D o c t o r a l ... U n i v e r s i t y ,...

Doctoral Thesis in Biochemistry at Stockholm
University, Sweden 2014
O rg a n i z a t i o n o f m i t o c h o n d r i a l
gene expression in yeast
Organization of mitochondrial gene
expression in yeast
Specific features of organellar protein synthesis
Kirsten Kehrein
©Kirsten Kehrein, Stockholm University 2014
ISBN 978-91-7447-985-0
Printed in Sweden by Universitetservice AB, Stockholm 2014
Distributor: Department of Biochemistry and Biophysics, Stockholm University
“I have not failed. I've just found
10,000 ways that won't work.”
- Thomas A. Edison -
Abstract
Mitochondria contain their own genetic system, encoding key subunits of the
enzyme complexes driving oxidative phosphorylation. These subunits are
expressed by an organelle-specific gene expression machinery that developed from the bacterial ancestor of the organelle and was significantly modified during evolution. Despite the importance of the mitochondrial genetic
system for cellular function, the organization and interplay of the factors
implicated in gene expression are still mysterious. My work revealed a number of fundamental aspects of mitochondrial gene expression and provides
evidence that this process is organized in a unique and organelle-specific
manner, which likely evolved to optimize protein synthesis and assembly in
mitochondria. Most importantly, improving the experimental handling of
ribosomes I could show for the first time that mitochondrial ribosomes are
organized in large assemblies that we termed MIOREX complexes. Ribosomes present in these MIOREX complexes organize post-transcriptional
gene expression by recruiting multiple factors required for RNA maturation/
degradation, translation and subunit assembly thus providing a close coupling between these steps. In addition, we could reveal mechanisms by
which ribosome-interactor complexes modulate and coordinate the expression and assembly of the respiratory chain subunits. For example we showed
that the Cbp3-Cbp6 complex binds to the ribosome in proximity to the tunnel exit to coordinate synthesis and assembly of cytochrome b. This location
on the mitochondrial ribosome perfectly positions Cbp3-Cbp6 for direct
binding to newly synthesized cytochrome b and permits Cbp3-Cbp6 to establish a feedback loop that allows modulation of cytochrome b synthesis in
response to assembly efficiency. Likewise the interaction of the membraneanchor proteins Mba1 and Mdm38 with the tunnel exit region enables them
to participate in the translation of the two intron-encoding genes COX1 and
COB in addition to their role in membrane insertion. In summary, work presented in this thesis shows that mitochondrial gene expression is a highly
organized and regulated process. The concepts and technical innovations
will facilitate the elucidation of many additional and important aspects and
therefore contribute to the general understanding of how proteins are synthesized in mitochondria.
List of Publications
I.
Bauerschmitt H, Mick DU, Deckers M, Vollmer C, Funes S,
Kehrein K, Ott M, Rehling P, Herrmann JM. Ribosome-binding
proteins Mdm38 and Mba1 display overlapping functions for
regulation of mitochondrial translation (2010) Mol. Biol. Cell.
21(12):1937-44
II.
Gruschke S, Kehrein K, Römpler K, Gröne K, Israel L, Imhof
A, Herrmann JM, Ott M. Cbp3-Cbp6 interacts with the tunnel
exit of yeast mitochondrial ribosomes to promote cytochrome b
synthesis and assembly (2011) J. Cell Biol. 193(6):1101-14
III.
Gruschke S, Römpler K, Hildenbeutel M, Kehrein K, Kühl I,
Bonnefoy N, Ott M.
The Cbp3-Cbp6 complex coordinates cytochrome b synthesis
with bc(1) complex assembly in yeast mitochondria (2012) J.
Cell Biol. 199(1):137-50.
IV.
Kehrein K, Schilling R, Vargas Möller-Hergt B, Wurm C, Jakobs S, Langer T, Ott M. Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies. submitted
Additional Publications
I.
Kehrein K, Ott M. Conserved and organelle-specific molecular
mechanisms of translation in mitochondria (2012). Organelle
Genetics: Evolution of Organelle Genomes and Gene Expression. Springer Verlag Berlin Heidelberg p. 401-428
II.
Kehrein K, Bonnefoy N, Ott M. Mitochondrial protein synthesis: Efficiency and accuracy (2013). Antioxid. Redox Signal.
19(16):1928-39
III.
Kehrein K, Israel L, Imhof A, Ott M. The translational activators Cbs2 and Pet111 interact with the ribosome independently
of their client mRNA. Manuscript
IV.
Kehrein K, Meza E, Suhm T, Petranovic D, Ott M. Impact of
dysfunction of mitochondrial translation on cellular stress response. Manuscript
Abbreviations
CcO
Cytochrome c oxidase
COB
Cytochrome b (gene and mRNA)
IMM
Inner mitochondrial membrane
IMS
Intermembrane space
MIOREX
Mitochondrial organization of gene expression
MRP
Mitochondrial ribosomal protein
mtDNA
mitochondrial DNA
MTS
Mitochondrial targeting signal
OMM
Outer mitochondrial membrane
OXPHOS
Oxidative phosphorylation
UTR
Untranslated region
Contents
Abstract .........................................................................................................vii
List of Publications ..................................................................................... viii
Additional Publications .................................................................................. ix
Abbreviations .................................................................................................. x
Introduction ................................................................................................... 13
Significance of mitochondria ................................................................... 13
The respiratory chain ................................................................................ 14
The mitochondrial genome – small but important ................................... 16
Mitochondrial gene expression ................................................................ 20
Early steps of gene expression – synthesis, processing and degradation of
mitochondrial mRNAs ............................................................................. 21
Transcription in yeast mitochondria ......................................................... 21
Processing and degradation of mitochondrial transcripts in yeast ........... 22
Late steps of gene expression – translation and the mitochondrial
ribosome ................................................................................................... 24
The mitoribosome .................................................................................... 24
Translation in mitochondria ..................................................................... 28
Assembly of the bc1 complex .................................................................. 38
Aims of the thesis ......................................................................................... 41
Summaries of the papers ............................................................................... 42
Conclusion and future perspectives .............................................................. 46
Sammanfattning på svenska .......................................................................... 53
Deutsche Zusammenfassung ......................................................................... 54
Acknowledgements ....................................................................................... 56
References ..................................................................................................... 59
Introduction
Significance of mitochondria
Mitochondria are the site of many important catabolic reactions such as the
TCA cycle, β-oxidation of fatty acids and the degradation of amino acids.
Reducing equivalents generated by those reactions feed electrons into the
respiratory chain of mitochondria that is the main supplier for chemical energy in form of ATP. Thus, mitochondria are crucial in maintaining a high
ATP/ADP ratio in the cell that is required to drive many biochemical reactions. Additionally, the TCA cycle generates numerous important metabolic
intermediates that are utilized by various anabolic pathways including the
biogenesis of carbohydrates, nucleotides, lipids, amino acids and heme.
Hence, mitochondria represent an important intersection for intermediates of
catabolic and anabolic reactions. Due to this and the fact that mitochondria
produce the main part of the cell ATP by oxidative phosphorylation, mitochondria have long been considered as biosynthetic and bioenergetic organelles. However, during the last years more and more studies indicate that
mitochondria have an additional important function as signaling organelles
(reviewed in [1]) that constantly communicate their fitness to the rest of the
cell; a process generally referred to “retrograde signaling” [2]. This signaling
involves the release of proteins/peptides (e.g. apoptosis, unfolded protein
response), ROS (e.g. stabilization of HIF1, autophagy, transcription of genes
involved in stress response), or the recruitment of proteins to the outer membrane to form a signaling complex (e.g. apoptosis, autophagy, immune response) [3-10]. Furthermore it has been shown that mitochondria are in close
contact with the endoplasmatic reticulum [11-14]. This inter-organelle communication is crucial for modulation of mitochondrial morphology and function as well as for processes like lipid synthesis, apoptosis and Ca2+ homeostasis (reviewed in [15, 16]). Rizzuto and co-workers estimated that approximately 20% of the mitochondrial surface is in close contact with the endoplasmatic reticulum [17]. These contact sites called MAMs (mitochondrial
associated ER membranes) help to enrich the key enzymes required for lipid
synthesis [18, 19] and Ca2+ metabolism thereby forming microdomains alleviating the transfer of lipids [20] and Ca2+ [17] between the two cellular
compartments. Considering the involvement of mitochondria in such a wide
variety of biochemical reactions and processes it is not surprising that, par-
13
ticularly with regard to the strong impact of mitochondrial function on human diseases and aging, this organelle came more and more to the fore of
interest.
The respiratory chain
Mitochondria evolved about 1.5-2 billion years ago [21] from an αproteobacterial ancestor that joined a primitive eukaryotic cell [22-24]. As a
result of their endosymbiontic origin, mitochondria are surrounded by two
membranes, forming two biochemically different compartments; the inter
membrane space (IMS) and the matrix. The inner membrane is heavily folded into so called cristae and accommodates the respiratory chain complexes
(Fig. 1). This fascinating machinery is typically composed of four complexes
of dual genetic origin that transport electrons from NADH and FADH2, to
the final electron acceptor O2 by employing different types of electroncarrying molecules with increasing electron affinities. Complex I (NADH
dehydrogenase), complex III (cytochrome c reductase, bc1 complex) and
complex IV (cytochrome c oxidase) are able to couple this transport of electrons with the extrusion of protons (H+) from the matrix to establish an electrochemical gradient that is used by the ATP synthase (complex V) to produce chemical energy in the form of ATP [25]. The reaction of the respiratory chain starts with the formation of ubiquinol by complex I that transfers
electrons from NADH to ubiquinone. Ubiquinol is freely diffusible in the
membrane and can shuttle electrons between complex I and III. These reducing equivalents are further channeled by the dimeric complex III to cytochrome c by the so called Q cycle. This cycle represents an important switch
between the two-electron carrier ubiquinone and the one-electron carriers –
cytochrome b562, b566, c1 and c of the bc1 complex. Similar to ubiquinone,
cytochrome c can travel between complex III and IV to transfer electrons.
Cytochrome c oxidase (CcO) finally produces water by transferring the delivered electrons from cytochrome c to O2.
14
Figure 1: Electron flow through the respiratory chain.
The inner membrane of mitochondria accommodates the respiratory chain and the
ATP synthase. Electrons from NADH and succinate are removed by complex I and
II, respectively, and collected in the form of ubiquinol (UQ) that shuttles electrons to
complex III. Cytochrome c reduced (light blue) by complex III moves to complex
IV were it releases its electron to get oxidized (dark blue). Complex IV catalyzes the
transfer of electrons to the final electron acceptor O2, thereby forming H2O. Complex I, III, and IV couple this flow of electrons with the transfer of protons (H+) into
the intermembrane space (IMS).These protons return into the matrix through the
ATP synthase thereby driving synthesis of ATP.
The protons “collected” in the IMS by these reactions can flow back to the
matrix along their electrochemical gradient through the membraneembedded Fo part of ATP synthase. This results in a rotation of the Fo oligomeric ring that is transmitted into conformational changes of the F1 subunits thereby catalyzing ATP synthesis [26]. The succinate dehydrogenase
(complex II) is the only respiratory chain complex that is not involved in the
transfer of protons to the IMS. However it participates in channeling reducing equivalents into the respiratory chain by removing electrons from succinate in the TCA cycle.
As the multimeric complex I is missing in Saccharomyces cerevisiae,
NADH oxidation is performed by NdeI and Ndi, two single enzymes that do
not couple the reaction with the transfer of protons across the membrane
[27-29].
15
The mitochondrial genome – small but important
The prokaryotic past of mitochondria is reflected by the presence of an additional genome within the mitochondrial matrix. In the course of evolution
more and more genes of the mitochondrial ancestor were transferred to the
nucleus. Consequently, mitochondrial genomes encode today only a small
number of genes universally involved in oxidative phosphorylation and mitochondrial translation, and depending on the species, in transcription, RNA
maturation and protein import [30] (Fig. 2).
In contrast to its genetic conservation, the mitochondrial genome is surprisingly diverse in structure, size and gene expression mechanisms [31, 32].
Organization of the mtDNA and the mitochondrial nucleoid
The size of most mtDNAs is between 16 and 80 kb [30]. However, the dimension can be extended to several 100 kb in plants [33], even if the gene
content is nearly constant in all species [31]. The extreme differences in size
are mainly caused by a varying amount of non-coding regions (Fig 2B). For
instance, in the relatively small mammalian mtDNA (16,5 kb), all genes are
arranged close to each other and are only separated by tRNA genes, resulting
in a tight packaging of the mitochondrial genome [34] (Fig 4). Conversely,
genes encoded by the mtDNA of S. cerevisia (75 kb) are separated by long
non-coding stretches. Additionally, some coding sequences are interrupted
by introns (COB, COX1, 21S rRNA) that in some cases code for maturases
involved in the processing of intron-containing transcripts (Fig 4, Tab 1) [35,
36]. Therefore, baker‟s yeast mtDNA is about five times larger than its
mammalian counterpart, even if encoding a slightly lower number of genes
(Fig 2).
It has long been assumed that mtDNA is generally a circular molecule.
However, in the 1990th several studies indicated that the mtDNA in yeast
consists primarily of linear molecules [37-39]. These molecules are extensively coated with proteins that package the DNA into compact nucleoprotein complexes, called nucleoids. Membrane association of the nucleoid has
been observed in mammalian cells [40] as well as in yeast [41] and is mediated by some of those proteins.
16
Figure 2: Genetic composition of the mitochondrial genome.
A) Classification of mitochondrial encoded genes from plants, humans and fungi.
Genes encoded in mtDNA are universally involved in oxidative phosphorylation and
mitochondrial translation. Some mitochondrial genomes encode additional genes
involved in related processes such as transcription or RNA maturation. B) Comparison of mitochondrial genome size and composition from plants, humans and fungi.
The size of the mtDNA can vary dramatically between species even when the number of encoded genes is nearly the same (see A). This size difference is mainly
caused by the number and size of non-coding regions. (Modified from [30])
Furthermore, some of the proteins identified as nucleoid associated components are clearly implicated in aspects of mtDNA metabolism such as the
RNA-Polymerase (transcription), Mgm101 (DNA maintenance and repair),
Rim1 (replication) or Abf2 (mtDNA packaging), whereas others have a primary role in cell metabolism (e.g. Aco1, Ilv5, Kgd1, Kgd2) or other mtDNA
unrelated processes [42]. Even if the distinct function of an association of
metabolic proteins with the mtDNA remains to be determined, it is suggested that this might be an adaption of the organelle to couple the inheritance
and organization of their genomes with cell metabolism [43]. Accordingly,
the size and number of nucleoids per cell varies depending on physiological
conditions [44, 45], although it is assumed that under aerobic conditions 1-2
DNA molecules form one nucleoid [43].
17
Genes encoded
The genes that remained in the mitochondrial genome, encode mainly hydrophobic membrane proteins of the respiratory chain complexes and the
ATP synthase (Fig 2A, 4). The mtDNA of bakers‟s yeast, the model organism most commonly used to investigate the regulation and molecular mechanisms of mitochondrial translation, encodes 7 subunits of the respiratory
chain complexes III (Cytb) and IV (Cox1, Cox2, Cox3) and the ATP synthase (Atp6, Atp8, Atp9) as well as several core components of the mitochondrial specific translation machinery required for their synthesis, including rRNAs, tRNAs, a subunit of RNase P and the ribosomal protein Var1
[46]. In contrast, mammalian mtDNA encodes exclusively components of
the oxidative phosphorylation system (OXPHOS) but also a full set of
tRNAs and the two RNAs of the mitoribosome [34] (Fig 4). Comparisons of
the gene content of different mitochondrial genomes suggest that the gene
transfer to the nucleus was rather a specific than random process that occurred in a gradual manner. More complex mitochondrial genomes encode
genes involved in the same processes as genes encoded by smaller and probably higher evolved mtDNAs. However, they encode additional genes, mainly implicated in mitochondrial gene expression such as ribosomal proteins or
the RNA polymerase. Interestingly, there are only 4 genes that are universally encoded by mitochondria, namely the two rRNAs, COB and COX1 [31].
Why mitochondria keep their own genome
As a consequence of the gene transfer to the nucleus, mitochondria encode
only about 1% of the more than 1000 organellar proteins [47, 48]. Consequently, most of the mitochondrial proteins are synthesized in the cytosol
and post-translationally imported into the organelle (Fig 3). A remarkable
fraction of those proteins is required for the expression of only 8 proteins
encoded by mitochondria in yeast [48]. It has long been speculated why mitochondria make such a huge effort to maintain and express an additional
genome.
18
Figure 3: The OXPHOS system is composed of subunits derived from two different genetic origins.
Most of the mitochondrial proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and subsequently imported through the TOM and the TIM complex
into mitochondria. A newly synthesized protein is recognized as mitochondrial component via a mitochondrial targeting signal (MTS). Within mitochondria the
OXPHOS complexes are assembled with the subunits encoded by the organelle and
synthesized on mitochondrial ribosomes (modified from [49]).
A number of hypotheses have been elaborated to explain this: As most mitochondria use an alternative genetic code, the transfer of a mitochondrial gene
to the nucleus would require a synchronistic evolutionary modification of
recoding. In most mitochondria the codon TGA codes for tryptophan while it
is usually used as a stop codon. Thus, the transfer of mitochondrial encoded
genes to the nucleus would most likely result in a defective protein. An alternative explanation is that the retention of genes in mitochondria can allow
for an organelle-specific regulation mechanism that couples synthesis and
assembly of the hydrophobic membrane proteins. Such a mechanism has
been elucidated for Cox1 [50, 51] and the ATPase subunits 6 and 8 [52] as
described in more detail below.
Furthermore, the extreme hydrophobicity of some proteins or the size of
some molecules might prevent their efficient import from the cytosol into
19
mitochondria. This is consistent with the observation that the genes universally encoded in mitochondria include the two large ribosomal RNAs as well
as the highly hydrophobic proteins cytochrome b and Cox1. Indeed, there is
experimental evidence that a recoded version of cytochrome b fused to a
mitochondrial targeting signal that is synthesized in the cytosol is prevented
from import by forming aggregates [53]. Accordingly, a mutant of a recoded
version of COX2 that exhibits reduced hydrophobicity can be posttranslationally imported into the organelle [54]. Also other subunits as Atp6
can become imported from the cytosol however with strongly reduced efficiency [55], providing further supportive evidence that the hydrophobicity of
mitochondrial encoded products prevents transfer of the corresponding genes
to the nucleus.
Mitochondrial gene expression
Mitochondrial gene expression depends mainly on nuclear encoded, general
as well as gene-specific factors that regulate these processes predominantly
at the post-transcriptional level. One striking feature of yeast mitochondrial
gene expression is the employment of specificity factors required for expression of only one gene. These factors are involved in splicing, stabilization
and translation of their client mRNA (for a review see [56]). To this end, the
surprising number of roughly 250 proteins implicated in these processes is
imported into yeast mitochondria, representing about one quarter of the
whole mitochondrial proteome [48]. However, how all this different factors
are organized within the organelle is currently not known. In spite of pioneering work in the field of mitochondrial gene expression during the last 30
years, many aspects are still very poorly understood. One difficulty in elucidating these processes is that deletion of many factors implicated in any
aspect of replication, transcription, RNA processing/degradation and translation result in the loss of mtDNA, indicating that gene expression and DNA
maintenance are tightly coupled in the organelle. This idea was supported by
several studies [57-59] (see also below). Despite the great divergence in the
organization of mitochondrial genomes, some general mechanisms of organellar gene expression are surprisingly conserved. However, others did
change dramatically and are often unique for a given species. In the following, the specific features of early and late gene expression steps in yeast are
discussed.
20
Figure 4: The mitochondrial genome of man and yeast.
The mitochondrial genomes of man and yeast differ from each other mainly by their
amount of non-coding regions. Genes in S. cerevisiae are interrupted by introns and
separated by long non-coding regions, making it much larger than the human counterpart. Transcription in S. cervisiae is initiated from multiple promotors, generating
several polycistronic transcripts. In contrast, the human mtDNA employs three promotor regions. Two of them (LSP; HSP2) result in long polycistronic transcripts
spanning almost the entire length of the genome. A third promotor on the heavy
stand (HSP1) is used for transcription of the two rRNAs (Modified from [60] and
[61]).
Early steps of gene expression – synthesis, processing and
degradation of mitochondrial mRNAs
Transcription in yeast mitochondria
Since mitochondria evolved from a bacterial ancestor one can assume that
organellar gene expression might employ prokaryotic mechanisms. This,
however, is not the case because key enzymes of transcription and replication of the mtDNA have been replaced by proteins of viral origin [62]. Yeast
mitochondria employ multiple promotor regions characterized by a con21
served nonanucleotide motif (ATATAAGTA) [63] that recruits a phage-like
RNA polymerase and the transcription factor Mtf1. Applying this motif, at
least thirteen transcription units have been identified in S. cerevisae [63], and
even up to nineteen functional initiation sites were reported in the strain
FY1679 [46]. This implies that most of the genes are transcribed as long
polycistronic precursor molecules (Fig 4) encoding two or more coding sequences. In comparison RNA synthesis of the mammalian mitochondrial
genome starts from only three promotors (HSP1, HSP2 and LSP). Transcription from HSP2 and LSP generates much longer polycistronic precursor
molecules than yeast that span almost the entire length of the genome (Fig
4). Compared to the complex regulation of transcription in the nucleus, mitochondrial transcription is a rather simple process. However, it has been
shown that the transcription rate in mitochondria can be modulated by the
availability of ATP thereby allowing adjustment of mitochondrial gene expression to cellular needs [64]. Likewise, attenuation of transcription in
combination with rapid RNA degradation was proposed to cause the observed differences in the transcription rates of proximal and distal genes on a
polycistronic transcript [65, 66].
Processing and degradation of mitochondrial transcripts in
yeast
The polycistronic transcripts are subjected to complex processing events,
including intron-splicing and 3‟- and 5‟- end processing in order to obtain
individual mRNAs, tRNAs and rRNAs (Tab 1). tRNAs are usually located at
the 5‟- and 3‟- end of the transcript and are liberated by the action of RNaseP
and RNase Z [67, 68]. A conserved dodecamer motif in protein-encoding
genes serves as processing point to generate mRNAs with stable 3‟- ends
[69].
5‟-end trimming is accomplished by tRNA cleavage and depends at least for
several mitochondrial transcripts on Pet127, shown by the accumulation of
precursor mRNAs in a pet127 mutant. However, this defect inhibits respiratory growth only at elevated temperatures indicating that mRNAs can still be
translated without proper processing of their 5‟- ends [70, 71]. After these
processing steps yeast mitochondrial mRNAs are flanked by long 5‟- and 3‟UTRs that play an important function for the regulation of translation (see
below).
22
As mentioned above some yeast mitochondrial genes are interrupted by introns and need additional maturation steps in order to generate the mature
transcript. Introns are removed by the combined action of intron encoded
maturases and nuclear encoded splicing factors that are either specific for
one mRNA or involved in general processing steps [72-74].
All these processes are assisted by other factors such as RNA helicases and/
or chaperones that have multiple important functions during mitochondrial
gene expression, including translation, RNA splicing/degradation and genome maintenance (for a review see [75]). One member of this family is
Suv3 that interacts with the RNase Dss1 to from the mitochondrial
degradosome (mtEXO) [76] that has a general function in RNA turnover and
RNA surveillance, thereby regulating mitochondrial gene expression [77,
78]
Table 1: Proteins involved in early steps of mitochondrial gene expression
Transcription
Rpo41
RNA polymerase
[79]
Mtf1
RNA polymerase specificity factor
[80]
tRNA cleavage
RNase P
5„-end processing of tRNAs
[81]
RNase Z
3„-end processing of tRNAs
[68]
3„-end processing
mtEXO (Suv3/Dss1)
RNA degradation and surveillance
[82]
5„-end processing
Pet127
Involved in stability and 5‟-end pro- [70]
cessing of RNAs
Cbt1
Involved in 5„ end processing of COB [83, 84]
and 15S rRNA; may also be involved in
3‟-end processing of COB
Splicing
Maturases (e.g. bI2, bI3, Removal of group I and group II introns
bI4, aI1,aI2, aI3, aI4, aI5α
, SceI)
Cbp2
Required for splicing of COB mRNA
[85]
Nam1 /Mtf2
General RNA processing factor; couples [86]
RNA transcription and translation
Nam2
Splicing of several group I introns
[87]
Mrs1
Required for splicing of the two mito- [88]
chondrial group I introns BI3 (COB)
and AI5β (COX1)
23
Pet54
Mss18
Mne1
Ccm1
Cox24
RNA helicases/chaperones
Suv3
Mrh4
Mss116
Irc3
RNA degradation
mtEXO (Suv3/Dss1)
Dual function in COX3 translation and
splicing of the AI5β intron
Required for efficient splicing of COX1
intron AI5β
Required for splicing of COX1 intron
AI5β
Maturation of COB and COX1 mRNA;
15S binding protein
Required for COX1 intron splicing
[89, 90]
Component of the mtEXO; involved in
splicing of COX1 intron AI5β by recycling of Mrs1
Required for the assembly of the large
ribosomal subunit
Required for efficient splicing of group
I and group II introns
Probably involved in DNA maintenance
and replication
[95]
RNA degradation and surveillance
[76]
[91]
[92]
[93]
[94]
[96]
[74] [97]
[75]
Late steps of gene expression – translation and the
mitochondrial ribosome
The mitoribosome
Most of our current understanding about the function of ribosomes has been
derived from studies of the bacterial machinery where a manipulatable in
vitro translation system is available. It has long been assumed that the structure and function of the mitochondrial counterpart closely resembles its bacterial ancestor. However, structural analysis of mammalian and yeast mitoribosomes revealed that the “heart” of the translation machinery changed dramatically in course of organellar evolution [49].
The most obvious alteration is the increasing reduction of RNA components
from yeast to mammalian mitoribosomes with simultaneously increase in
mitochondrial specific proteins (Fig 5). This is supported by the finding that
proteins of the large subunit of the yeast mitoribosome are surrounded by in
24
average 4.5 proteinous neighbors instead of about 1.5 in bacteria thereby
increasing the importance of protein-protein interactions in the mitoribosome
[98]. Consistently, many mitochondrial ribosomal proteins (MRPs) are extensively larger than their bacterial homologs, possessing N- and C-terminal
extensions. Some of those C-terminal ends show a high probability to form
coiled-coil structures, known to be involved in protein-protein interactions.
Importantly, ribosomal proteins with bacterial homologs are located at functionally and structurally conserved sites such as the peptidyltransferase center, indicating that the general mechanism of protein synthesis by the ribosome is highly conserved. This is supported by the sensitivity of mitoribosomes to the same antibiotics known to disturb efficiency and accuracy of
the bacterial particle.
It is still a challenging and interesting question why the mitoribosome recruited additional proteins. One explanation is that they compensate structurally and functionally for the missing RNA moieties. However, about 80%
of the lost RNA segments haven‟t been found to be replaced by mitospecific proteins [99]. Instead, most of the new structural components are
located on the surface of the mitoribosome, giving the particle a markedly
diverged overall shape. Recently, structural insights of the yeast mitoribosome revealed that the 21S rRNA exhibits expansion segments that protrude
to the surface to serve as a scaffold for the addition of these new mitospecific proteins [98]. It is supposed that the adopted proteins play an important role in regulation and organization of unique features of organellar
gene expression, for instance by recruiting essential factors involved in synthesis and assembly of the mitochondrial encoded genes. Alternatively, mitochondria-specific proteins could assume the binding and adjustment of the
mRNA on the ribosome since a bacterial-like Shine-Dalgarno sequence is
missing in the mitochondrial mRNAs. Indeed the mRNA entrance site of the
mammalian mitoribosome is mainly made up by mitochondrial specific protein mass whereas two homologes of the respective site in bacteria are missing [99] indicating that this evolved to bind the unusual leaderless mRNAs
of mammalian mitochondria.
25
Figure 5: Composition of the mitochondrial ribosome and its bacterial counterpart
Mitochondrial ribosomes diverged from their prokaryotic ancestor during evolution.
A striking difference is the reduction of RNA components and the simultaneous
evolution of mitochondrial specific ribosomal proteins (dark purple box). Most of
the proteins that occupy functional important sites in the bacterial particle have
homologues in the mitoribosome (light purple box). (Modified from [49])
The ribosomal tunnel exit and its interactors
The ribosomal tunnel exit is an important site for the future fate of a newly
synthesized polypeptide, because here it is exposed to a hydrophilic environment for the very first time. Thus, it is not surprising that this site of the
ribosome serves as an important platform for several biogenesis factors of
the newly synthesized proteins. Studies with bacterial ribosomes revealed
that these interactors can be sorted into three categories: 1. molecular chaperones, 2. enzymes involved in modification, 3. components involved in
targeting of the protein [100, 101]. The rim of the bacterial tunnel exit is
made up by mainly four proteins (L22, L23, L24, L29) that are highly conserved and found in all ribosomes. Structural analysis revealed that the tun26
nel exit of the mitoribosome is slightly displaced in respect to the eubacterial
exit site and in addition highly enriched with mitochondrial specific protein
mass [98]. These results are supported by studies in which such proteins
were specifically x-linked to the tunnel exit. [102]. Nevertheless the
knowledge about interactors of the ribosomal tunnel exit that are involved in
organelle-specific, post-translational steps is still very limited.
The membrane insertion machinery of mitochondria turned out to be much
simpler than in eubacteria as homologs of the SecYEG translocon could not
be identified [103]. Furthermore, a signal recognition particle in mitochondria is dispensable as mitoribosomes are permanently attached to the inner
membrane in both a translational active and an inactive state [104, 105]. It
has long been speculated which factors could tether the ribosome to the
membrane. One suggestion was that membrane-anchored proteins that perform the insertion of the respiratory chain subunits could be the key players
in this purpose. Oxa1 is a member of the YidC/Alb3/Oxa1 family found in
bacteria, chloroplasts and mitochondria and involved in the insertion, folding
and assembly of membrane proteins [106]. Oxa1 forms a homooligomeric
[107] clamp-like structure that was proposed to form a protein conducting
channel that releases the protein segments laterally into the inner membrane
[108]. Consistent with its function in posttranslational insertion and folding,
Oxa1 could be x-linked to the ribosomal tunnel exit in vicinity to the L24
(Mrpl40) and L23 (Mrp20) homologs [109]. This position allows early contact with the newly synthesized proteins as soon as they emerge from the
tunnel exit [110].
The co-translational insertion by Oxa1 is assisted by the peripheral membrane protein Mba1 that has a proposed role as a ribosome receptor that
helps to align the tunnel exit with the site of insertion [105]. Like for Oxa1, a
direct interaction of Mba1 with the tunnel exit protein Mrpl4 (L29) has been
experimentally shown by chemical cross-linking [102]. The simultaneous
deletion of Mba1 and the C-terminal domain of Oxa1 results in the accumulation of defective proteins resulting in a respiratory deficient phenotype.
Thus, an overlapping function of Mba1 and Oxa1 in membrane insertion was
proposed [105].
Some years ago Rehling and co-workers identified a third membrane receptor protein called Mdm38 that binds to the tunnel exit in the vicinity of
Mrp20 [111]. This integral membrane protein has an apparently dual function. On the one hand it is involved in ion homeostasis [112], on the other
hand it seems to be involved in a membrane insertion pathway independent
27
of Oxa1 [111]. However, the molecular function of Mdm38 as well as the
way of cooperation of Oxa1, Mba1 and Mdm38 in membrane insertion remains to be elucidated.
Susceptibility of the mitochondrial ribosome
As mentioned above the structure of the mitochondrial ribosome diverged
dramatically from its bacterial counterpart during evolution. In addition to
the enormous reduction of the RNA components in general, the stability of
the RNA folds is supposed to be strongly impaired by the drop of the guanine content of mitochondrial RNAs, the base involved in the strongest basepairing. Concomitant, mitochondrial RNAs contain similar amounts of cytosine resulting in a drop of base-pairing from 60% to 45% in mammalian
mitochondria [113]. Therefore it is supposed that the mitoribosome is much
more fragile and prone to degradation than the bacterial particle probably
resulting in loss of peripherally attached mitochondria-specific proteins during isolations. Importantly in yeast, RNA extensions are exposed to the surface of the ribosome making them easy targets for nucleases. This might be a
reason why structural and biochemical analyses of mitoribosomes and especially for the yeast mitoribosome are rather challenging.
Translation in mitochondria
The general steps of translation and the translation factors involved in initiation, elongation, termination and recycling are well conserved from bacteria
to mitochondria [61]. However, the organelle established some additional
specific features in order to translate its unusual mRNAs, optimizing protein
synthesis and assembly of the respiratory chain subunits.
One unusual feature of mitochondrial mRNAs is the absence of a ShineDalgarno sequence or a 5‟-cap structure that are used by other translation
systems for determination of the start site [114]. Indeed, Li and Tzagoloff
found that mitochondrial mRNAs have complementary sequences to the 15S
rRNA that might fulfill a similar function as the Shine-Dalgarno sequence in
bacteria [115]. However, these recognition sequences vary in distance from
the start codon and have been shown to be dispensable for its selection [116,
117]. Similarly, the usage of a scanning mechanism as used by the cytosolic
ribosome is rather unlikely, as most mitochondrial mRNAs have at least one
28
additional AUG upstream of the initiation site. This indicates that translation
initiation in mitochondria is accomplished by another mechanism, but how
this process is performed is still a big mystery. However, even if the detailed
mechanism of start codon recognition is not known so far there is clearly
stringent start site selection in mammalian [118] as well as yeast mitochondria [119].
Translational control in mitochondria
One specific feature of yeast mitochondrial protein synthesis is the employment of nuclear encoded gene-specific proteins that have mainly been identified by the pioneering work of Tom Fox and Alex Tzagoloff. These translational activators were initially discovered by mutations that specifically
block the accumulation of one gene product leading to a respiratory deficient
phenotype (Pet- phenotype). Spontaneous suppressor mutations resulting in
gene rearrangements of the 5‟ - UTR nicely demonstrated [120] that this
region of the mRNAs is crucial for translation activation by these regulatory
proteins. A powerful tool for the further investigation of translational control
was the establishment of biolistic transformation that allows the directed
manipulation of mitochondrial DNA [121].
For all mitochondrial encoded transcripts with exception of ATP8 and VAR1,
one or more transcript specific translation activators have been identified.
(Fig 6) (For a detailed description of the single translational activators see
[61]). In case of the employment of several activator proteins it seems that
some of them have an additional function upstream or downstream of translation activation in order to coordinate translation of nuclear and mitochodrially encoded subunits (Mss51, Cbp6, Atp25) [50, 122-124] or to adjust the
synthesis of subunits belonging to the same complex (Pet54) [89, 90, 125].
The dual function of some of these proteins will be described later in more
detail.
29
Figure 6: Translational control in mitochondria of S. cerevisiae.
Mitochondrial mRNAs are flanked by specific 5‟- and 3‟ untranslated regions (5‟-3‟UTRs) of varying length and depend on several nuclear encoded translational activators (X) for translation. These gene-specific proteins recognize the 5‟-UTR of their
client mRNA to activate translation. For ATP8 and VAR1 no clear translational activator has been identified so far (modified from [126]).
Because direct manipulation of the mammalian mitochondrial genome is not
yet possible, the knowledge about translation initiation and regulation in this
system lacks behind. As the mammalian mitochondrial mRNAs have just a
few if any nucleotides at their 5‟-end it is likely that translation initiation is
regulated in a different way than in S. cerevisiae. In accordance, the majority
of translation activators have no homologs in higher eukaryotes. The protein
LRPPRC has been identified as possible homolog of Pet309. However, in
contrast to the COX1 specificity of Pet309, LRPPRC seems to have multiple
functions in stabilization, storage, translation and polyadenylation of all
mRNAs although COX1 is one of the most sensitive targets [127]. As it is
not affecting transcription it is proposed that LRPPRC is a posttranscriptional regulator of mitochondrial gene expression [128]. However,
its detailed molecular function needs to be further elucidated. Only one
gene-specific translation activator in mammalian mitochondria has been
identified so far. TACO1 was found in a patient with a cytochrome c oxidase
(CcO) deficiency but normal levels of COX1 mRNA, indicating that TACO1
30
is specifically involved in translation activation of the COX1 transcript
[129]. But also in this case its mode of action is not understood so far.
Synthesis of cytochrome b
As my work concentrates mainly on the biogenesis of cytochrome b, I will
describe the synthesis of this subunit in more detail in the following section.
Cytochrome b is the only subunit of the bc1 complex encoded by the mitochondrial genome. Its expression depends on several nuclear genes either
required for translation or processing and maturation of the pre-transcript.
Cytochrome b is co-transcribed with tRNAGlu, resulting in a bi-cistronic,
intron-containing pre-transcript that undergoes multiple processing steps. As
described above, tRNAGlu is removed by the action of RNase P and RNaseZ
[67, 68] and the generated 5‟- end subsequently modified by Pet127 [71].
The cleavage by Pet127 is followed by binding of Cbp1, which protects the
COB mRNA from further degradation [71]. This was experimentally shown
by mutations of the CCG trinucleotide of the COB 5‟- leader that serves as
binding site for Cbp1 [130]. Accordingly, cells lacking CBP1 have strongly
reduced level of the COB precursor transcript and fail completely to accumulate the mature mRNA, which accounts for the respiratory deficient phenotype [131, 132]. An important role of Cbp1 for the stabilization of the COB
mRNA was experimentally verified by a gene rearrangement that fuses the
5‟- end of ATP9 to the COB coding sequence. This alteration suppressed the
∆cbp1 phenotype and led to the accumulation of the COB transcript [132].
Accordingly, stabilization of an ATP9 transcript flanked by the 5‟- UTR of
COB is dependent on Cbp1 [133]. These studies clearly indicate that Cbp1
specifically acts at the 5‟- end of the COB mRNA to prevent it from degradation.
Further maturation of the COB transcript requires several factors involved in
excision of introns (Tab 1). Three of the cytochrome b introns encode socalled maturases required for removal of the intron encoding them or processing of another intron-containing transcript. The ORFs of those introns
are in frame with the upstream exon and therefore depend on the proper and
accurate translation of the precursor mRNA [134]. One intron of the COB
gene encodes a maturase that is also required for splicing of the COX1 precursor [135]. Therefore mutants affected in COB translation often exhibit an
additional defect in the accumulation of mature COX1 mRNA. Additionally
several other factors have been shown to be essential for COB maturation;
31
they are either involved in splicing of all transcripts, such as Mss116, or are
specific for COB as shown for Cbp2 [72, 136] (Tab 2).
Translation of COB is controlled by the three translation activators Cbs1,
Cbs2 and Cbp6. Cells lacking Cbs1 or Cbs2 fail to synthesize apocytochrome b and accumulate intron-containing precursor forms [137, 138]
[139]. This argues for a role of both proteins in splicing of the COB precursor or/and its translation activation. However, a primary and direct involvement in splicing was not compatible with the observation that Cbs1 and
Cbs2 are still necessary for synthesis of cytochrome b in a strain devoid of
introns [139]. Furthermore, replacement of the COB leader with that of
ATP9 restored COB translation in a cbs1 and cbs2 deletion strain and confirmed that the COB transcript can be spliced even in the absence of both
proteins [137, 138]. Therefore the accumulation of intron-containing precursor forms of the COB mRNA is probably a secondary effect caused by the
inability to synthesize intron-encoded maturases of the pre COB mRNA in
the absence of Cbs1 and Cbs2. As shown for other proteins implicated in
translation regulation, the long 5‟- leader of COB seems to be the target site
for the translation activators. Accordingly, the synthesis of Cox3 was dependent on Cbs1 when bearing the COB leader but independent when
flanked by the authentic COX3 5‟- UTR [140]. However, it is still unknown
whether both translational activators interact directly with the 5‟ regulatory
region or whether the interaction is mediated by other factors. Nevertheless,
genetic analysis of the 954 bp long 5‟-UTR of COB could narrow the sites
important for translation activation by Cbs1 and Cbs2 to two sequences between -232 and -4 [116].
Besides Cbs1 and Cbs2 a third protein, called Cbp6, has been shown to be
involved in the synthesis of cytochrome b [124]. As observed for cbs1 and
cbs2 mutant strains, the absence of Cbp6 impairs the accumulation of cytochrome b resulting in a respiratory deficient phenotype. However, this is not
accompanied by a splicing defect as normal levels of mature COB mRNA
were detected [124]. Importantly, a replacement of the COB leader cannot
rescue the cbp6 phenotype as seen for Cbs1 and Cbs2 [141]. This observation argues for a dual function of Cbp6 in the biogenesis of cytochrome b
and suggests that the mode of action is distinct from that of the other translational activators. The authors proposed that Cbp6 might participate in the
formation of an initiation complex or by assisting the ribosome in start site
selection [124] even when an involvement in post-translational steps cannot
be excluded.
32
Possible functions of translational activators
Even after many years of research, there are numerous open questions about
the process of mitochondrial translation and especially the molecular mechanisms of translational activators. However, based on experimental data there
are several hypotheses, suggesting how these regulatory proteins act in translation:
1. Support of start site selection
One of the most popular ideas is that translational activators constitute a
specific feature, developed by the organelle to compensate for the missing
Shine-Dalgarno sequence. The strongest argument for this hypothesis was
the observation that translational activators are able to interact with the inner
membrane, the ribosome and probably with the 5‟-UTR of their client
mRNA [58, 142-147]. Therefore they could help to tether the translation
machinery and the mRNA close to the inner membrane and help to load the
mRNA onto the ribosome. Genetic studies on the 5‟- UTR of COB supported
the idea that the binding site of the translational activators on the 5‟- leader
might be crucial for correct start site selection. Deletion of the region -33
and -4 did not prevent translation of COB but resulted in a truncated protein
that was synthesized from the next in-frame AUG within the coding sequence [116]. This might indicate that this sequence provides the correct
spacing to the start codon and serves as possible binding site for the translational activators.
2. Stabilization of mRNA
Several studies reported the loss of an mRNA in the absence of its respective
translational activator, pointing to a role in stabilization of the transcript. As
described above Cbp1 has a well-documented role in the stabilization of the
COB mRNA [133, 148]. Likewise, an involvement in protection of the
mRNA was reported for Cbs1, Cbs2 and Pet309. However, the mature transcript was only destabilized in the absence of one of those proteins when
COB and COX1 contained introns, whereas the stability was unaffected in
intronless strains [139, 149]. This argues against an essential role of these
proteins in mRNA stabilization [150]. Likewise, a destabilization of intronless transcripts, such as COX2, has been reported in the absence of their
translational activators [151, 152]. Even if those mRNAs do not depend on
33
splicing events, it has been proposed that this phenotype might also be the
consequence of impaired translation rather than a direct role of translational
activators in stabilization of the transcript [152]. Due to the interdependence
of translation and transcript maturation it is difficult to determine whether
translational activators have in general a direct role in mRNA stabilization or
not. Furthermore it is supposed that mRNAs that cannot be translated are
generally prone to degradation. However, not all mutants affecting genespecific mitochondrial translation exhibit reduced stability of their respective
mRNAs, therefore it is still puzzling how stabilization of the single mRNAs
is accomplished.
3. Organization of translation
Due to their regulatory function in translation, translational activators are
suggested to organize this process in order to facilitate the assembly of the
respiratory chain subunits into the respective complexes. Experimental evidence for this hypothesis was provided by Tom Fox and co-workers who
found that the translational activators for the three mitochondrial encoded
subunits Cox1, Cox2 and Cox3 of CcO physically interact. As all those proteins are associated with the inner membrane it was supposed that this organization helps to co-localize the synthesis of the three subunits and thereby
facilitate assembly of the core complex of CcO [58]. Supportive evidence for
this model came from the finding that the ectopic expression of Cox2 and
Cox3 from the VAR1 locus did not prevent their synthesis but impaired their
efficient assembly into the respiratory chain [153]. Other studies proposed
that translational activators participate in organizing earlier steps of gene
expression by coupling mRNA transcription and translation. Accordingly,
Pet309 indirectly associates with the RNA polymerase via Nam1 suggesting
that transcription and translation are directly linked in mitochondria thereby
allowing a channeled transfer of the mRNA to a ribosome that is optimal
positioned by its translational activators to the site of insertion [57, 58]. Additionally, Pet309 was found in a large 900 kDa complex that also contained
Cbp1, indicating that this large complex is composed of several message
specific entities. This arrangement could permit a direct binding of the respective translational activator to their mRNA after transcription [154].
34
4. Regulation of translation
Last but not least, modulation in mitochondrial gene expression occurs at
least partially at the level of translation. Translational activators are generally expressed at low levels and at least for Pet111 and Pet494 it has been
demonstrated that their accumulation in the matrix limits the translation of
their respective mRNA [146, 155]. Moreover, it was reported that expression
of Pet494 is modulated in response to environmental changes such as high
glucose concentration [156]. The reduced expression level of Pet494 under
glucose-repression could therefore directly adjust mitochondrial translation
to environmental conditions [155]. A similar response has also been reported
for Cbs1 and Cbs2 [157], indicating that regulating the levels of genespecific proteins in the matrix is a general mechanism to directly modulate
gene expression in mitochondria.
Beside adaptation of mitochondrial function in response to environmental
changes, mitochondria also need to coordinate the assembly of the respiratory chain subunits originating from two different genetic systems. Because of
their vital role in translation regulation, membrane association and ability to
interact with the ribosome, translational activators are excellent candidates
for modulators of translation in response to the efficiency of respiratory
chain assembly. Such an auto-regulatory mechanism that couples synthesis
and assembly was also reported in chloroplast and has been described for all
complexes of the thylakoid membrane, pointing to a highly conserved mechanism to regulate organellar gene expression [158, 159]. Similar feedback
loops are well documented for the CcO and ATP synthase of mitochondria.
CcO assembly starts with the mitochondrially encoded subunit Cox1 that
contains two redox active co-factors required for transfer of electrons.
Hence, unassembled Cox1 may be harmful to the cell by the formation of
reactive oxygen species [160]. In order to avoid such a scenario, Cox1 synthesis is modulated by a feedback loop that prevents further synthesis of
Cox1 in case of disturbed assembly of CcO [161]. The key player in this
regulatory cycle is the bi-functional protein Mss51 that is essential for COX1
translation and its assembly by directly binding to newly synthesized Cox1
[50, 162]. In case CcO assembly is blocked, Mss51 is trapped in a Cox1
assembly intermediate and is not available to promote further translation of
the COX1 mRNA. Interestingly, Soto et al. have recently demonstrated that
Mss51 is also a heme-binding protein, therefore serving as a sensor that reduces Cox1 translation in case of heme unavailability [51]. This regulatory
35
circuit is further modulated by several other factors such as Cox14 and Coa3
that stabilize the Mss51-containing assembly intermediate [163-165].
Figure 7: Feedback regulation in mitochondria.
Translation in mitochondria is activated by translational activators (TA). Some of
these regulatory proteins have a dual function (TA2) and can also assist the assembly of the newly synthesized protein into the respiratory chain complex. Under normal conditions TA2 is released from an assembly intermediate when further structural subunits are integrated into the complex. The released protein can return to the
ribosome to stimulate new rounds of translation (green arrow). In case assembly is
blocked, TA2 is trapped in an assembly intermediate and prevented from serving as
a translational activator (red arrow) (modified from [126]).
The mitochondrially encoded subunits Atp6, Atp8 and Atp9 are part of the
proton-conducting channel (Fo) of the ATP synthase. Assembly of F1 and Fo
has to be carefully coordinated as the membrane embedded part would allow
unopposed proton flow back to the matrix in the absence of the hydrophilic
head domain resulting in the dissipation of the membrane potential. Some
years ago Rak and Tzagoloff showed that the synthesis of Atp6 and Atp8 is
strongly impaired in the absence of the F1 subunits α and β or their chaperones Atp11 and Atp12 [52]. The activating effect of F1 intermediates on
translation was supported by the observation that overexpression of Atp22
(the translational activator of Atp6) could restore the synthesis of Atp6. This
regulatory mechanism seems to be different from those described for Cox1
and chloroplasts, however it is not known how exactly the missing F1 intermediates impair translation.
36
Is mitochondrial gene expression organized in an expressosome-like
structure?
It is an ongoing question how the numerous factors implicated in mitochondrial gene expression are organized in time and space. But there is strong
experimental evidence that this process has dramatically diverged from gene
expression in other systems. In contrast to the nuclear genome, mtDNA has
no physical barrier that would separate transcription and translation. Accordingly, several studies suggested that transcription, RNA processing, translation and degradation might be coupled to form a large complex at the inner
surface of the membrane to allow an optimized transfer from transcription to
assembly, like in a construction line [57, 58, 154, 166]. This idea of an expressosome-like structure is supported by several recent findings in mammalian mitochondria that indicate that these processes occur in close proximity
to each other and the mitochondrial nucleoid. Foci of newly synthesized
RNA have been found in the vicinity of the mtDNA. These foci also associate with several factors involved in post-transcriptional steps such as RNA
binding proteins, methyltransferases, the degradosome or the 5‟and 3‟ processing factors RNase P and ELAC2 [167-171]. Additionally, some ribosomal proteins have been found to associate with the nucleoid in a transcription dependent manner [169]. Taken together these data provide evidence
that ribosome assembly, RNA processing/ modification and degradation
occur in close proximity to the nucleoid. Accordingly, He et al. reported an
interaction of C4orf14 (NOA1), a biogenesis factor of the small ribosomal
subunit, with the mitochondrial nucleoid [172]. This factor also binds to the
ribosome and other translation factors therefore probably connecting transcription and translation. Likewise, affinity purification of mtDNA binding
proteins revealed an association of a subset of MRPs and components required for protein synthesis with the mitochondrial nucleoid [173]. All these
data point to a higher order of mitochondrial gene expression. However, it is
not clear if functional ribosomes are also part of those structures found in the
vicinity to the nucleoid. Furthermore many constituents and mechanisms
involved in organellar gene expression still remain to be discovered.
37
Assembly of the bc1 complex
The bc1 complex is one of two complexes of the respiratory chain of S. cerevisiae participating in the formation of a proton gradient across the inner
membrane. Nine subunits imported from the cytosol have to be assembled
with the core subunit cytochrome b, encoded by the mitochondrial genome
[174]. Only three subunits (cytochrome b, cytochrome c1, Rieske iron-sulfur
protein (Rip1)) contain redox centers that qualify them to participate in the
transfer of electrons through the complex. The remaining seven subunits lack
any co-factors and their relevance for the functionality of the complex is
largely unknown. Interestingly, bacterial complex III lacks homologs to
those accessory subunits and is instead exclusively composed of the catalytic
core made up by the three redox subunits [175].
Using yeast mutants lacking structural components of complex III, it was
clearly shown that assembly occurs in a stepwise manner forming different
structural intermediates. [176-179]. Assembly of the bc1 complex starts with
the insertion of cytochrome b into the inner membrane. Cytochrome b is
subsequently joined by the nuclear encoded subunits Qcr7 and Qcr8 forming
the central core of the complex. Next, a sub-assembly composed of Cor1 and
Cor2 is added together with cytochrome c1. After incorporation of the intermembrane space subunit Qcr6, these seven subunits form a stable intermediate, called the 500 kDa complex [177]. Finally, assembly is completed by
incorporation of the last accessory proteins Qrc9 and Qcr10 and the catalytic
subunit, the Rieske iron-sulfur protein (Rip1) [179]. The mature bc1 complex
exists as homodimer. However, when and how its dimerization is accomplished is not known so far. Furthermore, the bc1 complex has been found to
be structurally and functionally connected to one or two copies of CcO forming so-called respiratory supercomplexes. This arrangement is supposed to
increase the efficiency of electron transfer between respiratory complexes by
restricting electron carrier diffusion [180, 181].
38
Figure 8: Assembly of the bc1 complex.
The bc1 complex is composed of nine nuclear encoded subunits and the mitochondrial encoded subunit cytochrome b. Assembly is initiated with the membrane insertion of cytochrome b that assembles with the small structural subunits Qcr7 and
Qcr8. This pre-complex is joined by cytochrome c1 and the core subunits Cor1 and
Cor2 as well as Qcr6 to form the 500 kDa complex (modified from [182]). Assembly is completed by incorporation of the last structural subunits Qcr9, Rip1 and
Qcr10. Mzm1 and Bcs1 have been shown to play a role in the insertion of Rip1. The
other assembly factors Cbp3, Cbp4 and Bca1 are implicated in earlier steps of assembly, but their distinct role is much less understood.
In addition to structural components, a functional complex depends on multiple assembly factors. These proteins assist the formation of the enzyme
complex but are not permanently associated with it. More than 30 factors
have been found to participate in assembly of CcO [161, 183, 184]. In contrast, only five assembly factors (Cbp3, Cbp4, Bca1, Bcs1, Mzm1) are
known so far that support assembly of the bc1 complex, tempting to speculate that many additional factors still remain to be discovered.
Mutations in Cbp3 and Cbp4 have been found to exhibit a similar phenotype
with specifically impaired bc1 complex activity as well as reduced cytochrome b stability [185, 186]. As both mutants accumulated normal levels of
COB mRNA and were able to synthesize cytochrome b it was postulated that
Cbp3 and Cbp4 are specific chaperones implicated in the assembly of the bc1
complex. Accordingly, two regions of Cbp3 have been identified to be crucial for complex III assembly [187].
In addition to cytochrome b, the accumulation of three other structural subunits was significantly impaired in cbp3 and cbp4 deletion strains, including
Qcr7, Qcr8 and Rip1. This might indicate that both proteins are involved in
the formation of the same sub-complex. Supportive, Cbp3 and Cbp4 were
detected in a high molecular weight complex of similar size and an interaction of both proteins has been shown by co-immunoprecipitation [188].
39
However, it is not known if this interaction occurs directly or if it is mediated by additional proteins. Importantly, the individual molecular function of
Cbp3 and Cbp4 still remains to be elucidated.
Another bc1 complex assembly factor, called Bca1, was recently identified
by a transcriptome-based screen [189]. Bca1 is only found in fungi and is a
single-membrane spanning protein with a large C-terminal domain exposed
to the intermembrane space. Cells lacking Bca1 have slightly reduced level
of cytochrome b and exhibit strongly impaired accumulation of Rip1. rip1
deletion mutants accumulate the 500 kDa intermediate that is strongly destabilized in case of a concomitant knock-out of Bca1. Therefore it has been
proposed that Bca1 is required for an early or intermediate assembly step
before the insertion of Rip1 [189].
Conversely, the assembly factors Bcs1 and Mzm1 function in the mitochondrial matrix and are involved in late assembly steps of bc1 complex formation by supporting the insertion of Rip1 [190, 191]. Bcs1 is a singlemembrane spanning protein with a highly conserved AAA domain that has
been shown to be required for Rip1 assembly [192]. Bcs1 binds to a bc1 precomplex and mediates Rip1 translocation in an ATP-dependent manner.
Ostojic et al. proposed that the availability of ATP is not only required for its
chaperone activity but might also exhibit a regulatory mechanism that allows
for coupling the rate of bc1 complex biogenesis with the metabolic state of
mitochondria [193]. Insertion of Rip1 by Bcs1 is assisted by Mzm1. This
recently identified assembly factor is required for stabilization of Rip1 before its membrane translocation by Bcs1 [191, 194].
40
Aims of the thesis
Mitochondria encode only a handful of proteins that are expressed by an
organelle-specific transcription and translation machinery. In contrast to the
knowledge on bacterial translation the general organization and regulation of
mitochondrial gene expression is hardly understood. Due to the prokaryotic
ancestry of mitochondria it has long been assumed that gene expression in
mitochondria closely resembles the bacterial counterpart. However, recent
work has revealed that mitochondrial protein synthesis structurally and functionally diverged substantially with many aspects carrying organelle-specific
features. Especially the mitochondrial ribosome was adapted to the need to
coordinate expression of the mitochondrially encoded subunits with their
assembly into respiratory chain complexes that consists of subunits produced
in two different compartments.
In this thesis I aimed to analyze the evolutionary changes that regulate and
organize protein synthesis and assembly in mitochondria of baker‟s yeast.
Starting from the ribosome as central component of the translation machinery, I was interested in determining interacting proteins and their specific
role during the biogenesis of mitochondrial encoded proteins.
41
Summaries of the papers
Paper I: Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial
translation
Mitochondrial translation occurs at the inner membrane where the ribosome
interacts with the protein insertion machinery to directly couple synthesis
with membrane insertion of the hydrophobic proteins. Mba1 and Mdm38
were postulated as ribosome receptors as they are involved in the biogenesis
of the respiratory chain and they interact with both the ribosome and the
membrane. In this paper we showed that simultaneously deletion of MBA1
and MDM38 causes a growth defect on non-fermentable carbon sources that
is accompanied by a defect in complex III and IV assembly. In vivo pulse
labeling of mitochondrial translation products demonstrated that this mutant
strain fails to synthesize Cox1 and cytochrome b, core subunits of both complexes. Interestingly, higher molecular transcripts of COB and COX1 accumulate in this strain, which shows that splicing is impaired. However, because the defect to synthesize Cox1 or cytochrome b was not observed in the
single mutants it was suggested that Mba1 and Mdm38 have an overlapping
function in expression of these two intron-containing genes. Supportive evidence for a specific role of Mba1 and Mdm38 in regulation of Cox1 synthesis was obtained by employing yeast cells with a genome lacking introns.
Also in this mutant a synthetic growth defect was observed indicating that
defective splicing cannot explain the growth phenotype of the double mutant. Based on the observation that Pet309 co-purifies with Mdm38 a model
was proposed that Mdm38 and Mba1 might influence translation by recruiting specific translational activators to the ribosome.
Paper II: Cbp3-Cbp6 interacts with the yeast mitochondrial
ribosomal tunnel exit and promotes cytochrome b synthesis
and assembly
Cbp3 was described as a bc1 complex-specific assembly factor [185] that we
found in this work to be a novel interactor of the ribosomal tunnel exit by
chemical x-linking experiments. The ribosomal tunnel serves as a platform
for proteins involved in early steps of protein biogenesis.
42
In this work we unravel that Cbp3 has two principal roles in the biogenesis
of cytochrome b. First, the location of Cbp3 at the ribosomal tunnel exit
allows an efficient and direct interaction of the protein with newly synthesized cytochrome b. In the absence of Cbp3 cytochrome b is rapidly degraded indicating that Cbp3 is essential for its stabilization. Labeling of mitochondrial translation products in a ∆cbp3 strain revealed reduced levels of
newly labeled cytochrome b that could be the result of both increased destabilization and reduced translation in the absence of Cbp3. In order to analyze
a possible dual function of Cbp3 in translation and assembly of cytochrome
b in a separate way, we used an artificial mitochondrial genome encoding a
recoded version of ARG8 that was used as reporter for COB expression
(cob::ARG8 mtDNA). Using this strain we could demonstrate a dual function of Cbp3 in translation and stabilization of cytochrome b. Furthermore
this study provided evidence that Cbp3 forms a stable complex with the cytochrome b specific translational activator Cbp6 [124]. Both proteins stabilize each other and their interaction is mandatory both for the binding to the
ribosome in order to stimulate cytochrome b translation and for the stabilization of newly synthesized protein. After binding to newly synthesized protein, Cbp3-Cbp6 is released from the ribosome to assist assembly of cytochrome b into the bc1 complex. The trimeric complex composed of Cbp3Cbp6 and cytochrome b recruits Cbp4, another assembly factor, to form an
assembly intermediate. In summary, our data indicate that the Cbp3-Cbp6
complex is required for efficient translation and assembly of cytochrome b
and thereby fulfills two important functions for its biogenesis. This organization might provide an optimal channeled and protected assembly of cytochrome b into the bc1 complex.
Paper III: The Cbp3-Cbp6 complex coordinates cytochrome
b synthesis with bc1 complex assembly in yeast mitochondria.
Based on our previous findings that the Cbp3-Cbp6 complex has a dual
function in the biogenesis of cytochrome b, we asked whether it might be
involved in a regulatory circuit that allows adjusting cytochrome b synthesis
in response to the efficiency of assembly as seen for the biogenesis of Cox1
[195]. In order to address this we deleted the individual structural subunits
and assembly factors of the bc1 complex leading to a block at different steps
of the assembly line. We observed that the steady-state levels of cytochrome
b were reduced in these mutants to varying extent. This can be caused by
either instability of the protein or a reduced translation rate. As a wild type
43
mitochondrial genome does not allow for differentiation between both possibilities, we employed artificially engineered mitochondrial genomes. The
cob::ARG8 genome, already used in Paper II, enabled us to show that only
Cbp3 and Cbp6 but no other structural component or assembly factor of the
bc1 complex are required for translation of COB mRNA. To address the
question whether cytochrome b translation is modulated in response to the
efficiency of assembly, we generated a novel mitochondrial genome by introducing the COB coding sequence flanked by the regulatory regions of
COX2 at a silent location in the yeast mitochondrial genome (cox2::COB
cob::ARG8 mtDNA). This allowed analyzing cytochrome b assembly and
synthesis separately from each other. By monitoring the Arg8 levels we
could show that cytochrome b synthesis is indeed modulated when cytochrome b assembly is blocked. Interestingly, expression of the COB mRNA
expression reporter was only reduced when the assembly line was stalled at
early or intermediate steps indicating that an impairment of translation is not
a general consequence of a blocked assembly of the bc1 complex. In addition, we could significantly refine the assembly line of the bc1 complex by
showing that it assembles through four different intermediates, starting with
a sub-complex composed of Cbp3-Cbp6/Cyt b/Cbp4 that we already investigated in Paper II. After addition of Qcr7 and Qcr8, Cbp3-Cbp6 is released
whereas Cbp4 stays attached, forming intermediate II. Addition of further
structural subunits creates intermediate III and IV until the mature complex
is formed after incorporation of Qcr9, Rip1 and Qcr10. When the assembly
is blocked at early or intermediate steps, intermediate I accumulates, sequestering Cbp3-Cbp6. In such a scenario Cbp3-Cbp6 is trapped in an assembly
intermediate and prevented from stimulating translation at the ribosome.
This model could be confirmed by simultaneous overexpression of Cbp3 and
Cbp6 that restored synthesis of Arg8 from the cob::ARG8 locus. This shows
that Cbp3-Cbp6 acts as a sensor for the efficiency of bc1 complex assembly
that provides a feedback regulatory mechanism to fine-tune the rate of cytochrome b synthesis according to the actual needs.
Paper IV: Organization of mitochondrial gene expression in
two distinct ribosome-containing assemblies
The effort to synthesize proteins in mitochondria is immense: Roughly one
quarter (250 proteins) of the mitochondrial proteome is implicated in any
step of mitochondrial gene expression. How mitochondrial gene expression
and the mitochondrial ribosome are organized within the organelle is cur44
rently not known, but this knowledge might be the key to unravel the molecular mechanisms of gene expression. In this study, we established conditions
for analysis of ribosomes and its interactors whereby the RNA and DNA are
stabilized and most protein interactions are kept. Using these conditions we
could show that the mitochondrial ribosome plays a previously unrecognized
role in the organization of gene expression by forming large complexes with
multiple proteins involved in post-transcriptional steps such as RNA maturation/ modification/ degradation, translation or assembly. To highlight that
these complexes are conceptually very much different from ribosomes exclusively involved in translation, we termed them MIOREX complexes
(MItochondrial ORganization of gene EXpression). Using biochemical approaches and STED microscopy we could demonstrate that a subset of these
complexes associates with the nucleoid thereby consolidating all steps of
mitochondrial gene expression from transcription to assembly. This organization likely allows channeled transfer of newly transcribed RNA from the
transcription to the translation machinery. In addition these large assemblies
likely allow an optimal assembly of the respiratory chain subunits by the
recruitment of factors supporting their biogenesis. The results of this study
demonstrate that gene expression in mitochondria is organized in a completely different and organelle-specific way probably in order to coordinate
expression and assembly of proteins encoded and synthesized in two different compartments.
45
Conclusion and future perspectives
I. Impact of the mitochondrial ribosome for the general
organization of gene expression
During the last years numerous studies revealed that many processes in mitochondria are organized in large highly ordered assemblies in order to make
them more efficient. Among those are for instance supercomplexes that allow for an optimized transfer of electrons between the respiratory complexes
or the ERMES complexes that are important for the inter-organellar communication between ER and mitochondria thereby playing an important role for
mitochondrial biogenesis and function. In this thesis, evidence is provided
that shows that mitochondrial gene expression is organized in similar highly
ordered assemblies in form of the MIOREX complexes with the mitochondrial ribosome as central component. Conversely, the ribosome serves as
platform for multiple factors involved in post-transcriptional steps to bring
the various steps of gene expression temporally and spatially in close proximity (Paper IV). By this, translation is coupled with mRNA maturation and
protein insertion. Importantly, the partial association of these ribosome containing complexes with the mtDNA (nucleoid-MIOREX complex) might
allow channeled transfer of mRNAs from the transcription to the translation
machinery. This assembly additionally couples transcription with posttranscriptional steps of gene expression and provides a protected pathway for
the newly transcribed mRNA. This hypothesis is supported by our observation that the mRNA is always bound to the ribosome or other large complexes and not present in a free form in the mitochondrial matrix (Fig 9). This
kind of organization differs dramatically from the cytosolic systems where
RNA maturation and translation occurs in a temporally and spatially separated manner. The close coupling of transcription, RNA maturation and translation by the ribosome might provide a mechanism to regulate gene expression
within the organelle where the ways to regulate this process on the level of
transcription is rather limited.
46
Figure 9: mRNAs are part of the MIOREX complex.
A) mRNAs co-migrate with the ribosome and are not found in free forms in the
mitochondrial matrix. After separation on a sucrose gradient, RNA was isolated
from each fraction and analyzed by Northern blotting and autoradiography. B) The
distribution of mRNA and rRNA in sucrose gradients under low salt conditions was
densiometrically determined. C) mRNA and rRNA co-migrate with mitochondrial
ribosomes even under increased ionic strength. Mitochondria were lysed in a buffer
containing 100 mM KOAc and RNA extracted and analyzed as described in 9 A. An
asterisk indicates a degradation product of COB mRNA D) The distribution of
mRNA and rRNA in sucrose gradients under intermediate salt conditions was densiometrically determined. T, total (representing 10% the starting material); MW, molecular weight.
II. Role of translation activators in translation initiation
It is still a big mystery how and when the mRNA is loaded onto the ribosome and how translation is initiated in mitochondria. It has long been proposed that translational activators might play a role during translation initiation, but clear evidence for a distinct role in this process is still missing. The
results of my work allow some new hypothesis how this process might be
accomplished: We observed that some translational activators have a higher
affinity to the ribosome than others. Likewise, the separation of the nucleoidMIOREX and the peripheral-MIOREX complexes revealed that the distribution of several translational activators between those two types of complexes
47
differs. It is conceivable that one of those factors with a higher affinity to the
nucleoid-MIOREX complex is involved in the transfer of the newly transcribed mRNA to the ribosome where it is matured and translated (Fig 11).
Additionally, I found that some translational activators can bind to the
MIOREX complex even in the absence of their mRNA whereas others seem
to be specifically recruited to the ribosome by their client mRNA. This observation rather supports the idea that translational activators have slightly
different functions and work in a sequential manner during translation. Importantly, our methodical improvement to stabilize ribosomal complexes as
well as mRNAs might allow us to follow the distribution of mRNAs between the two types of MIOREX complexes in several deletion mutants in
order to determine the time point of ribosome binding. Furthermore, it is
likely that the characterization of some of the uncharacterized ribosomal
interactors found by our analysis will lead to the identification of additional
proteins involved in translation initiation, for example for ATP8 or VAR1
where a clear translation activator is still missing.
The close association of proteins implicated in translation initiation and
RNA binding with the ribosomes, might be important for the coordination of
respiratory chain assembly as it might allow for a repeated reading of the
same mRNA once it is loaded onto the ribosome. This mechanism might be
particularly useful for subunits such as Atp9 that are required in multiple
copies in order to form the functional oligomeric Fo complex of the ATPase.
III. Model of mitochondrial gene expression exemplified by the
biogenesis of cytochrome b
The data presented in this thesis support a model for the way how gene expression is organized and particularly how cytochrome b expression might
be accomplished in mitochondria (Fig 11). Our results indicate that the cytochrome b-specific factors Cbs1, Cbs2, Cbp1 and Cbp3-Cbp6 probably act in
a sequential manner during cytochrome b biogenesis, but the detailed molecular functions of most of them are still ill-defined. As discussed above the
different affinities of translational activators for the ribosome and the two
types of MIOREX complexes might indicate an involvement of those factors
at different steps during translation activation. Accordingly, Cbs2 has a
much stronger affinity for the ribosome than Cbs1 even in the absence of the
COB mRNA (Fig 10 A+B) suggesting that Cbs2 might act prior to Cbs1 in
the translation process. Similarly, Cbs2 is enriched in the nucleoid-MIOREX
48
complex making it a possible factor for loading the mRNA on the ribosome
after transcription. However, it is likely that this process is assisted by other
factors such as Rmd9. In contrast, Cbs1 is specifically recruited to the ribosome by the COB mRNA (Fig 10 C+D) indicating that its interaction with
the ribosome is rather transient. This idea is supported by the observation
that co-purification of Cbs1 with the ribosome is rather inefficient. The
mRNA binding protein Cbp1 co-migrates strongly with the ribosome similar
to Cbs2 but was not enriched in the nucleoid-MIOREX complex. However,
it was also found in a higher molecular weight complex that was free from
the ribosome suggesting that it could be involved in a transient step between
transcription and translation (Paper IV). Unfortunately, our data does not
allow differentiation whether Cbp1 works before, after or simultaneously
with Cbs2.
In addition to proteins directly involved in the translation process we found
factors implicated in mRNA maturation and degradation as interactors of the
ribosome indicating that intron splicing occurs in direct vicinity to the translation machinery. Accordingly, the COB splicing factor Cbp2 interacts
strongly with the ribosome and was clearly enriched at the peripheralMIOREX complex. As maturation of intron-containing transcripts such as
COB depends additionally on the synthesis of intron encoded maturases the
accumulation of splicing factors on the ribosome organizes all maturation
processes in close proximity making this process more efficient.
After generation of the mature COB mRNA, it is translated and cytochrome
b emerges from the tunnel exit where it is immediately stabilized by interaction with the Cbp3-Cbp6 complex that plays an important role in synthesis
and assembly of cytochrome b (Paper II). We could show that the Cbp3Cbp6 complex exhibits a dynamic interaction with the mitoribosome providing the opportunity for a regulatory circuit that enables modulation of mitochondrial gene expression in response to assembly efficiency (Paper III). By
this organelle-specific organization, mitochondria are able to sense and coordinate expression of mitochondrially and nuclear encoded subunits. However, it remains an intriguing question how the Cbp3-Cbp6 complex located
at the ribosomal tunnel exit (Paper II) is able to influence the translation of
COB. Possible scenarios are that it does so by assisting mRNA binding and
start site selection on the ribosome, a direct interaction with the mRNA
or/and the other translational activators or a conformational change within
the ribosome triggered by the binding of Cbp3-Cbp6 that alleviates or prevents translation. Usage of artificial mitochondrial genomes and mutants
blocking different steps of COB biogenesis might allow for identification of
49
additional binding sites of Cbp3-Cbp6 on the ribosome. Furthermore the
determination of protein-RNA interactions by UV-cross-linking can confirm
a possible direct interaction of Cbp3-Cbp6 with the mRNA and might help
to understand how this complex cooperates with the other translational activators of COB. In case cytochrome b synthesis is no longer required, the
mRNA is degraded by the mitochondrial degradosome composed of Suv3
and Dss1. As we found the degradosome subunit Suv3 strongly enriched in
the nucleoid-MIOREX complex whereas Dss1 had a stronger affinity to the
ribosome, it is likely that the ribosome returns to the nucleoid to bring both
subunits of the degradosome together. After RNA decay in the vicinity of the
nucleoid, the ribosome can be directly loaded with a new pre-mRNA.
Figure 10: Translational activators can interact with the MIOREX complex in
the absence of their mRNA whereas others are specifically recruited.
A) Cbs2, a translation activator for COB mRNA is part of the MIOREX complex. B)
In absence of the COB mRNA (∆cbp1), Cbs2 is still able to bind to the MIOREX
complex however with reduced efficiency. C) Cbs1, a translation activator for COB
mRNA is part of the MIOREX complex. D) In absence of the COB mRNA
(∆cbp1), Cbs1 fails to migrate with the MIOREX complex, indicating that it is recruited by the COB mRNA to the ribosome.
50
IV. Dedicated ribosomes
My work demonstrated that mitochondrial gene expression occurs in a highly ordered and organized fashion. It has long been speculated how the membrane-bound ribosome coordinates the assembly of the mitochondrial encoded subunits into the respective complexes especially in case of the CcO or
the ATP synthase that contain three mitochondrial encoded subunits.
Figure 11: Model of cytochrome b biogenesis.
Ribosomes are loaded with a pre-mRNA on the nucleoid. Cbs2 that is enriched in
the nucleoid-MIOREX could be involved in loading the COB mRNA onto the ribosome. After processing of the 5‟-end COB mRNA is stabilized by Cbp1 and the
ribosome is released from the nucleoid by a still unknown mechanism. In the second
step, the peripheral-MIOREX complex recruits other COB specific factors (x) such
as Cbs1 and the Cbp3-Cbp6 complex to form a primed ribosome, specialized for the
synthesis of cytochrome b. After initiation of translation the pre-mRNA is matured
(removal of purple areas) by the combined action of splicing factors (such as Cbp2)
and intron encoded maturases. As soon as cytochrome b emerges from the tunnel
exit it is bound and stabilized by the Cbp3-Cbp6 complex that is afterwards released
from the ribosome to assist bc1 complex assembly. When cytochrome b synthesis is
no longer required, the ribosome returns to the nucleoid where the mRNA is degraded and the ribosome loaded with a new COB mRNA.
51
Due to the strict dependence of mitochondrial translation on gene-specific
factors it was proposed that their binding to the ribosome defines it for the
synthesis of only one mitochondrial encoded gene [100]. This organization
would allow for an optimal coordination of complex assembly. My data contribute supportive evidence to the model of “dedicated ribosomes” (Fig 11
“primed ribosome”). We found that some of the gene-specific factors have a
strong affinity to the ribosome even under high salt conditions (Paper IV).
These factors could prime the ribosome for the synthesis of a single mitochondrial encoded subunit and could serve as recognition sign for other
gene-specific factors with higher affinity to the nucleoid-MIOREX. The
model of dedicated ribosomes is further supported by our findings that ectopically expressed cytochrome b is normally synthesized but fails to accumulate to wild type level (Paper III). An explanation for this phenomenon
could be that cytochrome b when expressed under control of the COX2 regulatory regions is synthesized by a “wrong” ribosome that is not equipped
with the Cbp3-Cbp6 complex at the tunnel exit. This prevents the optimal
interaction of newly synthesized cytochrome b with Cbp3-Cbp6 therefore
increasing its degradation. Similar results were obtained with ectopically
expressed Cox2 and Cox3 [153]. The purification of specialized ribosomes
via gene-specific factors should allow the verification of this model. However, the low abundance and the separation of the nucleoid-MIOREX and the
peripheral-MIOREX complexes before the purification render this experiment rather challenging.
III. Final remarks
There are still many things to investigate concerning mitochondrial translation. The complicated and carefully organized assembly of the mitochondrial
gene expression system shown here might be the reason why the establishment of a mitochondrial in vitro translation system was not possible so far.
My findings and methodical improvements are a good starting point to further analyze the sequential steps of mitochondrial gene expression. Additionally, we identified many so far uncharacterized proteins as part of the
ribosomal interactome (Paper IV). The analysis of these proteins might lead
to the determination of other specificity factors involved in splicing, translation, recruitment (Paper I) and assembly of a certain mitochondrial gene. We
are clearly still far away from the complete understanding of mitochondrial
gene expression. It will be definitely a long and exciting way to unravel the
unique gimmicks that evolved to optimize protein synthesis in mitochondria.
52
Sammanfattning på svenska
Mitokondrierna har sitt eget genetiska system som kodar för viktiga
subenheter av enzymkomplex som utgör oxidativ fosforylering. Dessa
subenheter uttrycks av ett organell-specifikt system som utvecklats från den
bakteriella anfadern av organellen och som har förändrats väsentligt under
evolutionen. Trots det mitokondriella genetiska systemets stora betydelse för
cellulär function så är både organisationen och samspelet av de faktorer som
är inblandade i genuttrycket fortfarande okänt. Min forskning visadne ett
flertal grundläggande aspekter inom mitokondrie-genuttryck och att dessa
processer är organiserade i ett unikt och organellspecifikt sätt som sannolikt
utvecklats för att optimera proteinsyntes och sammansättning i mitokondrier.
Huvudsakligen, genom att ha förbättrat den experimentella hanteringen av
ribosomer, har jag upptäckt att mitokondriella ribosomer är organiserade i
stora samlingar, som vi betecknat MIOREX komplex. Ribosomerna i dessa
MIOREX komplex är organiserade i posttranskriptionssteg genom att
rekrytera flera faktorer som behövs för RNA mognad/nedbrytning,
translation och subenhetsamling vilka resulterar till en koppling mellan alla
dessa steg i genuttrycket. Dessutom kunde vi visa mekanismer genom vilka
ribosom-interaktorkomplex modulerar och samordnar uttrycket och
sammansättning av andningskedjans subenheter. Till exempel har vi kunnat
demonstrera att Cbp3 - Cbp6 komplexet binder till ribosomen i närheten av
tunnelmynningen för att samordna syntes och sammansättning av cytokrom
b. Här på den mitokondriella ribosomen, positionerar sig Cbp3 - Cbp6
utmärkt för direkt bindning till nyligen syntetiserade cytokrom b och låter
Cbp3 - Cbp6 att upprätta en återkoppling som gör att moduleringen av
cytokrom b -syntes som svar på sammansättning är effektiv. Likaså är
samverkan av membranförankringsproteinerna Mba1 samt Mdm38 med
tunnelmynnings- regionen möjlig för att delta i translationen av de två
intron -kodande generna COX1 och COB utöver sina roll i
membraninsättning.
Sammanfattningsvis så visar arbetet i denna avhandling att det
mitokondriella genuttrycket är en väldigt organiserad och reglerad process.
Dessa koncept och tekniska innovationer kommer att underlätta
klartläggningen av ett mångfald viktiga aspekter och därmed bidra till den
allmänna förståelsen för hur proteiner syntetiseras i mitokondrier .
53
Deutsche Zusammenfassung
Mitochondrien besitzen ein eigenes genetisches System, das wichtige
Untereinheiten der Atmungskettenkomplexe kodiert, die ATP durch
oxidative Phosphorylierung herstellen. Diese Untereinheiten werden von
einer Mitochondrien-spezifischen Expressionsmachinerie hersgestellt, die
einige Besonderheiten im Vergleich zum bakteriellen Vorfahren entwickelt
hat. Trotz der Wichtigkeit der mitochondrialen Proteinsynthese für die
Funktion der ganzen Zelle ist nur wenig darüber bekannt, wie die
verschiedenen Faktoren, die an diesen Prozessen beteiligt sind, organisiert
sind und miteinander wechselwirken. Meine Arbeit deckte einige
erstaunliche Aspekte der mitochondrialen Genexpression auf und zeigt, dass
dieser Prozess in einer einzigartigen und Organell-spezifischen Art und
Weise organisiert ist; ein Aufbau der sich höchstwahrscheinlich enwickelt
hat, um Proteinsynthese und –aufbau in Mitochondrien zu optimieren. Durch
Verbesserung der experimentellen Handhabung von Ribosomen konnte ich
zeigen, dass das mitochondriale Ribosom eine Schlüsselrolle in der
Organisation von post-transkriptionalen Schritten der Genexpression spielt,
indem es als Plattform für zahlreiche Faktoren dient, die für RNA
Maturierung/ Abbau, Translation und Aufbau benötigt werden. Diese
Organisation ermöglicht eine enge Verbindung zwischen allen Schritten der
Genexpression. Diese Arbeit zeigt, dass die Interaktion von zahlreichen
Faktoren mit dem Ribosom eine wichtige Organisation darstellt, um die
Expression und den Aufbau der Atmungskettenkomplexe zu koordinieren,
die in zwei unterschiedlichen zellulären Kompartimenten kodiert und
synthetisiert werden. Dementsprechend konnten wir zeigen, dass der an zwei
Funktionen beteiligte Cbp3-Cbp6 Komplex in der Nähe des ribosomalen
Tunnelausganges bindet um die Synthese und den Aufbau von Cytochrome
b zu koordinieren. Dies positioniert Cbp3-Cbp6 optimal für die sofortige
Bindung von neu-synthetisiertem Cytochrom b und erlaubt Cbp3-Cbp6 in
einer Rückkopplungsschleife zu wirken, die eine Anpassung der Cytochrom
b Synthese in Antwort auf die Effizienz des Komplex Aufbaus erlaubt. In
ähnlichem Sinne ermöglicht die Interaktion der Membrananker-Proteine
Mba1 und Mdm38 mit dem Tunnelausgang eine Beteiligung in der
Translation der zwei intron-enthaltenden Gene COX1 und COB zusätzlich zu
ihrer Rolle in der Membraninsertion. Meine Arbeit deutet klar darauf hin,
dass die mitochondriale Genexpression ein hoch organisierter und regulierter
Prozess ist. Die Erkenntnisse aus dieser Arbeit werden ein Startpunkt für die
54
Aufklärung von vielen zusätzlichen und wichtigen Aspekten sein, die helfen
werden zu verstehen, wie Proteine in Mitochondrien synthetisiert werden.
55
Acknowledgements
At the end I would like to take the opportunity to thank some people that
accompanied and supported me during the last 5 years of my PhD.
First of all, I want to thank Martin Ott for being my supervisor during the
last 5 years. Thank you, for being always available for questions and problems of all kind and all your ideas and advices you gave me. You have exposed me to many different, challenging and exciting scientific questions
and even if it was not always easy I really appreciate how much I learned
from you and I‟m thankful for the possibilities you offered me to learn from
others. Furthermore, I want to thank you for giving me the opportunity to
come with you to Stockholm, what was a really great chance and experience
for me.
Thank you, Nathalie Bonnefoy and Inge Kühl for having me in your lab.
Even if we unfortunately couldn‟t finish our project together, it was really
fun to work with you and to learn from you!
I would like to thank all our collaboration partners Axel Imhof, Lars Israel,
and Tobias Lamkemeyer who did the mass spectrometry for Paper II and
IV, respectively. Thank you, Stefan Jakobs, Christian Wurm and Thomas
Langer for your great input and discussions for Paper IV. I would like to
thank Nathalie Bonnefoy and Inge Kühl for their great expertise in manipulating the mitochondrial genome that was a crucial contribution for Paper III.
Heike Bauerschmitt, David Mick, Markus Deckers, Soledad Funes,
Hannes Herrmann and Peter Rehling it was a pleasure to work with you
on Paper I.
Furthermore, I‟m very grateful to Carol Dieckmann from the University of
Arizona and Alex Tzagoloff from Columbia University, New York for nice
and helpful discussions and sharing a lot of knowledge as well as unpublished data and material.
Also, I would like to thank Per Ljungdahl, Claes Andréasson and their
whole group for their input and critical discussions during our “yeast” seminar 
Many thanks to the whole Hannes Herrmann group, as it existed until
2011 when I came to Stockholm. It was a great pleasure to work with you
and to be part of your team and even when I struggled a long time after my
Diploma to stay in Kaiserslautern for my PhD, you guys made it a nice place
and it was even harder to decide to leave you after 2 years . Especially I
56
would like to thank Andrea and Simone for their great help and support in
the lab and all other organizational matters. Steffi H, Yvonne, Vanessa –
thank you for being the best Diploma students! It was really fun to work
with you! Thank you Martin P. for the exhausting squash lessons after work
 and Kerstin G, Steffi H, I‟m glad that you joined our group and I‟m very
happy to have you as friends !
Markus, Steffi G, Manfred, Ramon, Kathi R. – it was a great adventure to
move the lab 2011 to Stockholm with you! I‟m very thankful that I had you
around during the start here and for all the nice moments we spend together
inside and outside the lab!
After coming to DBB I would like to thank first of all Stefan Nordlund for
his great effort to make the change into the Swedish PhD system as smooth
as possible for us and for his support during the last three years. Furthermore
I want to thank him, as well as Gunnar von Heijne, Pia Ädelroth and
Elzbieta Glaser for all the nice and interesting discussion during the big
exam and all examination points. Many thanks to the whole DBB for the
very warm welcome here! In this respect I would like to thank especially
Beata, Pedro, Dominik, Candan, Catharina and Diogo for all the nice
occasions we had together and for your hospitality !
I was incredibly fortunate to work with many great colleagues over the last 5
years! Very big thanks go to all former and present members of my group!
Markus, Steffi, Martin P., Ramon, Kathi R, Steffi H, Kerstin G,
Manfred, Kathi S, Tamara, Braulio, Hannah, Jacob, Lorena, Alexandra, Roger and Mama, thank you so much for your scientific and private
support during the last years and creating such a familiar environment!! I
really enjoyed working with you and I will miss the time we spend together!
Thank you dear “the Drews” – Povilas, Aziz, Mathieu, Emmanuel and
Saba for making the 5th floor finally a bit more international  It was very
nice to have you around! Sorry for occupying your lab space for hours when
I was developing blots and thank you for always cheering me up after I got
the results . Thank you Kimmo, Hena, Ljubica and Amin for being such
nice and collegial floor mates!
Some nice people took the time to proof- read my thesis – thank you Martin, Tamara, Kathi S, Braulio, Povilas, you guys helped me a lot ! Furthermore a big “thank you” to Saba and Alexandra for helping me with the
Swedish summary!
Finally, I would like to thank also Håkan, Peter, Torbjörn, Ann, Haidi,
Alex T., Lotta, Malin and Anita for their help and advices in all aspects of
the daily worklife!
57
Andi, Jörg, Caro and Anne – it was always a pleasure to be with you!
Thank you for all the nice moments we spend together!
Last but not least I want to thank my dad, my sister and Thorsten for their
great support and their patience. Thank you that you always encouraged me
and built me up when I was down! I also want to thank my mom for inspiring the interest in natural science in me!
58
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