Biogenesis of the Bc1 Complex in Mitochondria

Katharina Stephan Biogenesis of the bc1 complex in mitochondria

Katharina Stephan Biogenesis of the bc 1 complex in mitochondria

Katharina Stephan

ISBN 978-91-7911-116-8

Department of Biochemistry and Biophysics

Doctoral Thesis in Biochemistry at Stockholm University, Sweden 2020

Biogenesis of the bc1 complex in mitochondria Katharina Stephan Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Thursday 11 June 2020 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.

Abstract Mitochondria perform a variety of tasks, but the function they are most prominent for is the energy conversion to form ATP, the universal energy equivalent of the cell. The majority of this ATP is created by the oxidative phosphorylation system, consisting of the respiratory chain and the ATP synthase. These elaborate machineries channel electrons through the respiratory complexes and thereby generate an electrochemical gradient across the inner mitochondrial membrane. This, so called proton motive force, is in turn utilized by the ATP Synthase to produce ATP. A particularity of the oxidative phosphorylation complexes is that their subunits are derived from two genetic sources. As a result, and the fact that the respiratory chain complexes contain redox cofactors, the biogenesis of these enzymes is challenging and involves multiple, highly coordinated and regulated assembly steps. For the obligate homodimeric bc1 complex, a handful of assembly factors are known and its assembly can be divided into distinct assembly intermediates. In this work we provided insights into the maturation of the catalytic subunit cytochrome b. We revealed that the insertion of the redox active heme b groups is sequential and that it depends on the interaction with the early assembly factor Cbp4. With successful insertion of both heme bs, the binding of the structural subunit Qcr7 is necessary for stabilization and further assembly. Furthermore, we were able to delineate the dimerization event in detail. We could establish that the interaction of the two matrix subunits, Cor1 and Cor2, with the bc1 complex assembly intermediate II, as well as the dissociation of Cbp4, are the triggering point for dimerization. In our subsequent work we investigated the roles of the fairly uncharacterized assembly factor Bca1 and its interplay with the structural subunit Qcr7. We could demonstrate that Bca1 interacts early and transiently during assembly and is an important factor for efficient assembly. Additionally, we could show that Qcr7 is not only a structural subunit but also serves as an assembly checkpoint for the maturation of the bc1 complex. With our work we could illustrate the necessity for basic biochemical research within the model organism yeast, as the fundamental molecular mechanisms are well conserved. This is exemplified by our work on UQCC3, the human orthologue of the bc1 complex assembly factor Cbp4.

Keywords: Respiratory chain, bc1 complex assembly, mitochondrial protein biogenesis, molecular biology, yeast.

Stockholm 2020 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-180531

ISBN 978-91-7911-116-8 ISBN 978-91-7911-117-5

Department of Biochemistry and Biophysics

Stockholm University, 106 91 Stockholm

BIOGENESIS OF THE BC1 COMPLEX IN MITOCHONDRIA

Katharina Stephan

Biogenesis of the bc1 complex in mitochondria

Katharina Stephan ©Katharina Stephan, Stockholm University 2020

ISBN print 978-91-7911-116-8 ISBN PDF 978-91-7911-117-5

Printed in Sweden by Universitetsservice US-AB, Stockholm 2020 List of publications

I. Hildenbeutel M, Hegg EL, Stephan K, Gruschke S, Meunier B, Ott M. Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation. (2014) J Cell Biol. 205(4):511-24. II. Stephan K and Ott M. Timing of dimerization of the bc1 complex during mitochondrial respiratory chain assembly. (2020) Biochim Biophys Acta Bioenerg. 1861(5-6):148177. III. Wanschers BF, Szklarczyk R, van den Brand MA, Jonckheere A, Suijskens J, Smeets R, Rodenburg RJ, Stephan K, Helland IB, Elkamil A, Rootwelt T, Ott M, van den Heuvel L, Nijtmans LG, Huynen MA. A mutation in the human CBP4 ortholog UQCC3 impairs Complex III assembly, activity and cytochrome b stability. (2014) Hum Mol Genet. (23):6356-65. IV. Stephan K, Singh AP, Römpler K, Hildenbeutel M, Meunier B, Ott

M. Role of Bca1 and the early structural subunit Qcr7 in bc1 complex biogenesis. Manuscript Additional publications

I. Möller-Hergt BV, Carlström A, Stephan K, Imhof A, Ott M. The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria (2018) Mol Biol Cell. (20):2386-2396.

Abbreviations

ATP adenosine triphosphate OXPHOS oxidative phosphorylation CII – CIV complex II – IV of the respiratory chain FAD/FADH2 flavin adenine dinucleotide NAD+/NADH nicotinamide adenine dinucleotide FeS iron-sulfur cluster IMM inner mitochondrial membrane IMS intermembrane space Q/QH2 ubiquinone/ubiquinol ROS reactive oxygen species SC supercomplex TM transmembrane COB cytochrome b (gene and mRNA) MRP mitochondrial ribosomal protein OMM outer mitochondrial membrane mitoribosome mitochondrial ribosome mtDNA mitochondrial DNA UTR untranslated region Cryo EM cryo electron microscopy pmf proton motive force Contents

List of publications ...... i Additional publications ...... ii Abbreviations ...... iii Introduction ...... 1 Mitochondria ...... 1 Oxidative phosphorylation...... 4 The mitochondrial F1Fo-ATP Synthase ...... 4 Respiratory chain complexes ...... 6 Complex I ...... 8 NADH dehydrogenases in yeast ...... 11 Complex II...... 12 Complex III...... 15 Complex IV ...... 21 Supercomplexes ...... 25 Functional significance ...... 25 Dual genetic origin...... 27 Why mitochondria keep their own genome ...... 28 Mitochondrial translation ...... 29 Synthesis of Cytochrome b ...... 31 Complex III assembly ...... 33 Assembly factors of the bc1 complex ...... 33 The assembly line of the bc1 complex...... 37 Aim of this thesis ...... 39 Summary of the papers ...... 39 Paper I: ...... 39 Paper II: ...... 40 Paper III: ...... 41 Paper IV:...... 42 Conclusion and remarks ...... 43 Populärwissenschaftliche Zusammenfassung...... 45 Populärvetenskaplig sammanfattning...... 47 Acknowledgments ...... 48 References ...... 51

Introduction

Mitochondria Mitochondria are organelles within eukaryotic cells that fulfill a plei- otropic variety of tasks and belong to the most studied organelles of the cell. Mitochondria are very dynamic and variable, they are able to form a network and can adapt to the needs of their cell. For example, the number of mitochondria in a cell of the baker’s yeast Saccharomyces cerevisiae depend on the growth conditions, or mammalian cells can have up to 104 mitochondria depending on the energy demand of the tissue they are found in [1]. Roughly 2 billion years ago mitochondria evolved [2, 3], due to the joining of an α-proteobacterial ancestor with a primitive eukaryotic cell, which resulted in forming an endosymbiotic lifestyle rather than being digested by the engulfing cell [4, 5]. A consequence of the mitochondrial endosymbiotic origin is that they are contain two membranes. The outer membrane is permeable to smaller molecules. The inner membrane is more impermeable. Hence, two biochemically different soluble compartments are created: the inter membrane space (IMS), with a more oxidizing environment [6], and the matrix, a hub for many different metabolic processes [7]. In addition to the two membranes, the prokaryotic past of mitochondria is reflected by the presence of an organellar genome within the mitochondrial matrix. During evolution more and more genes of the mitochondrial ancestor were transferred to the nucleus. Consequently, today mitochondrial genomes encode only a handful of genes, mainly involved in oxidative phosphorylation and mitochondrial translation, and depending on the species, in transcription, RNA maturation and protein import [8].

1 The inner mitochondrial membrane (IMM), which encloses the matrix, is heavily folded into so called cristae, thereby increasing the surface significantly. This membrane harbors not only channels/transport- ers, the protein translocation/insertion machinery and bio-synthesis proteins, but also the oxidative phosphorylation (OXSPHOS) system. The OXPHOS system, comprised of the respiratory chain complexes and the ATP synthase, converts chemical energy from metabolites first into a membrane potential and then utilizes this proton motive force to generate ATP, the common energy currency of the cell. This so formed ATP can in turn either be used in the matrix to fuel chemical reactions or is exported into the IMS or cytosol where it can be utilized. Furthermore, mitochondria harbor many important catabolic pathways such as the tricarboxylic acid (TCA) cycle, β-oxidation of fatty acids, and the degradation of amino acids. The reducing equivalents, like FADH2 or NADH, generated by those reactions feed electrons into the OXPHOS system to create ATP and thereby render the mitochondria, the main supplier for chemical energy in form of ATP. Additionally, these catabolic pathways generate numerous important metabolic intermediates and precursors that are in turn needed by various anabolic pathways harbored in mitochondria, including the biogenesis of carbohydrates, nucleotides, lipids, amino acids and redox cofactors like heme or iron-sulfur (FeS) clusters. Therefore, these organelles represent an important link for intermediates of the anabolic and catabolic metabolism. Consequently, and due to the fact that mitochondria produce the main part of the cells ATP by oxidative phosphorylation, mitochondria have long been considered as bioenergetic and biosyn- thetic organelles. Recently, an increasing number of studies shows that mitochondria have an additional important role in cell signaling [9]. Mitochondria constantly communicate their fitness state to the cell. In the literature this is commonly referred to as “retrograde signaling” [10, 11]. For example, if the mitochondria become dysfunctional they are able to induce organelle degradation (mitophagy) or even cell death (apoptosis) [12].The signaling modes range from the release of proteins/peptides (e.g. apoptosis, unfolded protein response (UPR)), over production and

2 release of reactive oxygen species (ROS) (e.g. autophagy, stress response), to the recruitment of proteins to the outer membrane to form a signaling complex (e.g. apoptosis, autophagy, immune response) [13- 18]. Furthermore, mitochondria are in close contact with the endoplasmic reticulum [19-22]. This inter-organelle interaction is not only important for mitochondrial morphology and function, but additionally links processes like lipid synthesis, apoptosis and Ca2+ homeostasis [23, 24]. Considering the involvement of mitochondria in such a wide variety of biochemical reactions and processes it is not surprising that, particu- larly with regard to the strong impact of mitochondrial function on human diseases and aging, this organelle receives such growing interest.

3 Oxidative phosphorylation The OXPHOS system is the main production site of ATP within the cell [25]. It is accommodated within the cristae of the IMM and is typ- ically composed of five complexes of dual genetic origin [26, 27]. The first four complexes, composing the respiratory chain, are able to couple proton pumping from the matrix to the IMS and electron transport from NADH and FADH2 to the final electron acceptor, molecular oxygen. Due to the proton movement across the IMM an electrochemical gradient is established. The proton gradient or proton motive force (pmf), is composed of two parts: a pH gradient and a charge separation. This gradient is in turn utilized by the fifth and last complex of the OXPHOS system, the mitochondrial ATP Synthase to generate ATP.

The mitochondrial F1Fo-ATP Synthase

The ATP synthase is located in the cristae bends where it forms functional dimers and is also involved in the membrane curvature and formation of cristae [26, 28]. In humans, this complex consists of 29 subunits that form two functional domains. The F1 domain harbors the ATP synthesis site and is situated in the mitochondrial matrix. It consists of a catalytic globular hexamer and the central stalk [29]. The Fo domain is located in the IMM and is responsible for the proton translocating activity. In eukaryotes it is formed by subunit Atp9/c (yeast/human), which forms a ring with eight identical subunits in human, or ten in yeast. The Atp6/a subunit is associated to this ring structure and forms the proton conduction pathway surface [30]. The two domains are connected through the F1 central stalk and the peripheral stalk [31]. The ATP Synthase uses the energy that is stored in the electrochemical gradient, to phosphorylate ADP to ATP. The protons that are exported into the IMS can flow through the ATP synthase back into the matrix inducing a rotation that leads to conformational changes in the head of the F1 sector, thereby catalyzing ATP synthesis [25, 31, 32].

4 As my work is mainly focusing on the respiratory chain, I will explain in more detail the respiratory chain complexes per se, their functions, architectures and assemblies in the next paragraph.

5 Respiratory chain complexes In the majority of eukaryotic organisms, the respiratory chain contains four complexes, namely NADH oxidoreductase (Complex I), succinate dehydrogenase (Complex II), cytochrome c reductase (bc1 complex or Complex III) and cytochrome c oxidase (Complex IV) Complex I, III and IV couple electron transport with the export of protons from the matrix to the IMS, thereby creating a electrochemical gradient across the IMM [33]. Electron transport by the respiratory chain starts with the formation of ubiquinol (reduced form of ubiquinone) by Complex I and II. These two complexes take up electrons from NADH and FADH2 and transfer them to the electron shuttle ubiquinone, also called coenzyme Q. Ubiq- uinol is freely diffusible in the membrane and can shuttle electrons between Complex I, II and III. The electrons are then transferred by the obligate homodimeric Complex III to the soluble electron shuttle cytochrome c, via a sophisticated mechanism termed Q cycle. This cycle represents an important switch between the two-electron carrier ubiquinone and the one-electron carrier cytochrome c, mediated by the redox centers of the bc1 complex: cytochrome bL, bH and c1. Cytochrome c can freely travel between Complex III and IV to transfer electrons. Cy- tochrome c oxidase finally reduces molecular oxygen to water by transferring the delivered electrons from cytochrome c. The composition of the respiratory chain and its complexes can vary between organisms, but the core subunits are highly conserved between all three kingdoms. For example, the multimeric Complex I is missing in Saccharomyces cerevisiae, therefore the NADH oxidation is performed by Nde1, Ndi1 and Ndi2, single enzymes that do not couple the electron channeling to the transfer of protons across the membrane [34-36]. They only participate indirectly to the pmf by funneling the electrons into the ubiquinol pool that is in turn used by Complex III and IV to generate the proton gradient (Fig 1).

6 Fig 1: Schematic illustration of the respiratory chain in the baker’s yeast S. cerevisiae. Reducing equivalents like NADH or succinate enter the respiratory chain and their electrons are transferred by NADH dehydrogenases or succinate dehydrogenase to ubiquinol (QH2). Ubiquinol is then, by the bc1 complex, re-oxidized to ubiquinone (Q) and the electrons are transferred to the soluble electron shuttle cytochrome c (Cyt). The electrons are further shuttled to cytochrome c oxidase, the last

Complex of the respiratory chain. Complex IV catalyzes the reduction of oxygen (O2) to water (H2O). The two latter complexes, Complex III and Complex IV, respectively, translocate protons (H+) across the IMM, establishing an electrochemical gradient.

7 Complex I Complex I, or NADH oxidoreductase, is the first enzyme complex of the mitochondrial respiratory chain. Furthermore, with a molecular mass of approximately 1 MDa, Complex I is the largest and most complicated enzyme within the respiratory chain. It has many subunits encoded in the nucleus as well as in the mitochondria. For example, it contains 45 subunits in human that need to be assembled properly to maintain cellular vitality. The overall reaction it catalyzes is the transfer of electrons from NADH to ubiquinone and translocates protons across the inner mitochondrial membrane. In the reaction, four protons are pumped into the IMS while one molecule of NADH is oxidized. Structure and function Complex I has an L-shaped architecture with a hydrophobic arm embedded in the IMM (P module) and a hydrophilic or peripheral arm located in the matrix (N module and Q module) [37, 38]. The catalytic reactions occur in the peripheral arm on the matrix side. The so called N- module contains a non-covalently bound flavoprotein (flavin mononucleotide, FMN) and eight, canonical iron-sulphur (FeS) clusters that ultimately channel the electrons from NADH through the complex to the ubiquinone binding site buried in the hydrophobic arm (Q module). Electron leakage during this transfer accounts for significant portion of mitochondrial ROS production [39]. The hydrophobic arm or P module of the assembled multi-subunit complex is a proton pump, translocating protons from the matrix to the IMM, generating an electrochemical gradient, but the precis pumping mechanism is not yet resolved.

Fig 2: NADH oxidoreductase. Left hand side the structure of the human NADH oxidoreductase (PDB: 4UQ8) [40]. In blue the N module, pink the Q module, and in yellow, embedded in the IMM, the P module. On the right hand side a schematic depiction of Complex I with all its cofactors. Complex I oxidizes NADH at its N module and channels the electron via a flavoprotein and eight canonical FeS clusters to its ubiquinone binding site located at the Q module. The transferred electrons are used to reduce ubiquinone (Q) to ubiquinol

(QH2). This electron transfer energizes the translocation of protons through the P module.

Assembly The complexity of Complex I has made the study of assembly rather challenging. Recent studies using high content proteomics and cryo- electron microscopy (cryo-EM) have revealed different complex organization and assembly states [41, 42]. Currently, there are approximately 15 assembly factors known that are involved in Complex I formation and maturation. A Complex I assembly pathway was proposed with a step-wise ap- pearance of pre-complexes with distinct compositions of subunits as well as assembly factors. By employing systematic gene deletion of Complex I accessory subunits, six distinct assembly modules could be identified [43]. Additionally, newly synthesized mitochondrial encoded Complex I subunits assemble into a number of distinct membrane arm intermediates and require significant chase times for their integration into the hol-

9 oenzyme. In contrast, nuclear encoded subunits interact with pre-existing Complex I subunits to form intermediates to assemble into holoen- zyme [44, 45]. Analyses of different Complex I mutations indicated that both matrix and membrane arm subunits are already found together in early‐stage intermediates of Complex I [44].

10 NADH dehydrogenases in yeast A particularity of the respiratory chain in the baker’s yeast S. cerevisiae is that it lacks Complex I. Therefore, it compensates the missing NADH oxidation by employing Ndi1, Nde1 and Nde2, three NADH dehydrogenases (alternative or NDH-2-type NADH dehydrogenases). These proteins are located in the periphery of the IMM and contain FAD cofactors in their active site [46]. Like Complex I, NADH dehydrogenases catalyze the transfer of electrons from NADH to the electron carrier ubiquinone, but unlike Complex I, they do not translocate protons across the IMM and do not participate in the formation of an electrochemical gradient. NADH is produced in the cytosol as well as the matrix, but the IMM is not permeable for these charged and large molecules. To maintain the redox balance, one NADH dehydrogenase, called internal NADH dehydrogenase (Ndi1), is facing the matrix [47], while the other two NADH dehydrogenases, called external NADH dehydrogenase (Nde1 and Nde2), face the IMS [36]. Combined with Complex II, these NADH dehydrogenases are the entrance site for electrons to the respiratory chain.

Fig 3: NADH dehydrogenases in yeast. Schematic illustration of the yeast mitochondrial NADH dehydrogenases. They catalyze the electron transfer of NADH to ubiquinol (QH2) with the help of its bound cofactor FAD. As NADH cannot pass through the IMM yeast employs one “internal” enzyme facing the matrix (Ndi1) and two “external” NDH dehydrogenases facing towards the IMS (Nde1 and Nde2).

11 Complex II In most eukaryotes, succinate dehydrogenase, or Complex II, is the only respiratory chain complex that does not participate in the transfer of protons across the IMM. However, it is able to channel reducing equivalents into the respiratory chain by removing electrons from succinate and transfer them to ubiquinone. This complex thereby directly links the TCA cycle to the respiratory chain. Another special feature of Complex II, compared to the other respiratory chain complexes, is that all its subunits are nuclear encoded and imported independently into mitochondria via the TOM/TIM machinery. Complex II is embedded in the IMM and has a large extension into the matrix. The enzyme contains three FeS clusters, a covalently attached flavin cofactor in the peripheral part, and a heme b embedded in the IMM. Despite its somewhat simple structure, Complex II requires several assembly factors for its biogenesis [48]. Structure and function Succinate dehydrogenase is a heterotetrameric complex composed of four subunits. Sdh1 and Sdh2 form the hydrophilic part of Complex II while Sdh3 and Sdh4 form the hydrophobic anchor (Fig 4). While the hydrophilic domain represents the catalytic core, the hydrophobic anchor serves as the ubiquinone binding site [49, 50]. Sdh1 is the catalytic subunit and facilitates the oxidation from succinate to fumarate. It contains a covalently bound FAD cofactor next to its succinate-binding site. Sdh2 harbors three FeS centers which serve essentially as a wire, transferring electrons through the complex from the FAD cofactor to ubiquinone. In addition to its important role in this process, Sdh2 also serves as the interface between the membrane anchor and Sdh1 [49, 50]. The membrane anchor domain harbors two potential ubiquinone binding sites. One is called the high affinity site (QP-proximal), which is located closer to the matrix side of the IMM. This is the dominant ubiquinone binding site. The other binding side is closer located to the IMS side of the IMM and is called lower affinity site (QD- distal) [51]. Until now, it is not clear why Complex II possesses two binding sites for ubiquinone. Additionally, the functional significance of the highly

12 conserved heme b is not fully understood [52, 53]. Since heme b is located between the two ubiquinone binding sites, it was speculated that it might transfer electrons between these two sites [52]. Another pro- posal is that heme b oxidizes ubisemiquinone to avoid ROS formation [50].

Fig 4: Succinate dehydrogenase. Left the structural overview of Complex II (PDB: 1ZOY) [49]. In pink and lilac the membrane anchor formed by Sdh3 and Shd4 and in blue the hydrophilic, catalytic active part formed by Sdh1 and Sdh2. On the right a schematic illustration of the succinate dehydrogenases and all its cofactors. Complex II catalyzes the oxidation of succinate to fumarate. Thereby it translocates an electron from a bound FAD via three FeS clusters to the ubiquinone (Q) binding site where ubiquinol (QH2) is produced. Assembly The first step in the assembly of Complex II is the maturation of Sdh1. It is imported as an apo-protein into the mitochondrial matrix. There it binds to Sdh5, a specific chaperone (assembly factor) that assists with the insertion and covalent attachment of its FAD cofactor [48, 54]. Interestingly, if Sdh5 is missing Complex II still assembles, but in an inactive complex. After flavinylation, the Sdh1-Sdh5 dimer dissociates, releasing the mature Sdh1, which binds to another assembly factor, Sdh8. This pre- complex formation prevents premature enzymatic reactions that would result in ROS production, and additionally supports the formation of the Sdh1-Sdh2 dimer [48].

13 Meanwhile, apo-Sdh2 also needs cofactor insertion. This process involves the insertion of three FeS clusters generated by the ISU and ISA complexes [48]. After insertion Sdh2 interacts with the assembly factors Sdh6 and Sdh7, which stabilize and protect exposed FeS clusters during the assembly process and assists furthermore with the association to the mature Sdh1 subunit, forming a heterotetrameric assembly intermediate. Finally, the Sdh1-Sdh2 hydrophilic head docks to the IMM via interactions with the Sdh3-Sdh4 membrane anchor domain, which may or may not preassemble at the IMM and the assembly factors are released. The assembly of the hydrophobic anchor remains more elusive. Sdh3 and Sdh4 are imported through the TOM complex, subsequently transferred to the TIM23 complex and laterally released to the inner membrane, like other α-helical transmembrane IMM proteins [55]. How- ever, it is unclear if chaperones are needed for Sdh3 and Sdh4 folding and dimerization. Nevertheless, it appears that the biogenesis of the hydrophobic anchor is dependent on the rest of the assembly process, as the membrane anchor is instable when Sdh1 and Sdh2 are missing. Per- haps the most intriguing question regarding the hydrophobic anchor is its cofactor: heme b. Regardless of its precise function, this cofactor is present across all species, suggesting it to be important for either electron transfer or complex stability. However, it is not yet known how it is assembled into Sdh3 and Sdh4.

14 Complex III

Ubiquinol-cytochrome c oxidoreductase, or Complex III, is the third complex within the respiratory chain that I referred to as the bc1 complex. This complex is an obligate homodimeric, integral multi-subunit enzyme that catalyzes the transfer of electrons from the reducing equivalent ubiquinol to the soluble electron shuttle cytochrome c (Fig 5) [56]. Additionally, the electron transfer energizes the translocation of protons across the IMM, thereby contributing to the electrochemical gradient, used for the synthesis of ATP by the mitochondrial F1-FO ATP synthase [57].

In S. cerevisiae, the bc1 complex has a molecular weight of around 670 kDa and exists only in a dimeric form [58]. Each monomer contains ten different subunits [59, 60]. Only one subunit, namely cytochrome b (yeast: Cytb/human: MTCYTB) is encoded by the mitochondrial DNA, whereas the other nine proteins are nuclear encoded. Cytb forms the central core of the bc1 complex and together with the Rieske iron-sulfur protein 1 (yeast: Rip1/human: UQCRFS1) and cytochrome c1 (yeast: Cyt1/ human: CYC1 or UQCR4) represents the catalytical core and redox center of this multi subunit complex. The other seven subunits are not directly involved in the catalytic function of the bc1 complex, but are needed for its stability and function. Only two of the supernumerical subunits, namely Qcr6 and Qcr10 are not required for activity. The bc1 complex couples electron transfer to proton shuttling by a mechanism known as the Q-cycle [61].

Structure and function of the subunits

The dimeric bc1 complex consists of two identical monomers that contain 10 subunits [62]. The majority of these subunits are accessory proteins and nine out of ten subunits are encoded in the nucleus and imported via the TOM/TIM machinery. Additional insight into the architecture and function of this enzyme have been made by recent structural analysis and high resolution cryo-EM [56, 62, 63]. The later study revealed that in yeast 12 phospholipid molecules, including four phos- phatidylcholines, four phosphatidylethanolamines, two cardiolipins,

15 and two 1,2-diacyl-sn-glycero-3-phoshocholines, are part of the dimeric bc1 complex. The suspicions arises that this membrane composition plays an important role in the complex architecture [63].

The catalytical subunits

The three catalytical subunits of the bc1 complex form the redox center of the complex. They catalyze electron transfer through the complex by channeling the electrons from ubiquinol through the two heme b cofactors, cytochrome c1 and the FeS-cluster to the soluble electron shuttle cytochrome c [64].

Cytochrome b This subunit is highly conserved throughout all kingdoms, and in eukaryotes this is the only subunit of the bc1 complex encoded within the mitochondrial genome. It forms the catalytical core of the dimeric complex. Cytb is a highly hydrophobic protein with eight transmembrane (TM) helices embedded in the IMM, which are co-translationally inserted with the help of the Oxa1/Mba1/Mrx15 machinery. Furthermore, this subunit harbors two redox centers: the high potential heme b562 (bH) and the low potential heme b565 (bL), which are located on distant positions in the membrane. Heme bL is located close to the IMS to accept electrons from ubiquinol at the Qo site, and heme bH is positioned closer to the matrix side of the enzyme, where it transfers electrons to ubiquinone or semiubiquinone at the Qi site [65, 66]. The two hemes are not covalently attached to Cytb, but rather coordinated by histidine residues in a four-helix bundle [59].

Cytochrome c1 This catalytic subunit is encoded in the nucleus, its precursor is imported into the mitochondrial matrix and processed once. After cofactor insertion, Cyt1 is processed a second time and joins the bc1 complex [67-69]. Cyt1 has one transmembrane domain that anchors the protein in the IMM and a globular, N-terminal domain within the IMS harbor- ing its redox center, a covalently attached heme c1. Cyt1 employs its

16 cofactor to accept electrons from Rip1 and to transfer them further to the soluble electron carrier cytochrome c.

Rieske iron-sulfur protein 1 This is the last catalytic subunit of the bc1 complex. Rip1 has a sim- ilar topology like Cyt1, with a mobile globular domain in the IMS har- boring the catalytic site, and a hydrophobic α helix bound to the IMM [59]. It is nuclear encoded and imported into the mitochondrial matrix, where it receives its cofactor, an FeS cluster. Then it is inserted into the IMM and its globular domain is translocated to the IMS [70-73]. Rip1 cycles between two conformational states to accept electrons from the ubiquinone bound in the Qo site and to transfer them to Cyt1 [74]. Important for this mechanism to work is the high flexibility of the IMS domain of Rip1 [28, 63, 75].

Interestingly, cytochrome c interacts with the homodimeric bc1 complex monovalently. Its binding induces a conformational change of the Rip1 head domain and subunit Qcr6 in one of the monomers, giving the dimer an asymmetric form [56, 74]. Furthermore, Rip1 physically cross-links the two functional monomers of the intertwined dimer and probably act as a regulatory element in the mechanistic alternating site model, with only one monomer of the bc1 complex active at a time [56, 62]. The supernumerary or accessory subunits

In addition to the three catalytic subunits, the bc1 complex contains seven nuclear encoded, structural subunits, which include Cor1, Cor2, Qcr6, Qcr7, Qcr8, Qcr9 and Qcr10. These proteins are not directly involved in the catalysis, neither in the electron transfer, nor in the proton translocation [76]. But all accessory subunits, except for Qcr6 and Qcr10, are essential for to maintain the complex functional. Cor1 and Cor2 are soluble matrix proteins and the two largest subunits of the bc1 complex [77]. Therefore, they were mistaken to form the core of the complex and were named core proteins. Cor1 is attached to the IMM, but mostly is exposed to the mitochondrial matrix, where it interacts with Cor2 and Qcr7. Additionally, Cor1 is part of the interaction surface with Complex IV to form supercomplexes [63, 78]. Within

17 the dimeric complex, Cor1 and Cor2 form a tetramer located in the matrix, where Cor2 forms an interaction surface within the dimeric structure [79, 80]. Cor2 is through Cor1 and Qcr7 connected to the complex [59, 63, 78]. Qcr6, a soluble protein located in the IMS, is an acidic subunit that stabilizes Cyt1 association with the complex [81-83]. Additionally, it assists cytochrome c binding to the homodimeric bc1 complex [56]. The protein is not essential and is only loosely associated with the complex. Indeed, qcr6 deletion mutants in yeast show only a marginal growth deficiency on a non-fermentable substrate [84]. Qcr7 is a small peripheral membrane protein at the matrix site of the IMM. It interacts with Cytb and is involved in stabilizing the early assembly intermediate II [85, 86]. Additionally, it interacts with Cor1 and helps connecting the two large matrix proteins to the membrane. Qcr8, Qcr9 and Qcr10 are small proteins that cross the IMM with a single hydrophobic α-helix and are located close to Cytb [87-90]While Qcr8 is assembled early onto the cytochrome b to stabilize it, Qcr9 is inserted in the later assembly steps, presumably stabilizing Rip1 within the complex [91, 92]. The structure and peripheral binding of Qcr10 could only be recently resolved in cryo-EM structure studies, as this subunit is only loosely attached to the bc1 complex [63, 78]. In the matrix, the extended N- terminus of Qcr10 interacts with Qcr7 of the other monomer, while in the IMS it interacts with Cyt1 [78]. Other studies also indicate that Qcr10 is interacting with Rip1 to hold its catalytic subunit in place [87, 93].

Fig 5: The bc1 complex. On the left side the structure of the yeast Complex III (PDB: 6GIQ) is shown [63]. The three catalytic subunits are colored. Red: cytochrome b

(Cytb); Orange: cytochrome c1 (Cyt1); Yellow: Rieske FeS cluster protein 1 (Rip1). The supernumerary subunits are shown in grey. On the right side a schematic depiction of the bc1 complex with all its redox factors is illustrated. The two electron carrier ubiquinol (QH2) is oxidized at the Qo site. One of the electrons is transferred via the

FeS cluster to heme c1 to the one electron carrier cytochrome c, which is located in the IMS. The other electron is channeled via heme bL and heme bH to the Qi site, were a ubiquinone (Q) gets reduced to ubisemiquinone. This bifurcated pathway is repeated twice so that the ubisemiquinone, which sits at the Qi site, gets fully reduced. This energy transfer powers the uptake of protons (H+) at the matrix site and their release at the IMS site, establishing an electrochemical gradient. Catalytic cycle- The Q cycle

The bc1 complex catalyzes the transfer of electrons from the two electron carrier ubiquinol to the one electron carrier cytochrome c by an elaborate process called the Q-cycle. This sophisticated mechanism employs a bifurcated pathway [61, 64]. The ubiquinol binds at the Qo site and one electron is transferred to Rip1. This electron is channeled through Cyt1 and subsequently transferred to the soluble cytochrome c (Fig 5, right panel). During electron transfer, the flexible globular domain of Rip1 undergoes a conformational shift [63, 74], moving towards the Qo site when

19 ubiquinol is binding, participating in the actual binding site. Next, the domain rotates to Cyt1 to transfer the electron. This conformational change of Rip1 is thought to be the rate-limiting step according to kinetic studies [80].

As only one electron has left, Qo site contains an ubisemiquinone that needs to be further oxidized to ubiquinone. Therefore, the second electron is transferred to the low potential heme bL, which channels the electron further to the high potential heme bH, and finally to the Qi site, where a ubiquinone accepts the electron to form ubisemiquinone. To reduce the formed ubisemiquinone in the Qi site this cycle needs to be repeated.

In the two cycles, necessary to fully generate ubiquinol at the Qi site, four protons are released to the IMS at the Qo site, while two protons are taken up from the matrix at the Qi site. Additionally, two cytochrome c molecules are reduced in the IMS [78].

As the main focus of my thesis lies on the assembly of the bc1 complex, I will describe the biogenesis in more detail in a dedicated chapter.

20 Complex IV Cytochrome c oxidase, or Complex IV, is a large transmembrane spanning enzyme composed of 12 subunits in yeast and concludes the respiratory electron transport chain. Complex IV receives electrons from the soluble electron shuttle cytochrome c, and transfers them to molecular oxygen converting it to two molecules of water. In this process, four cytochrome c molecules are oxidized, one dioxygen gets reduced, and by binding to four protons from the matrix, two water molecules are generated and released. During that process, another four protons get translocated, increasing the electrochemical gradient across the IMM (Fig 6). Complex IV has two catalytical core subunits, Cox1 and Cox2, and in most cases, these subunits together with Cox3, are encoded by the mitochondrial DNA. These subunits are highly conserved throughout the all kingdoms. The remaining structural subunits are encoded by nuclear genes, but the number of these varies between different organisms. These subunits are not involved in the function of the complex, but most of them are needed during assembly and for the stability of the complex [28, 94]. Structure and function The catalytic core is found in subunit Cox1, encoded in the mitochondrial DNA, which is a highly hydrophobic protein with twelve transmembrane spanning -helices. Furthermore, three out of the four redox active sites of the complex can be found within this subunit, heme a, heme a3 and CuB. The latter two form the active site, where oxygen binds and is reduced to water. Cox1 also harbors a conserved internal ring structure formed by five amino acids, which is thought to play an important role in the proper localization of the incoming oxygen [78, 95, 96]. Additionally, Cox1 contains the three proton translocation sites [63, 97, 98]. Cox2, the second mitochondrial encoded, catalytical core subunit has two TM helices, as well as a globular domain facing towards the IMS. This soluble domain harbors the fourth prosthetic group of the complex, a bimetallic CuA.

21 Cox3, the last mitochondrial encoded subunit is also a highly hydrophobic protein with seven transmembrane spanning domains. As it does not contain any redox cofactors, it is neither directly involved in the electron channeling, nor does it take part in the proton pumping. Its role is primarily to stabilize the two other catalytic subunits, Cox1 and Cox2, thereby prevent suicide inactivation of cytochrome c oxidase [99]. Fur- thermore, Cox3 is participating in the regulation of proton transfer [100]. Complex IV receives electrons from reduced cytochrome c and transfers them to the bimetallic CuA. This in turn channels them further to heme a and finally to the active site consisting of the binuclear center heme a3 and CuB. At the active site, molecular oxygen accepts the electrons and is reduced to water. During this reaction, four electrons are transferred and four protons are pumped to the IMS, while four additional protons (chemical protons) are utilized to form water. There are three proton channels in cytochrome c oxidase, but all four pumped protons take the same route. The other two channels transport the chemical protons that are needed for the formation of water [97].

Fig 6: Cytochrome c oxidase. On the left the structure of the yeast Complex IV (PDB: 6GIQ) [63]. In color the three catalytic and mitochondrial subunits. Green: Cox1; aq- uamarine: Cox2; light blue: Cox3. The structural subunits are shown in grey. On the

22 right side a schematic representation of the cytochrome c oxidase with all its redox factors is shown.

The reduced cytochrome c gets oxidized at the CuA site and the electron gets translocated to the catalytic site formed by heme a3 and CuB. The transfer is mediated via heme a. At the catalytic site, oxygen is reduced to water, which requires chemical protons. Additional to these protons the electron transfer energizes the translocation of protons across the IMM. In one cycle, four electrons are transferred, four protons are pumped into the IMS and four chemical protons are used for the production of two molecules of water.

Assembly

The assembly of the cytochrome c oxidase is a rather complex process and requires assistance at all stages beginning with the translation regulation of the mitochondrial encoded subunits via so called translational activators, over the insertion of these highly hydrophobic components, trough the maturation of the catalytical subunits, until the final joining of the different assembly modules and fine tuning of the complex [28, 101]. More than 30 assembly factors have been described for this complex and the list is continuously growing [102]. Additionally, cytochrome c oxidase assembly is partly modular, following the maturation of the three mitochondrial encoded subunits.

Cox1 contains two non-covalently bound heme a moieties (a and a3). They are synthesized from heme b, which is converted with the help of Cox10, a farnesyl transferase [103] and the Yah1-Cox15 complex, a monooxygenase [101, 104] to heme a. The mechanism of heme insertion into Cox1 remains elusive, however it depends on Cox10-Cox15 interaction [105-107] and additional assembly factors such as Coa2, Shy1 and Pet117 [108-110]. Copper is the other cofactor for Cox1. Copper does not reside freely in yeast and is therefore in complex with the copper chaperone Cox17. It delivers its copper to Cox11, which is an assembly factor involved in copper insertion into the Cox1 CuB site [111-113]. Also other soluble factors such as Cmc1, Cox19, and Cox23 are required for the maturation of the Cox1 CuB center [105, 114-118].

23 Cox1 is cotranslationally inserted into the IMM by the Oxa1/Mba1/Mrx15 insertase machinery [119-122]. After insertion, it is bound to the assembly factors Coa1, Coa3 and Cox14, forming an early assembly intermediate [94, 123-127]. This pre-complex then recruits Mss51, an assembly factor and translational activator of Cox1, as well as the first structural subunit Cox5. If bound to this complex, Mss51 is sequestered away from COX1 mRNA and is thereby unavailable to in- itialize a new round of translation. The maturing Cox1 sub-complex then recruits the structural subunits Cox6 and Cox8 forming the next assembly intermediate [94]. The succeeding complex intermediate is formed by the addition of Shy1, an assembly factor that has been implicated in heme insertion [125]. Shy1 is a multi TM spanning protein and is most likely one of the most fundamental proteins required for the assembly [128]. Cox2, the catalytical subunit with a CuA site and two TM domains, is the only mitochondrial encoded subunit that requires proteolytic processing before incorporation. The first 15 amino acids need to be cleaved off by Imp1 before association with Cox2 specific assembly factors occur [129, 130]. Cox20 as well as Cox18 assist with the precursor Cox2 maturation.

The binuclear CuA center formation depends on Sco1, a chopper chaperone that transfer its copper to the CuA site [131, 132]. Coa6 is an additional factor that is involved in the maturation of the CuA center [133, 134]. The structural subunit Cox16 seems to play a role in joining the Cox1 and Cox2 assembly modules [135-137]). Furthermore it is speculated, that the structural subunits, Cox9 and Cox12, associate with the Cox2 assembly module, based on structural studies, but its association with any assembly modules has not been observed [134, 138]. The last mitochondrial subunit, Cox3 associates with the structural subunits found in the final assembled enzyme (Cox4, Cox7, and Cox13) as well as Rcf1, a protein involved in supercomplex formation with complex III [138-141].

24 Supercomplexes The organization of the OXPHOS system was long strongly debated and different models [142-144] were discussed, but now the plasticity model is well established. It was already mentioned 1955 [145] that respiratory complexes could assemble into larger complexes, but only in 2000 when it was possible to demonstrated that the single respiratory complexes could be purified individually as well as in macromolecular supercomplexes, the plasticity model was widely accepted [146, 147]. The structures found in this way were named supercomplexes or respirasomes. In the plasticity model a dynamic equilibrium between the individual complexes and the supercomplexes was proposed, which depends on different physiological conditions and cellular demands [147-150]. The plasticity model is supported by structural as well as kinetic evidence [147, 151, 152]. It was proven that supercomplexes are functionally active and their formation is stable. The supercomplex assembly itself appears to be more dynamic and respiratory enzymes are able to alternate between participating in large supercomplexes and existing in an independent state [153]. It is not known what triggers changes in complex assembly, but it was revealed that the formation of supercomplexes is heavily dependent upon the lipid composition of the mitochondrial membrane. In particular cardiolipin, a unique mitochondrial lipid, is needed for stable supercomplex assembly [154-156]. While in yeast the supercomplexes are composed of one copy of the dimeric bc1 complex and either one or two copies of Complex IV, in mammals the supercomplex formations are more divers, also including Complex I [157-160].

Functional significance

Even if the existence of the supercomplexes is nowadays widely accepted, their functional significance is still under debate. It has been hypothesized that the organization of respiratory enzymes into supercomplexes allows a cross talk between the complexes that could reduce

25 oxidative damage (ROS production) and enhances metabolic efficiency for example by an improved electron flux. Additionally, the supercomplex formation could be important for the stability of the single respiratory complex, and it was also postulated that they could play a role in the regulation and modulation of respiratory chain activity [161-163].

26 Dual genetic origin Due to the endosymbiotic origin of the mitochondria, it has retained its own genome. As a result, mitochondria also harbor their own repli- cation, transcription and translation machinery. The genes that re- mained in the mitochondrial genome, encode mainly highly hydrophobic membrane proteins that play a key role in the respiratory chain complex and the ATP synthase. In S. cerevisiae, seven subunits of the OXPHOS system (Cytb, Cox1, Cox2, Cox3, Atp6, Atp8 and Atp9), one ribosomal protein (Var1), as well as several core components of the mitochondrial protein expression machinery, including rRNAs, tRNAs and a subunit of RNase P, are encoded in the mitochondrial genome [164]. In contrast, mammalian mitochondria encodes exclusively subunits of the OXPHOS system, but also a full set of tRNAs as well as the rRNAs needed for the mitoribosome [165]. Assessments of the different gene content of various mitochondrial genomes suggest that the gene transfer to the nucleus was not a random process, but occurred rather specific and in a gradual manner. Interest- ingly, there are only 4 genes that are universally encoded by mitochondria, namely the two rRNAs, COB and COX1 [166]. The size of mtDNAs is rather heterogeneous and varies manly between 16 and 80 kb between man and yeast [8]. However, also genomes with sizes over 100 kb are known, but mainly in plants [167]. The extreme differences in size depends mainly on a varying amount of non- coding regions. For instance, the mammalian mtDNA is rather small (16,5 kb), and the genes are quite packed only separated by tRNA genes [168]. However, genes encoded by the mitochondrial genome of S. cerevisiae (75 kb) are separated by long non-coding stretches. Addi- tionally, some coding sequences are interrupted by introns that in some cases code for maturases involved in the processing of intron-containing transcripts [169, 170]. Due to the lighter packing the mtDNA of yeast is considerably larger than its mammalian counterpart, even if lesser genes are encoded.

27 Why mitochondria keep their own genome A consequence of the gene transfer to the cell nucleus is that mitochondria encode merely for 1% of their own proteins [171, 172]. Sub- sequently, most of the mitochondrial proteins are synthesized in the cytosol and post-translationally imported into the organelle. A remarkable fraction of those proteins is required for the expression of only a handful of proteins encoded by mitochondria in yeast [171, 173]. It has long been speculated why mitochondria make such a huge effort to maintain and express an additional genome. 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. 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 [174-176]. Furthermore, the extreme hy- drophobicity of some proteins or the size of some molecules might prevent their efficient import from the cytosol into mitochondria.

28 Mitochondrial translation Our main understanding about the ribosomal function comes from bacterial studies and it was assumed for a long time that the organellar machinery resembles the prokaryotic, as they derived from a bacterial cell. However, structural analysis of mitoribosomes from different species revealed dramatic changes of the translation machinery in course of organellar evolution [173, 177]. The most pronounced difference is the decrease in the rRNA-protein ratio from bacteria (2:1) over yeast (1:1) to mammalian (1:2) (mito)ribosomes [177]. This ratio change increases the importance of protein- protein interactions in the mitoribosome [178]. Consistent with this observation is that many mitochondrial ribosomal proteins (MRPs) are ex- tensively larger than their bacterial homologs. Nevertheless, mitoribosomal proteins with bacterial homologs are located at conserved sites such as the peptidyltransferase center or accuracy center, indicating that the general mechanism of protein synthesis is highly conserved. Interestingly, instead of substituting for the lost rRNA, most of the new structural proteins of the mitoribosome are located on the surface, giving it a diverged overall shape. A supposed role of these additional proteins could be to regulate and organize organellar gene expression, for instance by recruiting essential factors needed for mitochondrial encoded gene expression. Alternatively, mitochondria-specific riboproteins could be involved in mRNA aligning onto the ribosome since a bacterial-like Shine-Dal- garno sequence is missing in the mitochondrial mRNAs. The general mechanism of translation as well as the involved translation factors are well conserved from bacteria to mitochondria [177]. However, the organelle established some additional specific features in order to compensate for its unusual mRNAs, and optimizing protein biogenesis of the respiratory chain subunits. As mentioned earlier, 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 [179]. Additionally, mitochondrial mRNAs have at least one additional

29 AUG upstream of their initiation site. This indicates that translation initiation in mitochondria is accomplished by another mechanism, but how remains elusive. However, even if the precise mechanism of start codon recognition is unknown, a clearly stringent start site selection in mammalian [180] as well as yeast mitochondria [181] is present. Translational control in mitochondria One specific feature of yeast mitochondrial protein synthesis is the employment of nuclear encoded gene-specific proteins, called translational activators. These regulatory proteins bind specifically to the 5’-UTRs of mitochondrial mRNAs demonstrating clearly that this region of the mRNAs is crucial for translation activation [182]. For all mitochondrial encoded genes with exception of ATP8 and VAR1, one or more translation activators have been identified [177]. The definite role of these regulatory proteins is still under debate and ranges from its involvement in start site localization, over mRNA stabilization to regulation and organization of protein biosynthesis. The knowledge about the mammalian mitochondrial translation initiation and regulation is still lacking, as mammalian mitochondrial mRNAs lack 5’-UTRs it is very 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.

30 Synthesis of Cytochrome b As my work mainly focuses on the biogenesis and assembly of the bc1 complex in yeast, I will describe the synthesis of Cytb, the only subunit of the bc1 complex encoded in the mitochondria, in more detail in the following section. Cytb expression depends on several nuclear and mitochondrial encoded genes either required for translation or processing and maturation of the transcript. Cytb is co-transcribed with tRNAE, resulting in a bi- cistronic, intron-containing pre-transcript that undergoes multiple maturation steps. tRNAE is dissociated by the action of RNase P and auxil- iary proteins [183-185]. After the pre-mRNA cleavage, catalyzed by Pet127, binding of Cbp1 occurs, which protects the COB mRNA from further degradation [185, 186]. Cells lacking CBP1 have strongly reduced levels of the COB precursor transcript and fail completely to accumulate the mature mRNA [187-189]. Cbp1 specifically acts at the 5’- end of the COB mRNA. Besides its stabilizing function, Cbp1 might also play a role in co-translational insertion of Cytb into the inner membrane [190]. Further maturation of the COB transcript requires several factors involved in the excision of introns. Three of the Cytb introns encode for maturases required for self-splicing or processing of another transcripts and are in frame with the upstream exon [191]. One of the mentioned introns encodes a maturase required for proper COX1 splicing [192]. Therefore mutants affected in COB translation often exhibit an additional defect in Cox1 synthesis. Additionally COB precursor RNA is processed by Mrs1 and Mss116, nuclear encoded proteins required for COB intron removal, however they are not exclusive for COB maturation. Mss116 is an RNA chaperone that belongs to the DEAD-box protein family, and it is required for efficient splicing of mitochondrial transcripts [193-195]. Furthermore, the translation of COB is controlled by the three translation activators Cbs1, Cbs2 and the Cbp3-Cbp6 complex.Cbs1 and Cbs2 interact with the 5′ end of COB mRNA as well as with the mitoribosome itself [196-199]. Cells lacking Cbs1 or Cbs2 fail to synthesize apo-cytochrome b and accumulate intron-containing precursor forms

31 [200-202]. This argues for a role of both proteins in splicing of the COB precursor and its translation activation. However, they are not primarily and directly involved in splicing as their presence was still needed for synthesis of cytochrome b in a strain lacking introns [202]. Further- more, it was shown that the COB transcript can be spliced even in the absence of Cbs1 and Cbs2 [200, 201]. Therefore the accumulation of intron-containing precursor forms of the COB mRNA seems to be a secondary effect. Besides Cbs1 and Cbs2 a third translational activator was described, Cbp3-Cbp6. These two proteins form a stable complex, which is involved in the synthesis and assembly of Cytb. Furthermore, stability and function of these two proteins dependent on each other [203-205]. As observed for cbs1 and cbs2 mutant strains, the absence of Cbp3- Cbp6 impairs the accumulation of Cytb, resulting in a respiratory defi- cient phenotype. Unlike the absence of the other two translational activators a deletion of CBP3or CBP6 result in normal levels of mature COB mRNA [203]. Importantly, a replacement of the COB leader cannot rescue the cbp3 or cbp6 phenotype as seen for Cbs1 and Cbs2 [206]. This observation argues for a dual function of Cbp3-Cbp6 in the biogenesis of Cytb and suggests a different mode of action. Cbp3-Cbp6 shows interaction sites with proteins at the mitoribosomal tunnel exit and the nascent polypeptide chain and not with the mRNA [68, 207- 209]. Cbp3- Cbp6 are not only translational activators of Cytb, but they additionally assist in the early assembly of the bc1 complex. Therefore a mechanism was proposed in which these two proteins are involved in a feedback loop required for the coordination of Cytb synthesis and assembly [203, 204]. In this feedback loop the Cbp3-Cbp6 complex associates with newly synthesized protein stabilizing it and assist the early steps of bc1 complex assembly [204]. Upon early failure in bc1 biogenesis, Cbp3-Cbp6 gets sequestered into the early assembly intermediate and is not available to start a new round of COB translation.

32 Complex III assembly

The bc1 complex is a multi-subunit transmembrane spanning enzyme, with subunits originating from two different genetic origins. Fur- thermore, three out of the then subunits in yeast contain redox centers needed for the catalytical function of the enzyme. The remaining subunits are not directly involved in catalysis and their relevance for the complex is largely unknown. Interestingly, bacterial Complex III is exclusively composed of the catalytic core subunits and lacks supernumerary subunits completely. Therefore it is not surprising that the assembly of the bc1 complex needs to be regulated and requires assistance from different assembly factors.

Assembly factors of the bc1 complex

Like the other complexes of the respiratory chain has the bc1 complex numerous assembly factors that are required for the correct formation of the enzyme. They are involved in the assembly, in the maturation of the catalytic subunits, as well as the proper insertion and topogenesis of the subunits [28, 67, 68]. Maturation of the catalytic subunits

Cytb is cotranslational inserted into the IMM with the help of the Oxa1/Mba1/Mrx15 insertion machinery. As mentioned earlier, the Cbp3-Cbp6 complex is not only involved in the translation of the COB mRNA but also in its early assembly [204]. After synthesis of Cytb, Cbp3-Cbp6, which binds to the tunnel exit of the ribosome and already interacts with the nascent polypeptide chain, dissociates and binds to the apo-cytochrome b stabilizing it [208]. This chaperone function of Cbp3-Cbp6 is a crucial step for Cytb maturation, as cbp3 or cbp6 cells are still able to translate COB mRNA at a reduced rate, but the protein is rapidly degraded [205]. Additionally, it was shown recently in another yeast wild type that Cbp3-Cbp6 are not required for translation but are indispensable for its maturation and stability [210]. These findings indicate that additional proteins could be involved in the translational regulation and feedback loop of Cytb. Recently, human

33 orthologues of Cbp3 and Cbp6 were found, UQCC1 and UQCC2 respectively. Like in yeast they bind to newly synthesized Cytb and stabilize it. Additionally, UQCC1 is required for UQCC2 function and vice versa [211]. Cbp4 is the next assembly factor that is required for the biogenesis of Cytb. This assembly factor has a transmembrane domain and a globular domain facing towards the IMS. Cbp4 stabilizes Cytb further and after its association to Cytb, the first supernumerary subunits bind and the Cbp3-Cbp6 complex dissociates and can induce a new round of COB mRNA translation. Cytb has two heme b moieties that need to be inserted for its maturation. These two heme groups are not covalently bound but rather coordinated by histidine moieties within a four helices bundle. The specific process of the hemylation of Cytb is investigated in paper I.

Fig 7: Maturation and insertion of cytochrome b. The newly synthesized cytochrome b dissociates together with Cbp3-Cbp6 from the ribosome. The next assembly factor Cbp4 binds and stabilizes this early pre-complex, named assembly intermediate I. Cbp3-Cbp6 dissociate and can induce a new round of translation via a feedback loop. After Cbp3-Cbp6 release the structural subunits Qcr7 and Qcr8 are added, forming assembly intermediate II. The precise reaction that triggers Cbp3-Cbp6 release is not yet understood. During these early assembly steps the heme b groups are incorporated.

Cyt1 is the second catalytical subunit that needs insertion of redox cofactors. The insertion of the covalently attached heme c1 into Cyt1 is

34 also dependent on regulatory proteins. As mentioned earlier Cyt1 possesses a bipartite signal sequence. The first part is cleaved by the mitochondrial processing peptidase (MPP) in the matrix after successful translocation. The second cleavage event is performed by Imp2, after the covalent insertion of heme c1 [212, 213]. The maturation of Cyt1 needs the assistance of Cyc2, a heme lyase. It catalyzes the covalent attachment of a heme b to apo-cytochrome c1 [213]. Cyc3, the heme lyase for the soluble cytochrome c electron carrier, is under special conditions also able to mature Cyt1 [214]. The human counterpart of Cyc2 is called HCCS and catalyzes in essence the same reaction, attachment of a heme group to the apo-protein.

Fig 8: Maturation of cytochrome c1. The apo-cytochrome c1 is imported and the N- terminal transmembrane segment is incorporated into the IMM. The mitochondrial processing peptidase complex (MPP) cleaves the matrix exposed part of the MTS, which is the first processing step. This triggers the insertion of the C-terminal TM domain. Subsequently, the heme ligase or holocytochrome c1 synthetase, Cyt2, mediates the covalent attachment of a heme b to the apo-cytochrome c1, forming a heme c in the catalytic site. Cofactor insertion causes a conformational changes that exposes a second cleavage site that is recognized by Imp2. The cleavage of the second signal peptide matures cytochrome c1 and its assembly into the bc1 complex can be completed

Last but not least Rip 1, the final catalytical subunit of the bc1 complex is inserted. This subunit harbors an iron-sulfur cluster in its globular IMS domain and two assembly factors, Bcs1 and Mzm1, are necessary for its maturation and insertion into the complex. Bcs1 binds to the bc1 pre-complex and mediates Rip1 insertion in an ATP-dependent manner. A hypothesis was proposed in which the availability of ATP is not only required but might also exhibit a regulatory mechanism that

35 allows a coupling of bc1 complex biogenesis with the energetic state of mitochondria [71]. Mzm1 resides in the matrix and stabilizes Rip1. It assists in the incorporation of the FeS cluster and shields the mature Rip1 from aggregation as well as degradation prior to Bcs1 association [70, 71]. Mzm1 has also a human counterpart, LYRM7 which is required for the stabilization of the human Rip1 (UQCRFS1) [215]. The second assembly factor of Rip1, Bcs1 is an AAA ATPase which mediates the translocation of the mature catalytical subunit. Thereby the globular domain of Rip1 with its bound FeS cluster is translocated to the IMS while the transmembrane domain is inserted into the IMM [73, 216]. Bcs1 has an N-terminal transmembrane domain spanning the

IMM. This domain is crucial for the assembly of Rip1 into the bc1 complex [217]. Additionally to the TM domain, Bcs1 has three regions: a non-canonical mitochondrial-targeting signal directly after the TM domain, an AAA ATPase and a highly conserved middle domain of unknown function [218, 219]. Bcs1 assembles into obligate homo-heptamers, which form two hydrophilic pockets, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane. These two cavities are separated by the seal-forming middle domain. Due to this unique architecture, an airlock-like translocation mechanism for Rip1 was proposed [220, 221].

Fig 9: Rieske iron sulfur protein 1 maturation and insertion. The precursor protein is imported into the mitochondrial matrix where it interacts with Mzm1. The assembly factor catalyzes the insertion of the iron sulfur cluster (FeS) into the apo-Rip1,

36 guards it from aggregation and escorts the mature Rip1 protein to the AAA- ATPase Bcs1. The later assembly factor translocates the globular domain with its bound FeS to the IMS and inserts the Rip1 into the bc1 complex

Finally, another bc1 complex assembly factor, called Bca1, was identified a few years back by a transcriptome-based screen [222]. Bca1 is only found in fungi and is a single-membrane spanning protein with a large C-terminal domain exposed to the IMS. Yeast cells lacking Bca1 show reduced Cytb and Rip1 levels but its specific function remains elusive. It was suggested that Bca1 is necessary in early or intermediate assembly steps of the bc1 complex, prior to Rip1 insertion [222].

The assembly line of the bc1 complex

The biogenesis of the bc1 complex occurs in a linear manner and can be divided into five distinctive intermediates [28, 68].

Assembly of the bc1 complex starts with the co-translational insertion of Cytb into the IMM where it associates with the Cbp3-Cbp6 complex [205]. Furthermore, Cbp4 binds to this early assembly complex forming intermediate I. This assembly intermediate builds a pool of ready-to-assemble Cytb, and the assembly factor Cbp3-Cbp6 can be sequestered into this pre-complex when the assembly line is disrupted, making this factor unavailable for its second task, the initiation of Cytb translation. After Cbp3-Cbp6 dissociation, the nuclear encoded structural subunits Qcr7 and Qcr8 bind and the next assembly intermediate, namely intermediate II is formed [65, 66, 204]. Subsequently, the assembly factor Cbp4 separates by a yet unknown mechanism and the two large supernumerary subunits Cor1 and Cor2 bind to the maturing bc1 complex. Additionally the matured Cyt1 together with the acidic Qcr6 subunits associate and intermediate III with a size of around 500kDa is formed [91, 204]. The addition of the structural subunit Qcr9 and the recruitment of the assembly factor Bcs1 create intermediate IV. The assembly is finalized by the incorporation of the last accessory protein Qcr10 and the catalytic subunit, Rip1 and the dissociation of Bcs1. Qcr9 interacts with the transmembrane segment of Rip1, integrating it firmly into complex [63].

37 The mature bc1 complex exists as homodimer. However, when and how it is dimerizes is not fully understood. It was speculated, that the dimerization is an early event during assembly as recent studies demonstrate that the late bc1 complex intermediate without Rip1 is already in its dimeric state, excluding a potential role of the late catalytic subunit in the dimerization process [223].

Fig 10: assembly line of the bc1 complex. The assembly line can be divided into five distinct assembly intermediates, beginning with the insertion of Cytb. The early assembly factors, Cbp3-Cbp6 and Cbp4, stabilize the newly synthesized Cytb, forming intermediate I. After Cbp3-Cbp6 release, assembly intermediate II is formed by in- serting the structural subunits Qcr7 and Qcr8. By adding the large matrix proteins Cor1 and Cor2 as well as the second catalytical subunit Cyt1 together with Qcr6, the next intermediate, namely assembly intermediate III is formed. The last assembly intermediate is formed by the 500kDa pre-complex together with the late assembly factor Bcs1 that helps insert the last catalytical subunit Rip1, forming intermediate V. After dissociation of Bcs1 the assembly is complete.

38 Aim of this thesis

The main mitochondrial function is energy conversion to form ATP. The majority of the mitochondrial ATP is formed via the respiratory chain and the ATP synthase, or short the OXPHOS system. To ensure the proper function and to tailor energy production to the needs of the cell many regulatory mechanisms are in place. Although many processes concerning the regulation, modulation and assembly of the OXPHOS system were resolved in the last few decades many open questions remain. This thesis focuses on the molecular mechanisms underlying the assembly of the bc1 complex. The major focuses were to understand the maturation of the mitochondrial encoded subunit Cytb and the role of the early assembly factors and supernumerary subunits in more detail. Additionally, we set out to tackle the question of the formation of the dimeric structure of the bc1 complex.

Summary of the papers In the following section I will summarize the papers that form the basis of my thesis and discuss the remaining open questions.

Paper I: Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation

In the first paper we investigated the maturation of the mitochondrial encoded subunit Cytb. To become catalytic active, this subunits needs two non-covalently bound heme b groups, one low-potential heme bL located close to the IMS side of the IMM, and one high-potential heme

39 bH at the matrix side of the membrane. Both heme b moieties are coordinated with the help of histidines within a four helix bundle inside the IMM. By employing yeast mitochondrial genetics and biochemical analyses, we were able unravel the sequence and timing of the incorporation of both heme b groups, and which factors are involved in this process. We could demonstrate that the hemylation is not coincidentally, but depends on a strict and sequential order. First bL, then bH, is inserted, and the involved assembly factors (Cbp3-Cbp6 and Cbp4) are able to rec- ognize the hemylation state of Cytb. We could demonstrate that Cbp4 binding is induced by hemylation of the bL site and Cbp3-Cbp6 release is triggered by insertion of the heme b in the bH site. Subsequently, Qcr7 binding is required to stabilize the fully hemylated Cytb. We could clearly show that the maturation of Cytb is directly coupled to its translation by a feedback loop of the assembly factor Cbp3-Cbp6. If hemylation fails, translation of Cytb is reduced, as the assembly factor is sequestered into the assembly line and cannot induce new rounds of translation.

Paper II:

Timing of dimerization of the bc1 complex during mitochondrial respiratory chain assembly

In this paper we addressed the question when the dimerization of the bc1 complex occurs during assembly and which subunits and factors could be involved. For a long time it was known that complex III is an obligate homodimeric complex, but it was not clear when during the assembly the dimerization occurred. Here we could, with the help of yeast genetics and biochemical analyses, determine the dimerization event. We could demonstrate that the dimerization is happening early during assembly and it depends on the formation of assembly intermediate II and a stable interaction between the two matrix proteins Cor1 and Cor2. Furthermore, we could show that the assembly factor Cp4 is preventing immature dimerization.

40 Additionally to the dimerization event, we could describe a new assembly module of the bc1 complex formed by the two structural subunits Cor1 and Cor2, and named it Cor1/Cor2 module. This assembly module is stable, even if no Cytb is expressed and, therefore, no bc1 complex assembly can take place. Moreover, this assembly module is formed independently of the Cytb assembly line and we were able to determine that the second catalytic subunit, Cyt1, incorporates independently of dimerization into the complex. This means that even if its insertion into the bc1 complex is simultaneous to the joining of the Cor1/Cor2 module and intermediate II (or the dimerization event per se), it has no role in dimerization, as its deletion does not prevent the formation of a dimeric pre-complex.

Paper III: A mutation in the human CBP4 orthologue UQCC3 impairs Com- plex III assembly, activity and cytochrome b stability

In a previous publication, our collaboration partners identified and characterized the human orthologues of the early assembly factors Cbp3 and Cbp6. Consequently, in paper III we described the human orthologue of Cbp4 and showed that the prior uncharacterized protein C11orf83, that was renamed UQCC3, is important for assembly activity and stability of the bc1 complex; like its counterpart in yeast. Addition- ally we were able to describe a homozygous missense mutation in a consanguineous patient diagnosed with isolated Complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and de- layed development without dysmorphic features. We could demonstrate that the mutation within the gene of UQCC3 causes a reduced Complex III activity and lower levels of the holocomplex and its subunits. The mutation is deleterious as no UQCC3 protein could be detected in the patient cells. Furthermore, we were able to show that UQCC3 is reduced in cells depleted for the assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the Complex III

41 assembly pathway downstream of UQCC1 and UQCC2, which is consistent with what is known in the yeast system. With this study we could underline the necessity of biochemical work within the model organism yeast as it would have been much more difficult to adequately address the function of this unknown protein without the prior work.

Paper IV:

Role of Bca1 and the early structural subunit Qcr7 in bc1 complex biogenesis

In the last paper of my thesis we focused on the assembly factor Bca1. This bc1 complex assembly factor was described first 2011 by Matieu et al [222].

In the study we analyzed the role of Bca1 on the assembly of the bc1 complex and could show that it aids in its efficiency. Bca1 is, unlikely the other assembly factors, not necessary for bc1 complex formation, but becomes important under stress conditions. The assembly factor interacts transiently during the early assembly and we were also able to detect a small pool of Bca1 protein bound to the ribosome. Furthermore, we could demonstrate that, when Cytb is not synthesized, Bca1 shifts towards the ribosome, like it is the case for Cbp3. This hints towards a feedback mechanism that also involves Bca1, but to a lesser extent than Cbp3-Cbp6. Moreover, we were able to reveal that Qcr7 is more than a stabilizing, structural subunit. In this study, we could establish that an excess of Qcr7 changes the distribution of the assembly intermediates and shifts it toward completion. Moreover, the assembly can be forced to continue even if cytochrome b is not matured, ending up with a fully assembled, non-functional bc1 complex. This provides us with the insight that Qcr7 interaction serves as a checkpoint for Cytb maturation and a signal to continue further with the assembly of the bc1 complex.

42 Conclusion and remarks With my thesis I aimed to elucidate the assembly of the respiratory chain complexes as well as its regulation. With my findings I contrib- uted significantly to resolve the molecular mechanisms behind the biogenesis of the bc1 complex. With my work, I could provide new insights into the maturation of the mitochondrial encoded subunit Cytb. I could define the role of the Bca1 assembly factor and determine the event that lead to the dimerization of the bc1 complex. Furthermore, I was able to show with my studies that the role of the assembly factor Cbp4 is conserved and I could aid to identify its human orthologue UQCC3. Nevertheless, there are still some open question concerning the biogenesis of the bc1 complex that remaining to be acknowledged. For example, the molecular mechanism behind the assembly factor Cbp4 is still not fully understood. It is known when this assembly factor is interacting in the assembly line and also that it is involved in maturation of Cytb. But it is not well understood which domains of this protein are involved in participating in these functions. Additionally, insight into the events that trigger the release of this factor are missing. Further- more, neither can it be excluded nor confirmed that Cbp4 has an important role in preventing immature dimerization. To answer these question structural analysis of the different assembly intermediates would be necessary. With the structural knowledge of interaction sites between the assembly factor and Cytb we could answer some of these questions. Furthermore, it would be interesting to investigate the interaction with the assembly factor Bca1 and the ribosome in more detail. Is Bca1, like the assembly factors Cbp3-Cbp6, sitting close at the polypeptide exit tunnel, or is it binding somewhere else? Another question, concerning the early biogenesis of the bc1 complex, is if there are other, yet unknown factors involved in the regulation of the synthesis and maturation of the mitochondrial encoded subunit Cytb. For example, a study shows that in different yeast wild types the double function of Cbp3-Cbp6 as a translational activator and assembly factor is not given [210]. Different wild type background in yeast imply that the expression level of certain proteins are altered. This could hint at the possibility that unknown factors are involved in this feedback loop. Furthermore,

43 the translational activation function of Cbp3-Cbp6 is not fully understood. Therefore, it would be prudent to identify additional interaction partners, for example through mass spectrometry analyses, to unravel this mechanism. Additionally it would be interesting which molecular changes trigger the release of Cbp3-Cbp6 from the assembly intermediate I. A conformational change, induced through the stepwise hemylation of Cytb, may lead to a dissociation. Alternatively, the interaction of Qcr7 out- competes Cbp3-Cbp6 binding to Cytb. Both scenarios are plausible. To address this it would be interesting to know the precise interaction site between Cbp3-Cbp6 and Cytb. Another interesting question is the protein quality control during the assembly of the bc1 complex and the fate of the single subunits. The half-life for some of the subunits is known and they differ. Additionally, the recycling of the cofactor needs to be considered. So far nothing or little is known about the proteins that are involved in these processes.

44 Populärwissenschaftliche Zusammenfassung

Jede Zelle benötigt Energie um zu überleben und ihre Aufgaben zu erfüllen. Der Großteil dieser Energie wird von den Mitochondrien bereitgestellt, in Form von ATP, dem universellen Energieträger der Zelle. Die Energie, die von Organismen in Form von Nahrung aufgenommen wird, muss in der Zelle durch Stoffwechselwege ab- und umgebaut werden. Die darin enthaltene chemische Energie wird dann größtenteils durch die Enzymekomplexe der Atmungskette sowie der ATP Synthase, die sich in der inneren mitochondriellen Membran befinden, in ATP umgewandelt. Eine Besonderheit dieser Enzyme ist, dass ihre Untereinheiten sowohl im Zellkern wie auch im Mitochondrium selbst codiert sind. Außerdem enthalten die Komplexe der Atmungskette auch reaktive Cofaktoren und daher ist es nicht weiter verwunderlich, dass der Aufbau der einzelnen Enzyme durch sogenannte Assemblierungsfaktoren überwacht und reguliert werden muss. Das Ziel der hier vorliegenden Arbeit ist es, die Zusammensetzung eines Enzymkomplexes der Atmungskette, oder etwas genauer gesagt der Cytochrome c Reduktase zu untersuchen. Wir konnten aufzeigen, wann während des Aufbaus des Proteinkomplexes, die Untereinheit Cytochrome b, seine Cofaktoren erhält und welche Assimblierungsfaktoren und Untereinheiten in diesen Prozess involviert sind. Auch konnten wir den Zeitpunkt bestimmen, an dem dieser Komplex seine dimerisierte Form einnimmt. Des Weiteren konnten wir zur Aufklärung des Aufbaus der Cytochrome c Reduktase beitragen, indem wir die Funktion des Assimblierungsfactors Bca1 beschreiben konnten. Dieses Protein trägt zur effektiven Herstellung des Atmungskettenkomplexes bei und interagiert früh während der Assemblierung. Wir konnten auch zeigen, dass die Proteinuntereinheit

45 Qcr7 ein Kontrollpunkt innerhalb der Aufbau Kette darstellt und dass durch ihre Interaktion die Zusammensetzung des Komplexes vorangetrieben wird. Ebenso konnte gezeigt werden, dass die molekularen Mechanismen hinter der Zusammensetzung des Proteinkomplexes innerhalb verschiedener Spezies konserviert sind, da der menschliche Assimblierungsfaktor UQCC3 die gleichen Aufgaben aufweist, wie sein Gegenstück Cbp4 in der Bäckerhefe Saccharomyces cerevisiae.

46 Populärvetenskaplig sammanfattning

Varje cell behöver energi för att överleva och göra sitt jobb. Det mesta av denna energi tillhandahålls av mitokondrierna i form av ATP, cellens universella energikälla. Den energi som organismer tar upp i form av mat måste brytas ned och omvandlas i cellens metabolism. Den kemiska energin som finns i den omvandlas sedan mestadels till ATP av enzymkomplexen i andningskedjan och ATP-syntaset, som är belägna i det inre mitokondriella membranet. En speciell egenskap hos dessa enzymer är att vissa av deras subenheter kodas i cellkärnan medan andra kodas i mitokondrierna själva. Dessutom innehåller komplexen i andningskedjan reaktiva kofaktorer och det är därför inte förvånande att uppbyggnaden för de enskilda enzymkomplexen måste övervakas och regleras av så kallade sammansättningsfaktorer. Syftet med studien var att undersöka sammansättningen av ett enzymkomplex i andningskedjan, eller mer exakt cytokrom c-reduktas. Vi kunde visa vid vilken tidpunkt cytokrom b-subenheten får sina kofaktorer samt vilka sammansättningsfaktorer och subenheter som är involverade i denna process. Vi kunde också bestämma den tidpunkt då detta komplex bildar sin dimeriserade form. Dessutom kunde vi belysa sammanfogningen av enzymkomplexet genom att beskriva funktionen för sammansättningsfaktorn Bca1. Detta protein bidrar till en effektiv produktion av andningskedjekomplexet cytokrom c-reduktas och interagerar tidigt under dess uppbyggnad. Vi kunde också visa att inkorporeringen av Qcr7-subenheten är en kontrollpunkt inom monteringskedjan och att dess interaktion driver komplexets sammansättning. Vi har också visat att de molekylära mekanismerna bakom proteinkomplexets sammansättning bevarats mellan olika arter, eftersom den mänskliga sammansättningsfaktorn UQCC3 har samma funktioner som dess motsvarighet Cbp4 i jästen Saccharomyces cerevisiae.

47 Acknowledgments

At the end I would like to take the opportunity to thank some people that accompanied and supported me during my journey to achieve this PhD. It was a hell of a ride!

First of all, I want to thank my supervisor Martin Ott for giving me the chance to do my PhD in his group. It was not always easy and we had some stepping stones, but we managed quite fine . Thanks, for being there if I had questions of all kind and all the ideas and advices you gave me. But also thank you for giving me the freedom to decide for myself when I need help and guidance and develop my own ideas.

I would like to thank my co-supervisor Dan Daley and my mentor Elzbieta Glaser for their support during the years.

A special thanks also goes to our former “yeast-seminar-groups” Per Ljungdahl, Claes Andréasson, Sabrina Büttner and their groups for their input and feedback during the meetings.

Additionally I would like to thank the “5th floor people”, especially Povilas, Aziz, Mathieu, Magnus and Saba for making my time at the 5th floor so much fun. It was very nice to have you around and it was really sad to leave you and move to the fourth floor.

A big thank goes to all the members of the DBB for all the fun lunch- break and hallway chats and of course the fikas! It was never boring and it was amusing to talk about scientific and non-scientific stuff.

Furthermore a big “thank you” to Anneli and Diana for helping me with the Swedish summary!

Finally, I would also like to thank the administrational stuff for all their help and advices in all aspects of the daily work life! Big thanks to all of you!

I would like to thank all our collaboration partners. Eric Hegg without you, we would have had much more trouble figuring out the hemylation. Your help with setting up the heme content measurements, for paper I, are very much appreciated. Thank you, Brigitte Meunier for your great input, the plasmids, and strains. Without them, Paper II and Paper IV wouldn’t have been possible. Furthermore, I am very grateful to Alex Tzagoloff from Columbia University, New York, for nice and helpful discussions and sharing a lot of knowledge as well as un-pub- lished data and material with us. A big thanks also goes to the Netherlands. Thanks to the late Leo Nijt- mans and Martijn Huynen for the great work on the human Cbp4! It is sad that Leo is no longer with us.

Some nice people took the time and made the effort to proof- read my thesis – thank you Martin and Hannah, you guys helped me a lot!

A huge thanks goes to all the past and present members of the Ott group. I was incredibly fortunate to work with many great colleagues over the past 8 years! I actually know all of the famous and infamous people that came to Stockholm all these years ago. So thanks for making this experience a notorious one: Markus, Steffi, Kirsten, Kathi R, Manfred, Tamara, Braulio, Hannah, Jacob (I just include you here, as well ), Lorena, Alex, Roger, Mama, Jens, Anneli, Magda, Andreas C., Abeer, Sorbhi, Carmela, Diana and Andreas A. I really appreciated all the scientific and private support during the last years and that you all created such a familiar environment. I enjoyed working with you and I will miss you!

I would also like to mention all the people who made the last years here in Sweden such a great experience. Thanks to all the “Stockholm people” Hannah, Jacob, Braulio, Caro, Kirsten, Tamara, Andy, for all the fun fikas, barbecues and get-to- gethers. It was great! But a big thanks goes also to all my friends in Uppsala. Thanks Kristina and Lars for being so nice landlords and friends. A big thanks also to Steffi, Jan Joshua, Kian, Ben, Anna and Emma, we shared so many great experiences together.

Last but not least, I could not have achieved all this without the constant Support of my family.

49 Mama und Papa, ihr seid immer für mich da und habt mich unterstützt. Ich weiß, ich kann immer auf euch zählen. Dieses Wissen gibt mir die Sicherheit und den Mut meine Träume zu verfolgen und nicht immer alles so schwarz zu sehen. Ihr habt mich zu dem Menschen gemacht der ich heute bin, ich hab euch lieb! Hasi und Mausi, auch als meine Zwillingsschwester Teresa und mein kleiner Bruder Max bekannt, ihr seid die Tollsten. Wir haben uns immer über eure Besuche gefreut und auch ihr wart immer da, wenn ich mich mal ausheulen musste. Danke Daniel das du auch ein Teil des bunten Haufens bist, der meine Familie ausmacht. Zu guter Letzt Jörg, wir haben so einiges geschafft die letzten Jahre und ich hätte es nicht ohne dich meistern können. Wir sind zusammen nach Schweden gezogen und haben uns hier ein Leben und eine Familie aufgebaut. Du gibst mir Halt und Zuversicht, hilfst mir so gut es geht und beruhigst mich immer, wenn ich mal wieder Gefahr laufe in Panik zu verfallen. Natürlich treibst du mich auch immer wieder in den Wahnsinn aber genau dafür Liebe ich dich! Du und unsere zwei kleinen Mädels Linnea und Melissa, danke, dass ihr mein Leben bunter macht!

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