Organization of Mitochondrial Gene Expression in Yeast

Home , 16S ribosomal RNA, Bacterial translation

Doctoral Thesis in Biochemistry at Stockholm Uni versity, Sweden 2014

Organization of mitochondrial 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. Ribo- somes 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 membrane- anchor 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, Jak- obs S, Langer T, Ott M. Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies. submit- ted

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 Expres- sion. 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 com- munication 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 approx- imately 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 alle- viating 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 α- proteobacterial 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 fold- ed 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 electron- carrying 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 de- livered electrons from cytochrome c to O2.

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. Com- plex 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 membrane- embedded 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 di- mension 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 exten- sively coated with proteins that package the DNA into compact nucleopro- tein 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.

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) Compari- son 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 organ- ism 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 oc- curred 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]. Conse- quently, 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.

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 post- translationally 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 eluci- dating 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.

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 con-

21 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). Transcrip- tion 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 Dual function in COX3 translation and [89, 90] splicing of the AI5β intron Mss18 Required for efficient splicing of COX1 [91] intron AI5β Mne1 Required for splicing of COX1 intron [92] AI5β Ccm1 Maturation of COB and COX1 mRNA; [93] 15S binding protein Cox24 Required for COX1 intron splicing [94] RNA helicases/chaperones Suv3 Component of the mtEXO; involved in [95] splicing of COX1 intron AI5β by recycling of Mrs1 Mrh4 Required for the assembly of the large [96] ribosomal subunit Mss116 Required for efficient splicing of group [74] [97] I and group II introns Irc3 Probably involved in DNA maintenance [75] and replication RNA degradation mtEXO (Suv3/Dss1) RNA degradation and surveillance [76]

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 ex- tensively 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 cen- ter, 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 mito- specific 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 mito- specific 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.

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 tun-

26 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 per- form 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 gua- nine content of mitochondrial RNAs, the base involved in the strongest base- pairing. Concomitant, mitochondrial RNAs contain similar amounts of cyto- sine 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 Shine- Dalgarno 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 mitochodri- ally 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.

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 post- transcriptional 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 so- called 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 gene- specific 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]. Ad- ditionally, 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 gene- specific 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 re- duces 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. Accord- ingly, 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].

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]). Assem- bly 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 specu- late 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 single- membrane spanning protein with a highly conserved AAA domain that has been shown to be required for Rip1 assembly [192]. Bcs1 binds to a bc1 pre- complex 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 Cbp3- Cbp6 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 in- troducing 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 investi- gated 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, seques- tering 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 cur-

44 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 ap- proaches 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 commu- nication 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 post- transcriptional 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.

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 nucleoid- MIOREX 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. Im- portantly, 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 peripheral- MIOREX 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 Cbp3- Cbp6 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. How- ever, 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. Howev- er, the low abundance and the separation of the nucleoid-MIOREX and the peripheral-MIOREX complexes before the purification render this experi- ment 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. Addi- tionally, 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 prob- lems 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 manipu- lating 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 un- published 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” semi- nar 

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, Alexan- dra, 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 Mar- tin, Tamara, Kathi S, Braulio, Povilas, you guys helped me a lot ! Fur- thermore 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!

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 inspir- ing the interest in natural science in me!

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