Jan, P.S., Esser, K., Pratje, E. and Michaelis, G. (2000) Som1, a third component of the yeast mitochondrial inner
membrane peptidase complex that contains Imp1 and Imp2. Mol Gen Genet, 263, 483-491.
Jarvis, P. and Soll, J. (2001) Toc, Tic, and chloroplast protein import. Biochim Biophys Acta, 1541, 64-79.
Jarvis, P., Chen, L.J., Li, H., Peto, C.A., Fankhauser, C. and Chory, J. (1998) An Arabidopsis mutant defective in
the plastid general protein import apparatus. Science, 282, 100-103.
Jelic, M., Sveshnikova, N., Motzkus, M., Horth, P., Soll, J. and Schleiff, E. (2002) The chloroplast import receptor
Toc34 functions as preprotein-regulated GTPase. Biol Chem, 383, 1875-1883.
Jensen, R.E. and Dunn, C.D. (2002) Protein import into and across the mitochondrial inner membrane: role of the
TIM23 and TIM22 translocons. Biochim Biophys Acta, 1592, 25-34.
Kadowaki, K., Kubo, N., Ozawa, K. and Hirai, A. (1996) Targeting presequence acquisition after mitochondrial
gene transfer to the nucleus occurs by duplication of existing targeting signals. EMBO J, 15, 6652-6661.
64
Kambacheld, M., Augustin, S., Tatsuta, T., Muller, S. and Langer, T. (2005) Role of the novel metallopeptidase
Mop112 and saccharolysin for the complete degradation of proteins residing in different
subcompartments of mitochondria. J Biol Chem, 280, 20132-20139.
Kaser, M. and Langer, T. (2000) Protein degradation in mitochondria. Semin Cell Dev Biol, 11, 181-190.
Kaser, M., Kambacheld, M., Kisters-Woike, B. and Langer, T. (2003) Oma1, a novel membrane-bound
metallopeptidase in mitochondria with activities overlapping with the m-AAA protease. J Biol Chem,
278, 46414-46423.
Kato, A., Sugiura, N., Saruta, Y., Hosoiri, T., Yasue, H. and Hirose, S. (1997) Targeting of endopeptidase 24.16 to
different subcellular compartments by alternative promoter usage. J Biol Chem, 272, 15313-15322.
Kennedy, A.P. and Lehninger, A.L. (1950) The products of oxidation of fatty acids by isolated rat liver
mitochondria. J Biol Chem, 185, 275-285.
Kerr, M.L. and Small, D.H. (2005) Cytoplasmic domain of the beta-amyloid protein precursor of Alzheimer's
disease: function, regulation of proteolysis, and implications for drug development. J Neurosci Res, 80,
151-159.
Kerscher, O., Holder, J., Srinivasan, M., Leung, R.S. and Jensen, R.E. (1997) The Tim54p-Tim22p complex
mediates insertion of proteins into the mitochondrial inner membrane. J Cell Biol, 139, 1663-1675.
Kessler, F. and Blobel, G. (1996) Interaction of the protein import and folding machineries of the chloroplast. Proc
Natl Acad Sci U S A, 93, 7684-7689.
Kessler, F., Blobel, G., Patel, H.A. and Schnell, D.J. (1994) Identification of two GTP-binding proteins in the
chloroplast protein import machinery. Science, 266, 1035-1039.
Kitada, S., Shimokata, K., Niidome, T., Ogishima, T. and Ito, A. (1995) A putative metal-binding site in the beta
subunit of rat mitochondrial processing peptidase is essential for its catalytic activity. J Biochem (Tokyo),
117, 1148-1150.
Klanner, C., Prokisch, H. and Langer, T. (2001) MAP-1 and IAP-1, two novel AAA proteases with catalytic sites on
opposite membrane surfaces in mitochondrial inner membrane of Neurospora crassa. Mol Biol Cell, 12,
2858-2869.
Kleffmann, T., Russenberger, D., von Zychlinski, A., Christopher, W., Sjolander, K., Gruissem, W. and Baginsky,
S. (2004) The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein
functions. Curr Biol, 14, 354-362.
Koehler, C.M., Merchant, S., Oppliger, W., Schmid, K., Jarosch, E., Dolfini, L., Junne, T., Schatz, G. and
Tokatlidis, K. (1998a) Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial
carrier proteins. EMBO J, 17, 6477-6486.
Koehler, C.M., Jarosch, E., Tokatlidis, K., Schmid, K., Schweyen, R.J. and Schatz, G. (1998b) Import of
mitochondrial carriers mediated by essential proteins of the intermembrane space. Science, 279, 369-373.
Kolodziejczak, M., Kolaczkowska, A., Szczesny, B., Urantowka, A., Knorpp, C., Kieleczawa, J. and Janska, H.
(2002) A higher plant mitochondrial homologue of the yeast m-AAA protease. Molecular cloning,
localization, and putative function. J Biol Chem, 277, 43792-43798.
Kominsky, D.J., Brownson, M.P., Updike, D.L. and Thorsness, P.E. (2002) Genetic and biochemical basis for
viability of yeast lacking mitochondrial genomes. Genetics, 162, 1595-1604.
Komiya, T., Rospert, S., Koehler, C., Looser, R., Schatz, G. and Mihara, K. (1998) Interaction of mitochondrial
targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid
chain' hypothesis. EMBO J, 17, 3886-3898.
Koonin, E.V., Makarova, K.S., Rogozin, I.B., Davidovic, L., Letellier, M.C. and Pellegrini, L. (2003) The
rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by
multiple ancient horizontal gene transfers. Genome Biol, 4, R19.
Kouranov, A. and Schnell, D.J. (1997) Analysis of the interactions of preproteins with the import machinery over
the course of protein import into chloroplasts. J Cell Biol, 139, 1677-1685.
Kouranov, A., Chen, X., Fuks, B. and Schnell, D.J. (1998) Tic20 and Tic22 are new components of the protein
import apparatus at the chloroplast inner envelope membrane. J Cell Biol, 143, 991-1002.
Kovermann, P., Truscott, K.N., Guiard, B., Rehling, P., Sepuri, N.B., Muller, H., Jensen, R.E., Wagner, R. and
Pfanner, N. (2002) Tim22, the essential core of the mitochondrial protein insertion complex, forms a
voltage-activated and signal-gated channel. Mol Cell, 9, 363-373.
Krimm, I., Gans, P., Hernandez, J.F., Arlaud, G.J. and Lancelin, J.M. (1999) A coil-helix instead of a helix-coil
motif can be induced in a chloroplast transit peptide from Chlamydomonas reinhardtii. Eur J Biochem,
265, 171-180.
65
Krojer, T., Garrido-Franco, M., Huber, R., Ehrmann, M. and Clausen, T. (2002) Crystal structure of DegP (HtrA)
reveals a new protease-chaperone machine. Nature, 416, 455-459.
Kuchler, M., Decker, S., Hormann, F., Soll, J. and Heins, L. (2002) Protein import into chloroplasts involves redoxregulated proteins. EMBO J, 21, 6136-6145.
Kunkele, K.P., Heins, S., Dembowski, M., Nargang, F.E., Benz, R., Thieffry, M., Walz, J., Lill, R., Nussberger, S.
and Neupert, W. (1998) The preprotein translocation channel of the outer membrane of mitochondria.
Cell, 93, 1009-1019.
Kurland, C.G. (1992) Evolution of mitochondrial genomes and the genetic code. Bioessays, 14, 709-714.
Kurland, C.G. and Andersson, S.G. (2000) Origin and evolution of the mitochondrial proteome. Microbiol Mol Biol
Rev, 64, 786-820.
Kuroda, H. and Maliga, P. (2003) The plastid clpP1 protease gene is essential for plant development. Nature, 425,
86-89.
Kurz, M., Martin, H., Rassow, J., Pfanner, N. and Ryan, M.T. (1999) Biogenesis of Tim proteins of the
mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main
import pathway. Mol Biol Cell, 10, 2461-2474.
Lancelin, J.M., Bally, I., Arlaud, G.J., Blackledge, M., Gans, P., Stein, M. and Jacquot, J.P. (1994) NMR structures
of ferredoxin chloroplastic transit peptide from Chlamydomonas reinhardtii promoted by trifluoroethanol
in aqueous solution. FEBS Lett, 343, 261-166.
Lang, B.F., Gray, M.W. and Burger, G. (1999) Mitochondrial genome evolution and the origin of eukaryotes. Annu
Rev Genet, 33, 351-397.
Langer, T. (2000) AAA proteases: cellular machines for degrading membrane proteins. Trends Biochem Sci, 25,
247-251.
Lee, K.H., Kim, S.J., Lee, Y.J., Jin, J.B. and Hwang, I. (2003) The M domain of AtToc159 plays an essential role in
the import of proteins into chloroplasts and chloroplast biogenesis. J Biol Chem, 278, 36794-36805.
Lensch, M., Herrmann, R.G. and Sokolenko, A. (2001) Identification and characterization of SppA, a novel lightinducible chloroplast protease complex associated with thylakoid membranes. J Biol Chem, 276, 3364533651.
Leonhard, K., Guiard, B., Pellecchia, G., Tzagoloff, A., Neupert, W. and Langer, T. (2000) Membrane protein
degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface.
Mol Cell, 5, 629-638.
Leonhard, K., Herrmann, J.M., Stuart, R.A., Mannhaupt, G., Neupert, W. and Langer, T. (1996) AAA proteases
with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent
degradation of inner membrane proteins in mitochondria. EMBO J, 15, 4218-4229.
Li, Y., Dudek, J., Guiard, B., Pfanner, N., Rehling, P. and Voos, W. (2004) The presequence translocase-associated
protein import motor of mitochondria. Pam16 functions in an antagonistic manner to Pam18. J Biol
Chem, 279, 38047-38054.
Lindahl, M., Spetea, C., Hundal, T., Oppenheim, A.B., Adam, Z. and Andersson, B. (2000) The thylakoid FtsH
protease plays a role in the light-induced turnover of the photosystem II D1 protein. Plant Cell, 12, 419431.
Lindahl, M., Tabak, S., Cseke, L., Pichersky, E., Andersson, B. and Adam, Z. (1996) Identification,
characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of
higher plants. J Biol Chem, 271, 29329-29334.
Lister, R., Mowday, B., Whelan, J. and Millar, A.H. (2002) Zinc-dependent intermembrane space proteins stimulate
import of carrier proteins into plant mitochondria. Plant J, 30, 555-566.
Lowe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W. and Huber, R. (1995) Crystal structure of the 20S
proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science, 268, 533-539.
Lu, Y. and Beavis, A.D. (1997) Effect of leader peptides on the permeability of mitochondria. J Biol Chem, 272,
13555-13561.
Lu, H., Allen, S., Wardleworth, L., Savory, P and Tokatlidis, K. (2004) Functional TIM10 chaperone assembly is
redox-regulated in vivo. J Biol Chem, 279, 18952-18958.
Lubeck, J., Soll, J., Akita, M., Nielsen, E. and Keegstra, K. (1996) Topology of IEP110, a component of the
chloroplastic protein import machinery present in the inner envelope membrane. EMBO J, 15, 42304238.
Luciano, P., Geoffroy, S., Brandt, A., Hernandez, J.F. and Geli, V. (1997) Functional cooperation of the
mitochondrial processing peptidase subunits. J Mol Biol, 272, 213-225.
66
Lustbader, J.W., Cirilli, M., Lin, C., Xu, H.W., Takuma, K., Wang, N., Caspersen, C., Chen, X., Pollak, S., Chaney,
M., Trinchese, F., Liu, S., Gunn-Moore, F., Lue, L.F., Walker, D.G., Kuppusamy, P., Zewier, Z.L.,
Arancio, O., Stern, D., Yan, S.S. and Wu, H. (2004) ABAD directly links Abeta to mitochondrial toxicity
in Alzheimer's disease. Science, 304, 448-452.
Ma, Y., Kouranov, A., LaSala, S.E. and Schnell, D.J. (1996) Two components of the chloroplast protein import
apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of
precursor proteins at the outer envelope. J Cell Biol, 134, 315-327.
Macasev, D., Newbigin, E., Whelan, J. and Lithgow, T. (2000) How do plant mitochondria avoid importing
chloroplast proteins? Components of the import apparatus Tom20 and Tom22 from Arabidopsis differ
from their fungal counterparts. Plant Physiol, 123, 811-816.
Macasev, D., Whelan, J., Newbigin, E., Silva-Filho, M.C., Mulhern, T.D. and Lithgow, T. (2004) Tom22', an 8-kDa
trans-site receptor in plants and protozoans, is a conserved feature of the TOM complex that appeared
early in the evolution of eukaryotes. Mol Biol Evol, 21, 1557-1564.
Mann, N.H., Novac, N., Mullineaux, C.W., Newman, J., Bailey, S. and Robinson, C. (2000) Involvement of an FtsH
homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC
6803. FEBS Lett, 479, 72-77.
Marc, P., Margeot, A., Devaux, F., Blugeon, C., Corral-Debrinski, M. and Jacq, C. (2002) Genome-wide analysis of
mRNAs targeted to yeast mitochondria. EMBO Rep, 3, 159-164.
Margulis, L. (1970) Origin of eukaryotic cells. Yale University Press, New Haven, Connecticut.
Martin, A.P. and Palumbi, S.R. (1992) Body size, metabolic rate, generation time, and the molecular clock. Proc
Natl Acad Sci U S A, 90, 4087-4091.
Martin, T., Sharma, R., Sippel, C., Waegemann, K., Soll, J. and Vothknecht, U.C. (2006) A protein kinase family in
Arabidopsis phosphorylates chloroplast precursor proteins. J Biol Chem, 281, 40216-40223.
Martin, W. and Herrmann, R.G. (1998) Gene transfer from organelles to the nucleus: how much, what happens, and
Why? Plant Physiol, 118, 9-17.
Matouschek, A., Azem, A., Ratliff, K., Glick, B.S., Schmid, K. and Schatz, G. (1997) Active unfolding of precursor
proteins during mitochondrial protein import. EMBO J, 16, 6727-6736.
Matsuzaki, K., Murase, O., Fujii, N. and Miyajima, K. (1996) An antimicrobial peptide, magainin 2, induced rapid
flip-flop of phospholipids coupled with pore formation and peptide translocation. Biochemistry, 35,
11361-11368.
May, T. and Soll, J. (2000) 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants.
Plant Cell, 12, 53-64.
McQuibban, G.A., Saurya, S. and Freeman, M. (2003) Mitochondrial membrane remodelling regulated by a
conserved rhomboid protease. Nature, 423, 537-541.
Meinecke, M., Wagner, R., Kovermann, P., Guiard, B., Mick, D.U., Hutu, D.P., Voos, W., Truscott, K.N.,
Chacinska, A., Pfanner, N. and Rehling, P. (2006) Tim50 maintains the permeability barrier of the
mitochondrial inner membrane. Science, 312, 1523-1526.
Mesecke, N., Terziyska, N., Kozany, C., Baumann, F., Neupert, W., Hell, K. and Herrmann, J.M. (2005) A disulfide
relay system in the intermembrane space of mitochondria that mediates protein import. Cell, 121, 10591069.
Mihara, K. and Omura, T. (1996) Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF
and hsp 70. Trends Cell Biol, 6, 104-108.
Mitchell, P. (1974) A chemi-osmotic molecular mechanism for proton-translocating adenosine-triphosphatases.
FEBS Lett, 43, 189-194.
Moberg, P., Nilsson, S., Stahl, A., Eriksson, A.C., Glaser, E. and Maler, L. (2004) NMR solution structure of the
mitochondrial F1beta presequence from Nicotiana plumbaginifolia. J Mol Biol, 336, 1129-1140.
Mokranjac, D., Popov-Celeketic, D., Hell, K. and Neupert, W. (2005) Role of Tim21 in mitochondrial translocation
contact sites. J Biol Chem, 280, 23437-23440.
Murcha, M.W., Elhafez, D., Millar, A.H. and Whelan, J. (2004) The N-terminal extension of plant mitochondrial
carrier proteins is removed by two-step processing: the first cleavage is by the mitochondrial processing
peptidase. J Mol Biol, 344, 443-454.
Muto, T., Obita, T., Abe, Y., Shodai, T., Endo, T. and Kohda, D. (2001) NMR identification of the Tom20 binding
segment in mitochondrial presequences. J Mol Biol, 306, 137-143.
67
Mzhavia, N., Berman, Y.L., Qian, Y., Yan, L. and Devi, L.A. (1999) Cloning, expression, and characterization of
human metalloprotease 1: a novel member of the pitrilysin family of metalloendoproteases. DNA Cell
Biol, 18, 369-380.
Neupert, W. (1997) Protein import into mitochondria. Annu Rev Biochem, 66, 863-917.
Neupert, W. and Brunner, M. (2002) The protein import motor of mitochondria. Nat Rev Mol Cell Biol, 3, 555-565.
Neupert, W. and Herrmann, J.M. (2007) Translocation of Proteins into Mitochondria. Annu Rev Biochem, 76, 6.16.27.
Nicolay, K., Laterveer, F.D. and van Heerde, W.L. (1994) Effects of amphipathic peptides, including presequences,
on the functional integrity of rat liver mitochondrial membranes. J Bioenerg Biomembr, 26, 327-334.
Nugent, J.M. and Palmer, J.D. (1991) RNA-mediated transfer of the gene coxII from the mitochondrion to the
nucleus during flowering plant evolution. Cell, 66, 473-481.
Nunnari, J., Fox, T.D. and Walter, P. (1993) A mitochondrial protease with two catalytic subunits of nonoverlapping
specificities. Science, 262, 1997-2004.
Obara, K., Sumi, K. and Fukuda, H. (2002) The use of multiple transcription starts causes the dual targeting of
Arabidopsis putative monodehydroascorbate reductase to both mitochondria and chloroplasts. Plant Cell
Physiol, 43, 697-705.
Oblong, J.E. and Lamppa, G. K. (1992) Identification of two structurally related proteins involved in proteolytic
processing of precursors targeted to the chloroplast. EMBO J, 11, 4401-4409.
Ohnishi, S. and Takano, K. (2004) Amyloid fibrils from the viewpoint of protein folding. Cell Mol Life Sci, 61,
511-524.
Ostersetzer, O. and Adam, Z. (1997) Light-stimulated degradation of an unassembled Rieske FeS protein by a
thylakoid-bound protease: the possible role of the FtsH protease. Plant Cell, 9, 957-965.
Park, S. and Rodermel, S.R. (2004) Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid
membrane biogenesis. Proc Natl Acad Sci U S A, 101, 12765-12770.
Pearce, D.A. and Sherman, F. (1995) Degradation of cytochrome oxidase subunits in mutants of yeast lacking
cytochrome c and suppression of the degradation by mutation of yme1. J Biol Chem, 270, 20879-20882.
Peeters, N. and Small, I. (2001) Dual targeting to mitochondria and chloroplasts. Biochim Biophys Acta, 1541, 5463.
Peltier, J.B., Ripoll, D.R., Friso, G., Rudella, A., Cai, Y., Ytterberg, J., Giacomelli, L., Pillardy, J. and van Wijk,
K.J. (2004) Clp protease complexes from photosynthetic and non-photosynthetic plastids and
mitochondria of plants, their predicted three-dimensional structures, and functional implications. J Biol
Chem, 279, 4768-4781.
Peltier, J.B., Ytterberg, J., Liberles, D.A., Roepstorff, P. and van Wijk, K.J. (2001) Identification of a 350-kDa ClpP
protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana. J Biol Chem,
276, 16318-16327.
Perry, S.E. and Keegstra, K. (1994) Envelope membrane proteins that interact with chloroplastic precursor proteins.
Plant Cell, 6, 93-105.
Rassow, J., Dekker, P.J., van Wilpe, S., Meijer, M. and Soll, J. (1999) The preprotein translocase of the
mitochondrial inner membrane: function and evolution. J Mol Biol, 286, 105-120.
Rawlings, N.D., Morton, F.R. and Barrett, A.J. (2006) MEROPS: the peptidase database. Nucleic Acids Res, 34
(Database issue), D270-272.
Rebeille, F., Macherel, D., Mouillon, J.M., Garin, J. and Douce, R. (1997) Folate biosynthesis in higher plants:
purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin
pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria. EMBO J, 16, 947-957.
Richter, S. and Lamppa, G.K. (1999) Stromal processing peptidase binds transit peptides and initiates their ATPdependent turnover in chloroplasts. J Cell Biol, 147, 33-44.
Richter, S. and Lamppa, G.K. (2002) Determinants for removal and degradation of transit peptides of chloroplast
precursor proteins. J Biol Chem, 277, 43888-43894.
Richter, S. and Lampaa, G.K. (2003) Structural properties of the chloroplast stromal processing peptidase required
for its function in transit peptide removal. J Biol Chem, 278, 39497-39502.
Roise, D., Horvath, S.J., Tomich, J.M., Richards, J.H. and Schatz, G. (1986) A chemically synthesized pre-sequence
of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial
phospholipid bilayers. EMBO J, 5, 1327-1334.
68
Rotanova, T.V., Melnikov, E.E., Khalatova, A.G., Makhovskaya, O.V., Botos, I., Wlodawer, A. and Gustchina A
(2004) Classification of ATP-dependent proteases Lon and comparison of the active sites of their
proteolytic domains. Eur J Biochem, 271, 4865-4871.
Rudhe, C., Chew, O., Whelan, J. and Glaser, E. (2002a) A novel in vitro system for simultaneous import of
precursor proteins into mitochondria and chloroplasts. Plant J, 30, 213-220.
Rudhe, C., Clifton, R., Whelan, J. and Glaser, E. (2002b) N-terminal domain of the dual-targeted pea glutathione
reductase signal peptide controls organellar targeting efficiency. J Mol Biol, 324, 577-585.
Sakamoto, W. (2006) Protein degradation machineries in plastids. Annu Rev Plant Biol, 57, 599-621.
Sanchez, H., Fester, T., Kloska, S., Schroder, W. and Schuster, W. (1996) Transfer of rps19 to the nucleus involves
the gain of an RNP-binding motif which may functionally replace RPS13 in Arabidopsis mitochondria.
EMBO J, 15, 2138-2149.
Santagata, S., Bhattacharyya, D., Wang, F.H., Singha, N., Hodtsev, A. and Spanopoulou, E. (1999) Molecular
cloning and characterization of a mouse homolog of bacterial ClpX, a novel mammalian class II member
of the Hsp100/Clp chaperone family. J Biol Chem, 274, 16311-16319.
Sarria, R., Lyznik, A., Vallejos, C.E. and Mackenzie, S.A. (1998) A cytoplasmic male sterility-associated
mitochondrial peptide in common bean is post-translationally regulated. Plant Cell, 10, 1217-1228.
Schleiff, E., Soll, J., Kuchler, M., Kuhlbrandt, W. and Harrer, R. (2003) Characterization of the translocon of the
outer envelope of chloroplasts. J Cell Biol, 160, 541-551.
Schmitz, U.K. and Lonsdale, D. M. (1989) A yeast mitochondrial presequence functions as a signal for targeting to
plant mitochondria in vivo. Plant Cell, 1, 783-791.
Schneider, A., Oppliger, W. and Jeno, P. (1994) Purified inner membrane protease I of yeast mitochondria is a
heterodimer. J Biol Chem, 269, 8635-8638.
Schnell, D.J., Kessler, F. and Blobel, G. (1994) Isolation of components of the chloroplast protein import
machinery. Science, 266, 1007-1012.
Seedorf, M., Waegemann, K. and Soll, J. (1995) A constituent of the chloroplast import complex represents a new
type of GTP-binding protein. Plant J, 7, 401-411.
Selkoe, D.J. (1999) Translating cell biology into therapeutic advances in Alzheimer's disease. Nature, 399, A23A31.
Serizawa, A., Dando, P.M. and Barrett, A.J. (1995) Characterization of a mitochondrial metallopeptidase reveals
neurolysin as a homologue of thimet oligopeptidase. J Biol Chem, 270, 2092-2098.
Shen., G., Yan, J., Pasapula, V., Luo, J., He, C., Clarke, A.K. and Zhang, H. (2007) The chloroplast protease subunit
ClpP4 is a substrate of the E3 ligase AtCHIP and plays an important role in chloroplast function. Plant J,
49, 228-237.
Shen, Y., Joachimiak, A., Rosner, M.R. and Tang, W.J. (2006) Structures of human insulin-degrading enzyme
reveal a new substrate recognition mechanism. Nature, 443, 870-874.
Sherman, E.L., Go, N.E. and Nargang, F.E. (2005) Functions of the small proteins in the TOM complex of
Neurospora crasssa. Mol Biol Cell, 16, 4172-4182.
Sickmann, A., Reinders, J., Wagner, Y., Joppich, C., Zahedi, R., Meyer, H.E., Schonfisch, B., Perschil, I.,
Chacinska, A., Guiard, B., Rehling, P., Pfanner, N. and Meisinger, C. (2003) The proteome of
Saccharomyces cerevisiae mitochondria. Proc Natl Acad Sci U S A, 100, 13207-13212.
Silva-Filho, M.C. (2003) One ticket for multiple destinations: dual targeting of proteins to distinct subcellular
locations. Curr Opin Plant Biol, 6, 589-595.
Silva-Filho, M.D., Wieers, M.C., Flugge, U.I., Chaumont, F and Boutry, M. (1997) Different in vitro and in vivo
targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. J Biol Chem,
272, 15264-15269.
Sinvany-Villalobo, G., Davydov, O., Ben-Ari, G., Zaltsman, A., Raskind, A. and Adam Z. (2004) Expression in
multigene families. Analysis of chloroplast and mitochondrial proteases. Plant Physiol, 135, 1336-1345.
Sirrenberg, C., Bauer, M.F., Guiard, B., Neupert, W. and Brunner, M. (1996) Import of carrier proteins into the
mitochondrial inner membrane mediated by Tim22. Nature, 384, 582-585.
Sjögren, L.L., Stanne, T.M., Zheng, B., Sutinen, S. and Clarke, A.K. (2006) Structural and functional insights into
the chloroplast ATP-dependent Clp protease in Arabidopsis. Plant Cell, 18, 2635-2649.
Small, I., Wintz, H., Akashi, K. and Mireau, H. (1998) Two birds with one stone: genes that encode products
targeted to two or more compartments. Plant Mol Biol, 38, 265-277.
69
Smith, P.M., Mann, A.J., Goggin, D.E. and Atkins, C.A. (1998) AIR synthetase in cowpea nodules: a single gene
product targeted to two organelles? Plant Mol Biol, 36, 811-820.
Sohrt, K. and Soll, J. (2000) Toc64, a new component of the protein translocon of chloroplasts. J Cell Biol, 148,
1213-1221.
Sokolenko, A., Pojidaeva, E., Zinchenko, V., Panichkin, V., Glaser, V.M., Herrmann, R.G. and Shestakov, S.V.
(2002) The gene complement for proteolysis in the cyanobacterium Synechocystis sp. PCC 6803 and
Arabidopsis thaliana chloroplasts. Curr Genet, 41, 291-310.
Soll, J. and Schleiff, E. (2004) Protein import into chloroplasts. Nat Rev Mol Cell Biol, 5, 198-208.
Spiess, C., Beil, A. and Ehrmann, M. (1999) A temperature-dependent switch from chaperone to protease in a
widely conserved heat shock protein. Cell, 97, 339-347.
Ståhl, A., Moberg, P., Ytterberg, J., Panfilov, O., Brockenhuus Von Lowenhielm, H., Nilsson, F. and Glaser, E.
(2002) Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades
targeting presequences in plants. J Biol Chem, 277, 41931-41939.
Ståhl, A., Pavlov, P.F., Szigyarto, C. and Glaser, E. (2000) Rapid degradation of the presequence of the f1beta
precursor of the ATP synthase inside mitochondria. Biochem J, 349, 703-707.
Stahl, T., Glockmann, C., Soll, J. and Heins, L. (1999) Tic40, a new "old" subunit of the chloroplast protein import
translocon. J Biol Chem, 274, 37467-37472.
Stahlberg, H., Kutejova, E., Suda, K., Wolpensinger, B., Lustig, A., Schatz, G., Engel, A. and Suzuki, C.K. (1999)
Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits.
Proc Natl Acad Sci U S A, 96, 6787-6790.
Stoebe, B and Maier, U.G. (2002) One, two, three: nature's tool box for building plastids. Protoplasma, 219, 123130.
Suissa, M. and Schatz, G. (1982) Import of proteins into mitochondria. Translatable mRNAs for imported
mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes. J Biol
Chem, 257, 13048-13055.
Suzuki, C.K., Suda, K., Wang, N. and Schatz, G. (1994) Requirement for the yeast gene LON in intramitochondrial
proteolysis and maintenance of respiration. Science, 264, 891.
Sveshnikova, N., Grimm, R., Soll, J. and Schleiff, E. (2000) Topology studies of the chloroplast protein import
channel Toc75. Biol Chem, 381, 687-693.
Takechi, K., Sodmergen, Murata, M., Motoyoshi, F. and Sakamoto, W. (2000) The YELLOW VARIEGATED
(VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis. Plant Cell
Physiol, 41, 1334-1346.
Tanzi, R.E., Moir, R.D. and Wagner, S.L. (2004) Clearance of Alzheimer's Abeta peptide: the many roads to
perdition. Neuron, 43, 605-608.
Taylor, A.B., Smith, B.S., Kitada, S., Kojima, K., Miyaura, H., Otwinowski, Z., Ito, A. and Deisenhofer, J. (2001)
Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import
signal sequences. Structure (Camb), 9, 615-625.
Taylor, S.W., Fahy, E., Zhang, B., Glenn, G.M., Warnock, D.E., Wiley, S., Murphy, A.N., Gaucher, S.P., Capaldi,
R.A., Gibson, B.W. and Ghosh, S.S. (2003) Characterization of the human heart mitochondrial proteome.
Nat Biotechnol, 21, 281-286.
Teichmann, U., van Dyck, L., Guiard, B., Fischer, H., Glockshuber, R., Neupert, W. and Langer, T. (1996)
Substitution of PIM1 protease in mitochondria by Escherichia coli Lon protease. J Biol Chem, 271,
10137-10142.
Thomas, P. and Fenech, M. (2006) A review of genome mutation and Alzheimer’s disease. Mutagensis, 22, 15-33.
Thorsness, P.E. and Fox, T.D. (1993) Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA
from mitochondria to the nucleus. Genetics, 134, 21-28.
Urantowka, A., Knorpp, C., Olczak, T., Kolodziejczak, M. and Janska, H. (2005) Plant mitochondria contain at least
two i-AAA-like complexes. Plant Mol Biol, 59, 239-252.
Urban, S., Lee, J.R. and Freeman, M. (2001) Drosophila rhomboid-1 defines a family of putative intramembrane
serine proteases. Cell, 107, 173-182.
Van der Bliek, A.M. and Koehler, C.M. (2003) A mitochondrial rhomboid protease. Dev Cell, 4, 769-770.
van der Laan, M., Chacinska, A., Lind, M., Perschil, I., Sickmann, A., Meyer, H.E., Guiard, B., Meisinger, C.,
Pfanner, N. and Rehling, P. (2005) Pam17 is required for architecture and translocation activity of the
mitochondrial protein import motor. Mol Cell Biol, 25, 7449-7458.
70
van der Laan, M., Rissler, M. and Rehling, P. (2006) Mitochondrial preprotein translocases as dynamic molecular
machines. FEMS Yeast Res, 6, 849-861.
Van Dyck, L. and Langer, T. (1999) ATP-dependent proteases controlling mitochondrial function in the yeast
Saccharomyces cerevisiae. Cell Mol Life Sci, 56, 825-842.
Van Dyck, L., Pearce, D.A. and Sherman, F. (1994) PIM1 encodes a mitochondrial ATP-dependent protease that is
required for mitochondrial function in the yeast Saccharomyces cerevisiae. J Biol Chem, 269, 238-242.
van Wilpe, S., Ryan, M.T., Hill, K., Maarse, A.C., Meisinger, C., Brix, J., Dekker, P.J., Moczko, M., Wagner, R.,
Meijer, M., Guiard, B., Honlinger, A. and Pfanner, N. (1999) Tom22 is a multifunctional organizer of the
mitochondrial preprotein translocase. Nature, 401, 485-489.
van't Hof, R. and de Kruijff, B. (1995) Transit sequence-dependent binding of the chloroplast precursor protein
ferredoxin to lipid vesicles and its implications for membrane stability. FEBS Lett, 361, 35-40.
van't Hof, R., Demel, R.A., Keegstra, K. and de Kruijff, B. (1991) Lipid-peptide interactions between fragments of
the transit peptide of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast membrane lipids.
FEBS Lett, 291, 350-354.
Verner, K. (1993) Co-translational protein import into mitochondria: an alternative view. Trends Biochem Sci, 18,
366-371.
von Heijne, G. (1986a) Why mitochondria need a genome? FEBS Letter, 198, 1-4.
von Heijne, G. (1986b) Mitochondrial targeting sequences may form amphiphilic helices. EMBO J, 5, 1335-1342.
von Heijne, G., Steppuhn, J. and Herrmann, R.G. (1989) Domain structure of mitochondrial and chloroplast
targeting peptides. Eur J Biochem, 180, 535-545.
Waegemann, K. and Soll, J. (1991) Characterization of the protein import apparatus in isolated outer envelopes of
chloroplasts. Plant J, 1, 149-158.
Waegemann, K. and Soll, J. (1995) Characterization and isolation of the chloroplast protein import machinery.
Methods Cell Biol, 50, 255-267.
Waegemann, K. and Soll, J. (1996) Phosphorylation of the transit sequence of chloroplast precursor proteins. J Biol
Chem, 271, 6545-6554.
Waegemann, K., Paulsen, H. and Soll, J. (1990) Translocation of proteins into isolated chloroplasts requires
cytosolic factors to obtain import competence. FEBS Lett, 261, 89-92.
Wang, J., Hartling, J.A. and Flanagan, J.M. (1997) The structure of ClpP at 2.3 A resolution suggests a model for
ATP-dependent proteolysis. Cell, 91, 447-456.
Wang, X. (2001) The expanding role of mitochondria in apoptosis. Genes Dev, 15, 2922-2933.
Wang, Y., Zhang, Y. and Ha, Y. (2006) Crystal structure of a rhomboid family intramembrane protease. Nature,
444, 179-180.
Watanabe, N., Che, F.S., Iwano, M., Takayama, S., Yoshida, S. and Isogai, A. (2001) Dual targeting of spinach
protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame
initiation codons. J Biol Chem, 276, 20474-20481.
Weber, E.R., Hanekamp, T. and Thorsness, P.E. (1996) Biochemical and functional analysis of the YME1 gene
product, an ATP and zinc-dependent mitochondrial protease from S. cerevisiae. Mol Biol Cell, 7, 307317.
Wienhues, U., Becker, K., Schleyer, M., Guiard, B., Tropschug, M., Horwich, A.L., Pfanner, N. and Neupert, W.
(1991) Protein folding causes an arrest of preprotein translocation into mitochondria in vivo. J Cell Biol,
115, 1601-1609.
Wienk, H.L., Wechselberger, R.W., Czisch, M. and de Kruijff, B. (2000) Structure, dynamics, and insertion of a
chloroplast targeting peptide in mixed micelles. Biochemistry, 39, 8219-8227.
Wischmann, C. and Schuster, W. (1995) Transfer of rps10 from the mitochondrion to the nucleus in Arabidopsis
thaliana: evidence for RNA-mediated transfer and exon shuffling at the integration site. FEBS Lett, 374,
152-156.
Yan, S.D. and Stern, D.M. (2005) Mitochondrial dysfunction and Alzheimer's disease: role of amyloid-beta peptide
alcohol dehydrogenase (ABAD). Int J Exp Pathol, 86, 161-171.
Yano, M., Terada, K. and Mori, M. (2003) AIP is a mitochondrial import mediator that binds to both import receptor
Tom20 and preproteins. J Cell Biol, 163, 45-56.
Young, J.C., Hoogenraad, N.J. and Hartl, F.U. (2003) Molecular chaperones Hsp90 and Hsp70 deliver preproteins
to the mitochondrial import receptor Tom70. Cell, 112, 41-50.
71
Young, L., Leonhard, K., Tatsuta, T., Trowsdale, J. and Langer, T. (2001) Role of the ABC transporter Mdl1 in
peptide export from mitochondria. Science, 291, 2135-2138.
Zhang, X.P. and Glaser, E. (2002) Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70
molecular chaperone. Trends Plant Sci, 7, 14-21.
Zhang, X.P., Sjoling, S., Tanudji, M., Somogyi, L., Andreu, D., Eriksson, L.E., Graslund, A., Whelan, J. and Glaser,
E. (2001) Mutagenesis and computer modelling approach to study determinants for recognition of signal
peptides by the mitochondrial processing peptidase. Plant J, 27, 427-38.
Zheng, B., Halperin, T., Hruskova-Heidingsfeldova, O., Adam, Z. and Clarke, A.K. (2002) Characterization of
Chloroplast Clp proteins in Arabidopsis: Localization, tissue specificity and stress responses. Physiol
Plant, 114, 92-101.
Zheng, B., MacDonald, T.M., Sutinen, S., Hurry, V. and Clarke, A.K. (2006) A nuclear-encoded ClpP subunit of the
chloroplast ATP-dependent Clp protease is essential for early development in Arabidopsis thaliana.
Planta, 224, 1103-1115.
Zhong, R., Wan, J., Jin, R. and Lamppa, G. (2003) A pea antisense gene for the chloroplast stromal processing
peptidase yields seedling lethals in Arabidopsis: survivors show defective GFP import in vivo. Plant J, 34,
802-812.
72