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T r a n s l o c a t i o n o f p r o t e i n s i n t o a n d a c r o s s t h e b a c t e r i a l a n d m i t o c h o n d r i a l i n n e r m e m b r a n e s

Salomé Calado Botelho

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Translocation of proteins into and across the bacterial and mitochondrial inner membranes

Salomé Calado Botelho

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Cover illustration ”Transmembrane helix checkmate”. Test-helix ”king” in orange will be ”checked”/tested for membrane insertion by the SecYEG translocon, represented by the chess queen, and by the TIM23 complex, represented by the chess king. © Salomé Calado Botelho, Stockholm University 2012 ISBN 978-91-7447-600-2 pp. i-86 Printed in Sweden by Universitetsservice AB, Stockholm 2012 Distributor: Department of Biochemistry and Biophysics, Stockholm University

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Aos meus Pais

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List of publications

I. Öjemalm K*, Botelho SC*, Studle C, von Heijne G (2012) Quantitative analysis of SecYEG-mediated insertion of transmembrane α-helices into the bacterial inner membrane. Manuscript

II. Botelho SC, Österberg M, Reichert AS, Yamano K, Bjorkholm P, Endo

T, von Heijne G, Kim H (2011) TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane. EMBO J 30: 1003-1011

III. Österberg M, Botelho SC, von Heijne G, Kim H (2011) Charged

flanking residues control the efficiency of membrane insertion of the first transmembrane segment in yeast mitochondrial Mgm1p. FEBS Lett 585: 1238-1242

 IV. Botelho SC, Takashi T, von Heijne G, Kim H (2012) Dislocation by the

m-AAA protease increases the threshold hydrophobicity for retention of transmembrane helices in the inner membrane of yeast mitochondria. Manuscript

V. Park K, Botelho SC, Hong J, Österberg M, Kim H (2012) Dissecting

stop-transfer vs. conservative sorting pathways for mitochondrial inner membrane proteins in vivo. J Biol Chem. Nov 26 Epub ahead of print.

* these authors contributed equally

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Abstract

The process of inserting proteins into biological membranes nearly always involves translocation machineries, so-called translocons. Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons found in the endoplasmic reticulum or bacterial membranes are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening. This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively.

These two translocons have been challenged in vivo with designed polypeptide seg-ments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the mem-brane. Natural transmembrane segments have also been analyzed using the same experimental set-ups.

A closer analysis of the mitochondrial system has made it possible to investigate not only the process of membrane integration mediated by the TIM23 complex but also membrane dislocation mediated by the m-AAA protease. It is shown that the thresh-old hydrophobicity required for a transmembrane segment to remain in the mito-chondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease.

Finally, an experimental approach is presented to distinguish between different pro-tein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria.

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Contents

List of publications ......................................................................................... vi

Abstract ......................................................................................................... vii

Contents ....................................................................................................... viii

Abbreviations .................................................................................................. x

Introduction ................................................................................................... 11 1. The biological membrane ..................................................................... 11

1.1 General features of the biological membrane ................................ 11 1.2 Various types of biological membranes ......................................... 12 1.3 Proteins in or at the bilayer ............................................................ 13

1.3.1 Protein-lipid interactions ........................................................ 15 1.3.2 Folding/Oligomerization of α-helical MPs in the lipid bilayer .................................................................. 17

2. Protein trafficking - targeting and transport ......................................... 18 2.1 Targeting to the bacterial and the ER membranes ......................... 19

2.1.1 Co-translational protein targeting: the SRP-dependent Sec pathway ............................................ 19 2.1.2 Post-translational protein targeting – Sec pathway ................ 20

3. Protein translocase machineries ........................................................... 21 3.1 The Sec translocon ......................................................................... 21

3.1.1 Additional/Accessory components of the Sec translocon ...... 24 3.1.1.1 Bacterial SecDFYajC and YidC ..................................... 24 3.1.1.2 TRAM, Calnexin, Calreticulin, OST, PDI in eukaryotic cells ........................................................... 24

3.1.2 Integration of α-helical MPs into the lipid bilayer ................. 25 3.1.2.1 Characteristics of the α-helical TM segments ................ 25 3.1.2.2 Topogenesis of α-helical MPs ........................................ 26

4. Protein import into mitochondria ......................................................... 28 4.1 Maintenance of mitochondrial DNA ............................................. 28 4.2 Mitochondrial protein import machineries .................................... 30

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4.2.1 Precursor protein targeting and transport to mitochondria .... 30 4.2.2 Translocase of the outer membrane ....................................... 31 4.2.3 The intermembrane space ...................................................... 34 4.2.4 Translocase of the inner membrane ....................................... 34

4.2.4.1 The TIM22 complex - the carrier import pathway ......... 34 4.2.4.2 The TIM23 complex ....................................................... 35 4.2.4.3 The OXA translocase ..................................................... 39 4.2.4.4 Stop-transfer vs. Conservative sorting ........................... 40

4.3 Mitochondrial protein import and the effect of lipids .................... 42 4.4 Mitochondrial protein import and the effect of MICOS/MINOS/MitOS ............................................................ 42

5. Membrane protein maturation – focus on protein processing and membrane dislocation .................................................................... 44 6. Quantitative studies of membrane partitioning .................................... 48

6.1 Hydrophobicity scales ................................................................... 48 6.1.1 A “biological” hydrophobicity scale ...................................... 49

Projects .......................................................................................................... 52 7. Aims ..................................................................................................... 52 8. Summary of the papers ......................................................................... 53 9. Conclusions .......................................................................................... 61

Sammanfattning på svenska .......................................................................... 63

Acknowledgements ....................................................................................... 64

References ..................................................................................................... 67

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Abbreviations

ATPases Associated with diverse cellular Activities

AAA Phosphatidylcholine PC

Cardiolipin CL Phosphatidylethanolamine PE Contact sites CS Phosphatidylglycerol PG Cristae junctions CJ Presequence translocase

associated motor PAM

Endoplasmic reticulum ER Proton motive force PMF ER–mitochondria encounter structure

ERMES Ribosome-nascent chain-SRP

RNC-SRP

Inner membrane IM Sorting and assembly Machinery

SAM

Intermembrane space IMS Secretory Sec Leader peptidase Lep Signal recognition particle SRP Membrane protein MP SRP receptor SR Mitochondrial DNA mtDNA Translocase of the IM TIM Mitochondrial IMS Assembly MIA Translocase of the OM TOM Outer membrane OM Transmembrane TM Amino acids 3 - letters 1 - letter

Alanine Ala A Asparagine Asn N Arginine Arg R Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

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Introduction

1. The biological membrane

The compartmentalization permitted by the formation of membranes in an aqueous environment, 3.8 billion years ago, allowed the development of what we know today as life (3). The evolutionary invention of biological membranes, together with a regulated flow of matter and energy through the membrane, has led to the appearance of specific biochemical environments that eventually formed the bacterial, archaeal and eukaryotic cells (4).

1.1 General features of the biological membrane

The biological membrane as we know today is composed of lipids, proteins and carbohydrates. It is commonly represented as a bilayer formed by lipids organized in two ~ 50 Å thick leaflets with their polar headgroups along the two surfaces and their acyl chains forming the nonpolar core in between. Proteins are either embedded in the lipid bilayer (integral mem-brane proteins) or associated to the membrane surface (peripheral membrane proteins). As for carbohydrates, they are externally localized in glycopro-teins and glycolipids. This description of biological membranes provided by the “Fluid Mosaic Model” by Singer and Nicolson in 1972 and forty years of membrane biolo-gy research have revealed the membrane as a crowded, heterogeneous, dy-namic structure (5,6). The visual image of the membrane resembles a patchwork carpet of hetero-geneous proteolipid niches, with proteins mostly in an oligomeric state, forming functional complexes, Fig. 1. Membranes generally have an asym-

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metric distribution of proteins and lipids along the two leaflets. The thick-ness and shape of the membrane results from the lipids phase, their move-ments in the bilayer (transversal and laterally) and the constant protein activ-ity on its surface (7).

One of the most important characteristics of the biological membrane is its semipermeable character (8). The strong interactions between the membrane components (proteins and lipids) create a barrier that only small, uncharged, nonpolar compounds can pass across. Protein machineries are required for other molecules to be able to enter or leave a certain membrane-delimitated compartment. Membrane proteins forming channels, carriers or pumps allow this selective communication between chemically distinct environments.

1.2 Various types of biological membranes

Different membrane systems with various heterogeneous composi-tions of lipids and proteins are found in the bacterial and eukaryotic cell. Archaeal membranes will not be considered here since they may differ from the bilayer model above described. The well-known Gram-negative bacterium Escherichia coli possesses two types of membranes, the outer and the inner membrane. The outer leaflet of the outer membrane consists mainly of lipopolysaccharides, while the inner leaflet has a lipid composition similar to that of the inner membrane (IM). The latter is composed of 20-30% of lipids (75% of phosphatidylethanola-

Figure 1. A biological membrane. Cartoon representation of a lipid bilayer populated with proteins (grey), membrane proteins with determined crystal structure (PDB ID from left to right: 2W1P, 3N5K, 2A79), and carbo-hydrates groups (black).

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mine (PE), 20% of phosphatidylglycerol (PG), 5% cardiolipin (CL)) and 70% of proteins (9). In eukaryotic cells, in addition to the plasma membrane that surrounds the whole cytoplasm, other subcellular membrane environments are present: the endoplasmic reticulum (ER), the mitochondria, chloroplasts, nucleus, perox-isomes, and lysosomes. The lipid content in each membrane of these orga-nelles varies, reflecting diverse biological roles. There are more than 1,000 different lipids in any eukaryotic cell (hundreds in prokaryotes), with restricted synthesis locations. The ER is the principal organelle involved in the synthesis of phospholipids and cholesterol (ergos-terol in yeast), however as the latter is rapidly transported to other orga-nelles, the ER membrane is loosely packed with mostly phosphatidylcholine (PC), PE and PI, and an overall protein/lipid composition of 70/30 in the rough ER and 50/50 in the smooth ER. The mitochondria are another rele-vant place for lipid synthesis. The IM of the mitochondria is enriched in PG, PE and CL with low sterol content and the protein to lipid ratio is about 80/20 (10). The high protein concentration in many membranes and the ex-istence of strong protein-protein interactions that exclude lipids, favor the formation of protein complexes. As for the plasma membrane, it is built to promote stability with higher content of sphingolipids and cholesterol, con-tains no detectable cardiolipin and has lower content of proteins (40%) (11).

1.3 Proteins in or at the bilayer

Membrane proteins (MPs) can be either peripheral or integral. Pe-ripheral proteins crowd the surface of the membrane through the establish-ment of electrostatic or hydrophobic interactions with lipids and mainly pro-teins and can be detached from the membrane using relatively mild treat-ments such as high salt or high pH. However, some proteins at the surface are covalently bound to membrane lipids (e.g., GPI-anchored proteins) and are released from the membrane with the same techniques used for integral MPs (see below). Integral proteins are firmly embedded in the lipid bilayer by hydrophobic interactions between the lipid hydrocarbon tails and the hydrophobic do-

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mains of the protein, and can only be removed using detergents (non-polar solvents that disrupt these interactions and form micelle-like clusters around the protein). Some integral membrane proteins have a hydrophobic part that penetrates into but not across the lipid bilayer (monotopic), some span the membrane once (bitopic), while others cross it multiple times (multispan-ning). Data from known structures have shown that two structural motifs predomi-nate in the transmembrane part of MPs: α-helical bundles and β-barrels, Fig. 2. This is dictated by the process of export, assembly of the protein in the bilayer and how a polypeptide chain surrounded by lipids, having no water with which to hydrogen bond, maximizes interchain H-bonds to obtain the most stable conformation (12,13) - stability of the embedded protein. Figure 2. A) Structure of the α-helical membrane protein GlpG from E. coli refined to 1. 7 Å resolution (PDB ID 2XTV) (1). B) Structure of the β-barrel membrane protein OmpA with 2.5 Å resolution (PDB ID 1BXW) (2). The membrane bilayer defined by the space in between the two opposing horizontal black lines. α-helical MPs are folded in more or less complicated bundles and have in general longer and more hydrophobic transmembrane (TM) segments than do the β-barrels. The latter usually have an even number of antiparallel, tilt-ed β-strands, and the barrel is closed by the first and last strand (Fig. 2). De-spite the structural differences, both α-helical bundles and β-barrels share common surface features: a band of hydrophobic (mainly aliphatic) residues flanked by two opposed rings constituted by aromatic residues, Trp and Tyr. The latter are located in the water/lipid interface region at the bilayer, inter-

A B

was carried out to investigate

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acting with the lipid head groups, while the hydrophobic belt is in contact with the hydrocarbon tails (14). In a typical genome, 20-30% of all open reading frames (ORFs) are predict-ed to encode α-helical integral membrane proteins, being distributed in all cellular membranes (15-17). The identified β-barrel MPs are only found in the outer membranes of Gram-negative bacteria, mitochondria and chloro-plasts. They are predicted to represent 2-3% of the E. coli proteome; in mito-chondria, only a handful of β-barrel MPs are known (18). This thesis focuses on the biogenesis of α-helical MPs and therefore β-barrel MPs, unless speci-fied, will not be further considered. The relevance of MPs is not only due to their abundance but mostly to their diverse and essential functions, e.g., in regulating the transport of molecules and the flow of information across the cell membranes (19). Determination of a high-resolution structure of a given MP is an important step in comprehending its functional mechanism and regulation at the mo-lecular level. This is not, however, a trivial achievement, as to this date (October 19th, 2012), there are only 367 unique MP structures (20), 0.5% of the 79,959 structure entries that are deposited in Protein Data Bank (21). This great disparity comes from the practical problems of working with MPs - specifically, difficulties in expression, purification and crystallization, ow-ing to their amphipathic nature (22). A considerable number of the known MP structures show tightly bound li-pids (23). This provides new insights into the importance of specific protein-lipid interactions, as summarized below.

1.3.1 Protein-lipid interactions

The lipid bilayer provides much more than a simple matrix for the MPs. In addition to the tight protein-lipid interaction that is required to main-tain the diffusion barrier and to keep the membrane electrochemically sealed, lipids and proteins work in concert, allowing all the features necessary for cell division, intracellular trafficking, and formation of specific microenvi-ronments (patches/rafts) with roles in signal transduction and membrane transport. Protein-lipid interactions are specific, and numerous biochemical

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and biophysical studies have demonstrated their importance for folding, structural integrity and proper function of membrane proteins (23).

To form a bilayer, the lipid should have a cylindrical shape with the radius of the headgroup approximately matching the radius of the hydrocarbon tails, e.g., the bilayer forming PC represents more than 50% of the phospholipids in most eukaryotic membranes. In some lipids like PE and CL, the hydrocar-bon tails splay out, so they cannot form a bilayer themselves. These “non-bilayer” lipids can be incorporated into a bilayer if they mix with bilayer-forming lipids. This leads to a shape mismatch and a curvature elasticity that can affect MP bilayer insertion and folding (11,24). Lipid interacts with MPs through three types of binding modes: the annular shell of lipids bound to the protein surface, the nonannular surface lipids that are found immersed in cavities and clefts of the protein surface, and the inte-gral protein lipids that reside within a MP or MP complex (23). Membranes with a specific lipid constitution have been shown to be neces-sary for structural integrity and proper function of MPs: the bacterial SecYEG translocon functions optimally in the presence of anionic (PG) and nonbilayer lipids (25), while in the mitochondrial IM, PE and CL are funda-mental for maximum activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential (26). Most MPs have specific requirements with regard to membrane fluidity, thickness, lateral surface pressure, or other membrane properties (27). How-ever, MPs reside in a variety of different lipid environments during their lifetime, showing a certain tolerance or adaptability in the face of these ma-jor changes. It is common that the hydrophobic surface of the MP is thicker or thinner than the hydrocarbon core of the bilayer – so-called hydrophobic mismatch. To avoid the unfavorable exposure of hydrophobic surfaces to a hydrophilic environment, either the protein can adjust to match the bilayer by oligomerization, aggregation, helix tilting. The bilayer can also deform to match the protein by stretching the acyl chains or even assemble into another type lipid phase, disrupting the bilayer organization (28,29). Hydrophobic mismatch has important consequences for MP function, sorting and orga-nelle localization. Indeed, Sharpe et al. have recently confirmed that the TM regions of plasma membrane proteins are in general longer than those of the ER and the Golgi complex (30). The correct sorting of proteins in the secre-

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tory pathway seems to be related to the sterol (cholesterol) content in the different membranes (31).

1.3.2 Folding/Oligomerization of α-helical MPs in the lipid bilayer

The secondary structure and topology of a MP is determined during the membrane insertion process. Following (or during) insertion, the MP begins to acquire its native conformation. Individual TM helices recognize each other to form helix bundles (intramolecular interactions). TM helix-packing interfaces require the matching of helix surfaces: a common oli-gomerization motif is the GXXXG (X denotes any amino acid), where the small glycine residues allow a close approach of the helices (32). Local distortions in TM helices such as kinks and short stretches of Π or 310 helices are also prevalent and important for the overall folding, imparting certain flexibility that allows functionally important structural adjustments and movements. Apolar side chains dominate TM helices, and van der Waals forces are very important in stabilizing interactions in the hydrophobic core of the lipid bilayer. H-bonds are also found but in the interior of MPs, where they must be judiciously formed to avoid inappropriate interactions in the crowded membrane environment (28). Intermolecular interactions between subunits lead to the formation of oligo-meric MP complexes. One of the largest known complexes is the mitochon-drial cytochrome c oxidase that is composed of 13 subunits, 10 of which span the membrane one or more times to give a total number of 28 TM heli-ces (33).

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2. Protein trafficking - targeting and transport

With the exception of proteins encoded in the mitochondrial and chloroplast genomes, all proteins are synthesized in the cytoplasm of the cell, and roughly half are transported into or across a membrane. The correct localization of a protein is a sine qua non feature of cell’s functionality and is possible due to the existence of targeting sequences and the concerted actions of protein transport machineries (34). Targeting signals are often N-terminal extensions to the polypeptide chain that are recognized by a cytoplasmic or organelle-bound receptor. Inte-restingly, 25% of randomly generated peptides can function as (inefficient) signal sequences for the ER, mitochondrial or bacterial membranes (34). The transport of proteins from the cytoplasm is, in the majority of the cases, me-diated by a translocon, a molecular gatekeeper that allows nascent polypep-tide chains to pass across or integrate into lipid membranes (35). A soluble non-cytoplasmic protein or a MP with a specific signal sequence can be targeted to the translocon in either a co- or a post-translational fash-ion. During co-translational targeting, the preprotein is directed to the trans-locon while being synthesized by the ribosome, whereas in the post-translational mode, the preprotein is synthesized to its full length prior to targeting (36). All these concepts are a result of more than 50 years of research. A field paved in the 1960s by Palade with the characterization of the secretory pathway in eukaryotes (37). Followed by Blobel and Sabatini in 1971 with the proposal of the “signal sequence hypothesis” for co-translational target-ing of ribosome-nascent chain complexes to the ER membrane (38), and finally by Blobel and Dobberstein, who in 1975 proposed that an aqueous pore formed by MPs allowed the movement of secretory proteins across the ER membrane (39). Below, I will focus on the processes that target proteins to the bacterial IM, the ER membrane and the mitochondrial IM.

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2.1 Targeting to the bacterial and the ER membranes

Targeting of polypetides to the bacterial IM or, in eukaryotes, to the ER membrane is the starting point of a journey that may guide the protein for secretion into the external milieu, or to one of the other intracellular compartments. This pathway, commonly known as the secretory (Sec) path-way is utilized by different substrates that range from very hydrophobic to very hydrophilic proteins. In bacteria, inner membrane proteins employ mainly the co-translational pathway, while the post-translational mechanism is utilized by proteins that are secreted across the IM (40). In mammals, tar-geting to the ER membrane is mostly co-translational (a few small secreted proteins might engage in a post-translational pathway), while in yeast both co- and post-translational pathways are used (36,37).

2.1.1 Co-translational protein targeting: the SRP-dependent Sec pathway

Co-translational protein targeting occurs in every cell and its molec-ular mechanism is strikingly similar for α-helical MPs that are targeted to the bacterial IM or the ER membrane. When the N-terminus of a protein that emerges from the exit channel of the ribosome presents the features of a signal sequence – a few N-terminal basic residues, followed by a stretch of 7 to 15 hydrophobic residues (41) – it is recognized by the signal recognition particle (SRP, Ffh in bacteria) (42). In eukaryotes, SRP binding induces a transient translational arrest (43). The ribosome-nascent chain-SRP (RNC-SRP) complex binds then to the membrane-bound SRP receptor (SR, FtsY in bacteria), a protein that dynam-ically associates with the translocon (44). GTP hydrolysis on both the SRP and the SR leads to transfer of the RNC to the translocon, and as translation resumes, the nascent chain passes through the translocon channel. TM seg-ments that are of sufficient length and hydrophobicity exit the channel later-ally and insert into the lipid bilayer during translocation (34,45,46). The N-terminal signal sequence is cleaved off by the signal peptidase, gen-erating the mature form of the protein. However, the majority of bacterial and eukaryotic membrane proteins do not contain a cleavable signal se-

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quence. Instead, the first hydrophobic TM segment is able to function as a signal for membrane targeting (47-49). Protein export is not a spontaneous process and there are different energetic requirements for protein translocation across or into the bacterial and ER membranes. In bacteria the SecA protein functions as an ATP-dependent stepping motor that drives unfolded proteins across the IM (50,51). SecA cycles between the cytosol where it associates with preproteins, and the membrane where it binds to the translocon. Another driving force is the membrane potential across the IM (proton motive force, PMF), Δψ and ΔpH (positive and acidic, respectively on the periplasmic side) that stimulate pro-tein translocation, e.g., driving the translocation of negatively charged resi-dues across the membrane (52). The PMF can drive rapid translocation once it has been initiated at the expense of ATP hydrolysis and SecA is no longer associated with the translocating preprotein (53). In the ER membrane, pro-tein synthesis by the ribosome may be sufficient to drive co-translational translocation towards the ER lumen, although some studies suggest that a soluble lumenal chaperone, BiP, might be necessary for optimal co-translational translocation (54).

2.1.2 Post-translational protein targeting – Sec pathway

In general, in both bacteria and yeast, proteins with less hydrophobic signal sequences are translocated via the post-translational pathway (42). Fully synthesized proteins are kept in a translocation-competent state, i.e., a loosely folded, non-aggregated state, by cytoplasmic chaperones (such as SecB in E. coli (55)) that also help targeting to the membrane. At the level of the translocon, SecB binds with high affinity to SecA which itself is bound to the translocon. In the case of eukaryotes, specifically yeast, in addition to the translocon, proteins such as Sec62p, Sec63p, Sec71p, Sec72p and the yeast homolog of the mammalian Bip, Kar2p (56), are required for post-translational targeting and translocation (36,57).

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3. Protein translocase machineries

In general, translocons manage the targeting of topologically distinct classes of proteins (membrane vs. soluble proteins), mediate targeting to different subcellular compartments, and respond to stress and development cues. Furthermore, they are highly coordinated with downstream events of protein folding, modification and assembly, providing a direct link between protein targeting and maturation (34). All translocons have docking sites for the targeting signals of the translocat-ing substrates, form selectively permeable protein-conducting channels that mediate translocation, and are coupled with a translocation driving force. In most cases, processing and folding take place in the final destination com-partment. Some translocons, however, translocate proteins that are already folded. These import machineries have a pore of variable size that accom-modates the folded precursor proteins. They can be found in the nuclear envelope, the peroxisomal membrane, the bacterial IM and the thylakoid membrane (58-62), e.g., the bacterial TAT translocon exports folded proteins bearing a unique twin-arginine motif (63,64).

3.1 The Sec translocon

In the bacterial IM and the eukaryotic ER membrane, the critical membrane protein component where the above-mentioned co- and post-translational targeting pathways converge is the Sec translocon. Conserved across all three domains of life, its core consists of a heterotrimeric protein complex designated as Sec61αβγ in eukaryotes, SecYEG in bacteria and SecYEβ in archaea (65). The Sec61 γ-subunit is homologous to SecE, how-ever Sec61β is not homologous to SecG in either structure or function (66). The Sec61α homologous to SecY, has 10 TM helices whereas the β and γ subunits typically have one TM helix (E. coli SecE has 3 TM helices and SecG has 2 TM helices, however) (67). SecY/61α and SecE/61γ are essential for viability and protein translocation, contrary to SecG/61β that is dispensa-ble (68).

In the yeast Saccharomyces cerevisiae there are two homologous Sec sys-tems: the essential Sec61p complex composed by Sec61p (Sec61α), Sbh1p

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(Sec61β), and Sss1p (Sec61γ), and a second non-essential complex consist-ing of Ssh1p (Sec61α), Sss1p (Sec61γ) and Sbh2p (Sec61β) that is thought to be involved only in co-translational protein translocation (69). Interesting-ly, a second SEC61 gene has been found in mammals, pointing to existence of a possible second Sec61 complex. Additionally, SecY, SecE and SecA homologues have been characterized in the thylakoid membrane of chloro-plasts, and SecY homologues have been found in cyanobacteria and certain mitochondria of primitive protists (65 and references therein). Analysis of the high resolution X-ray crystal structure of the SecYEβ com-plex of Methanococcus jannaschii at 3.2 Å, together with cryo-EM recon-structions of the E. coli SecYEG complex reveal a structure resembling an hourglass (70,71), Fig. 3A. The 10 TMs of the main subunit SecY form a clamshell in which TM segments 1-5 and 6-10 are hinged at the cytoplasmic loop between TM5 and TM6. The SecE protein embraces the two SecY halves, stabilizing the structure and preventing lipids from contacting the interior of SecY. The Secβ subunit makes only a few contacts with SecY, which possibly explains why it is dispensable and less conserved (72). Cross-linking experiments have confirmed that elongating nascent chains pass through the channel formed by a single copy of SecY (73), and that TM2B and 7 in SecY form the only possible opening from the interior to-ward the lipid bilayer (74,75). Additionally, electrophysiological measure-ments (76) and experiments using environment-sensitive fluorescent probes (46,77) indicate that the pore has an aqueous interior. This suggests that the SecY/Sec61 complex forms an interface between a proteinaceous and a lipid environment. Oligomers of the SecY/Sec61 complex have been isolated, suggesting either a ‘front-to-front’ (71) or a ‘back-to-back’ (78) orientation of two SecY mol-ecules. It has been proposed that SecA might bind to one of the two SecYs, feeding the polypeptide chain through the neighboring SecY copy (73,79). A recent study, however, showed that a single copy is sufficient for transloca-tion (80), so the question of the stoichiometry of the functional Sec trans-locon remains unclear.

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Figure 3. Structure of SecYEG/β translocon (PDB ID 1RHZ). A) Membrane cross view and B) cytosolic view of SecYEβ translocon from M. jannaschii (70). SecE (blue) embraces the two halves of SecY TM segments 1-5 in dark-pink and TM segments 6-10 in green that form the lateral gate to the lipid bilayer. The pore is closed at the periplasmic side by a short α-helix that folds back as a plug (purple) and is constricted at the center by a ring of hydrophobic residues (pore ring). SecG (Secβ) in light-green, is peripherally bound to SecY. Adapted from (81). Interestingly, the narrowest part of the hourglass shaped pore is made up by a constriction ring composed of six hydrophobic residues (the “hydrophobic collar”) that is believed to act as a seal around the elongating chain. Just below the hydrophobic collar, a short helix (TM2A) forms a plug on the periplasmic/lumenal side blocking the passage of small molecules through the translocon when it is in the closed state, Fig. 3B (82). During the handing-over of the co- or post-translationally targeted preprotein to the Sec translocon, the first hydropobic signal sequence or TM domain interacts with the Sec complex, displacing the plug and intercalating between the lateral gates helices TM2 and TM7 (83). Apparently, the binding of Se-cA to SecYEG induces a preopen state with partial plug displacement (50). A recent crosslinking analysis has suggested that the lateral gate has to open up at least 8 Å to support protein translocation (84). Conflicting studies on the size of the active translocating pore propose diameters that range from 16 Å (85), 22-24 Å (86) up to 40-60 Å (87). According to experimental data, the translocating peptide may be in an extended conformation or can possi-bly form an α-helix, depending on the amino acid sequence (88).

SecA/ribosome-associa!ng groups

Cytosol

Periplasm

Plug

SecE

SecY TMSs 6-10

SecY TMSs 1-5

SecG(Secβ)

Late

ral g

ate

Plug

Pore ring

A B

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Both in the resting state and when translocating a polypeptide, the Sec trans-locon must maintain a membrane barrier for ions and small molecules. This is particularly important in prokaryotes because of the membrane potential, but also in the ER membrane to prevent a free flow of Ca2+ ions. The interac-tion of the plug with the pore ring seems to be responsible for sealing the pore in the resting state, and in the active channel, once the plug is displaced the pore forms a gasket-like seal around the translocating polypeptide chain. With the termination of translocation, the plug returns and reseals the chan-nels (89,90).

3.1.1 Additional/Accessory components of the Sec translocon

3.1.1.1 Bacterial SecDFYajC and YidC

SecDFYajC is a MP complex that associates in a transient fashion with the bacterial Sec translocon. Although SecD and SecF are not essential, their inactivation in E. coli results in severe defects in growth and protein secretion (91). SecDF have been implicated in the cycling of ATP in SecA and also in the dependence of membrane potential for preprotein transloca-tion (92). YidC plays an essential role in the insertion of a subset of MPs (93). Cross-linking studies suggest that YidC transiently interacts with the SecYEG translocon during the insertion of MPs and that this interaction may involve SecDF (94). YidC is also an insertase on its own, however the number of substrates identified to date is limited, mostly membrane subunits of large respiratory complexes (95). Several MPs depend on YidC for folding rather than membrane insertion; one prominent example is the F1FO-ATPase com-plex (96,97).

3.1.1.2 TRAM, Calnexin, Calreticulin, OST, PDI in eukaryotic cells

One component of the mammalian translocon, the translocation-associated membrane protein, TRAM, was found to be required for the trans-location or integration of most proteins. The heterotrimeric Sec61αβγ and TRAM are the core components of the co-translational translocon and are sufficient to reconstitute translocation activity in vitro (67).

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Nascent polypeptides crossing the ER membrane are co-translationally N-glycosylated at the motif Asn-X-Thr/Ser (X represents any amino acid ex-cept Pro) by the oligosaccharyl transferase complex (OST) that remains ad-jacent to the translocon throughout translocation. N-linked oligosaccharides increase the solubility and stability of many proteins, contributing to the proper folding of both secretory and membrane proteins (98). Calnexin and calreticulin (a soluble homologue of calnexin) act as chaper-ones during the folding of nascent membrane glycoproteins and have been crosslinked to nascent chains (99). Protein disulfide isomerase (PDI) also interacts with the nascent chain co-translationally, promoting correct for-mation of disulfide bonds (100).

3.1.2 Integration of α-helical MPs into the lipid bilayer

The Sec translocon is an active participant in the process of MP in-tegration into the lipid bilayer, and is directly involved in the recognition, orientation, lateral movement, and insertion of α-helical TM segments into the membrane (101). A TM segment in a nascent chain might be expected to be recognized only after it reaches the membrane, yet it was recently discov-ered that a TM sequence in a nascent chain can be first detected by the ribo-some (102,103). The appearance of a TM stretch initiates a series of events that prepare the translocon for MP integration, including the closing of the periplasmic/lumenal end of the pore and the subsequent opening of the tight ribosome-translocon seal (104).

3.1.2.1 Characteristics of the α-helical TM segments

The actual membrane insertion process has been shown to depend on the properties of the TM segment. Typical TM helices consist of 20-25 most-ly apolar residues in a helix conformation, spanning the hydrocarbon core of the membrane (105). This compact secondary helical structure has been pro-posed to form already inside the ribosomal exit tunnel (106).

In terms of amino acid composition, TM helices are enriched in hydrophobic residues in the core part. Aromatic residues, particularly Tyr and Trp, are

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preferentially found at the edges of the TM helix near the phospholipid head group regions of the bilayer (107,108), being important for the precise posi-tioning of MPs (109). Although prolines, when located near the center of a TM segment, induce a kink in the α-helix, they are frequently found in the helix core where they might have a structural or functional role (110). Charged residues appear to be excluded from the non-polar environment and preferentially positioned in interface regions and in non-membrane domains (111). Statistical studies of MPs in bacteria have shown that positively charged residues are up to four times more likely to be present within cytoplasmic compared to periplasmic loops. This is known as the “positive-inside rule”; positively charged residues flanking the TM segments are topology determi-nants and remain in the cytosol during biogenesis. This rule is applicable to many organelles and organisms, although for proteins inserted into the ER the charge rule seems to be less strict than for bacterial proteins. Reasons for this specific charge effect might be related to interactions with anionic charged lipids, the membrane electrochemical potential found in the bacteri-al IM, and charged residues in the Sec translocon (112). Heinrich et al., demonstrated that the Sec61 channel allows the TM seg-ments to dynamically equilibrate between the aqueous phase and the hydro-phobic interior of the membrane, and that hydrophobic sequences exit the channel by partitioning more rapidly into the lipid phase than less hydropho-bic ones (113). Efficient membrane partitioning can occur for TM segments as short as 11 residues if their sequence is sufficiently hydrophobic (114). In addition to the hydrophobic character of the TM segments, there are other signals that regulate orientation and partitioning of TMs into the bilayer (115).

3.1.2.2 Topogenesis of α-helical MPs

In terms of the orientation of TM segments in the lipid bilayer, there are several topologies that can be attained. Single-spanning membrane pro-teins, i.e., proteins having only one TM segment, can exist with Nexo/Ccyt or Ncyt/Cexo (cytoplasmic N- and exoplasmic C-terminus). Type I MPs (Nexo/Ccyt) are initially targeted to the bacterial IM or to the ER membrane by

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a cleavable signal peptide and are then anchored in the membrane by the TM segment. Type II proteins (Ncyt/Cexo) have no cleavable signal sequence, but possess a signal-anchor sequence and translocate their C-terminus across the membrane. The opposite happens for type III proteins that have a reverse signal-anchor sequence, translocating the N-terminus across the membrane (112). Multi-spanning MPs can be considered as consisting of a series of alternat-ing start- and stop-transfer sequences inserted into the bilayer. According to the simplest model, the initial signal defines its own orientation, and the following TM segments simply follow suit, exiting the translocon in a se-quential fashion. In some cases, however, it has been shown that TM seg-ments cooperate during insertion, as a less hydrophobic TM segment exits the translocon to the lipid bilayer with the aid of a more hydrophobic TM segment. Competition between TM segments with the same preferred orien-tation, or the length of the loops in between the TMs, are also important to regulate the actual partition into the bilayer and the final topology of the MP. Whether the channel pore is able to accommodate several TM segments at the same time, or if it releases them one-by-one, is not well understood. Overall, there seem to be different pathways of insertion for different MPs (81). The “positive-inside rule” mentioned above seems to be extremely important in controlling membrane protein topology. Seppälä et al. have found that the topology of an E. coli inner membrane protein with four or five TM helices could be controlled by a single positively charged residue placed in different locations throughout the protein, including the very C-terminus (116).

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4. Protein import into mitochondria From the proteins synthesized in the cytosol of eukaryotic cells,

roughly 1000-1500 (117) are targeted to the mitochondria. Comparing the bacterial and the ER protein targeting mechanisms on one hand and the mi-tochondrial protein uptake pathways on the other, common principles emerge: the requirement of organelle-specific targeting signals, efficient transport to the target membrane by cytosolic factors, recognition by recep-tors on the organelle/membrane surface, transport into/across the membrane by a translocon, and energy-driven transport/translocation (34,118). Below follows a description of the mitochondrial protein import machineries, with special focus on the TIM23 translocon.

4.1 Maintenance of mitochondrial DNA

Mitochondria are ubiquitous in all eukaryotic cells, having crucial functions in energy conversion, amino acid, haem, lipid and fatty-acid me-tabolism, iron-sulphur cluster assembly and apoptosis regulation (117). Orig-inating from an α-protebacterium that was engulfed by a primordial eukary-otic cell 1.5-2 billion years ago, this bacterial symbiont - according to the endosymbiotic theory - exported a large portion of its genome to the host nucleus, or perhaps dispensed with it and in parallel acquired novel host´s proteins (119,120).

Sequenced mitochondrial genomes code for 0.1 to 1% of the cellular prote-ome, plus 22 tRNAs and 2 rRNAs in mammals (121). Protein import ma-chineries, therefore, had to be created de novo and established in the mito-chondrial membrane to allow protein interchange with the cytoplasm (122). The human mitochondrial DNA (mtDNA) encodes only 13 proteins (121), whereas in the yeast S. cerevisiae 8 proteins, namely: cytochrome b, Cox1, Cox2, Cox3, Atp6, Atp8 and Atp9 are encoded in the mtDNA and synthe-sized in the organelle (123). Over 100 nuclear genes are, however, dedicated to the maintenance of the mtDNA (117). Several reasons have been suggested to explain the retention of DNA in the mitochondrion: mitochondrial codon usage is different from the cytoplasmic system, e.g., UAG codes for Trp in mitochondria, rather than for a stop co-

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don, which would lead to premature synthesis termination of mitochondrial-ly encoded proteins if mitochondrial genes were moved to the nucleus (124). All mitochondrially encoded proteins represent inner membrane subunits of the respiratory chain, and the mtDNA has been shown to be hypervariable. Therefore, keeping these genes linked in the non recombinant mtDNA in-creases the chances of coevolution and selection of possible mutations as a functional unit (125). The physical separation of specific genes is, perhaps, important in regulating the assembly process of the respiratory chain com-plexes and controlling formation of products such as ROS (reactive oxygen species) (126). It has also been hypothesized that if the mitochondrially en-coded proteins were expressed in the cytoplasm, they would aggregate, be mistargeted to other organelles or hinder import through the mitochondrial protein translocases, as a consequence of their high hydrophobicity (127,128). The latter possibility is especially appealing from the point of view of pro-tein targeting. The presence of genes of bacterial origin, such as SecY in the mitochondrial genome of the protist Reclinomonas americana (129), or the fact that Oxa1 is a member of the YidC family of bacterial MP chaperones (130) and that mitochondrial Hsp70s are derived from DnaK-type Hsp70s (131), among other examples, clearly suggest that co-translational SRP-dependent protein targeting pathway was already established before the mi-tochondrial protein import machineries evolved (127). Analysis of mitochondrially encoded proteins by von Heijne (127) revealed that the majority are predicted to be intrinsic MPs with long, hydrophobic membrane-spanning regions near the N-terminus and all throughout their sequences. Comparing these features with the ones recognized by the SRP suggests that the majority of the analyzed proteins would not escape this pre-existing pathway. Interestingly, encoding markedly hydrophobic mitochon-drial inner membrane proteins, the atp6 (132), atp8 (133), and nad4 (134) genes have been recoded, introduced in the nuclear genome and successfully imported into the mitochondrial IM, albeit inefficiently. This argues against the hypothesized interferences between co-translation SRP protein targeting and the mitochondrial protein import system for highly hydrophobic mito-chondrial proteins. However, it should be mentioned that trials for reengi-neering and importing the two exceptionally hydrophobic proteins encoded by all mtDNAs, subunit I of cytochrome c oxidase and cytochrome b, into mitochondria have so far failed (135). Additionally it was shown that a sin-

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gle substitution in the first TM (W56R) of the yeast mitochondrially encoded COX2 enabled allotopic expression (136) and increasing the hydrophobicity of TM segments of certain nuclear-encoded mitochondrial proteins such as Tom5 or Tom20 targets these variants to the ER (137,138). These facts fur-ther support the hydrophobicity of the mitochondrially encoded proteins hypothesis as one possible cause for the existence of mtDNA. Clearly the mitochondrially encoded proteins show a higher hydrophobicity than their nuclearly encoded counterparts. Whether this is the reason for the maintenance of a mitochondrial genome, or an indirect effect resulting from the fact that retaining these genes in the mitochondria for some other reason lessens the selective pressure for mutations reducing their hydrophobicity, is still an open discussion.

4.2 Mitochondrial protein import machineries

4.2.1 Precursor protein targeting and transport to mitochondria

It is generally accepted that the vast majority of mitochondrial pro-teins are nuclearly encoded, synthesized in the cytoplasm and post-translationally imported (139). These precursor proteins possess specific signals, either a cleavable or an internal non-cleavable signal sequence, re-quired for targeting to the organelle. The cleavable presequences, named matrix targeting sequence (MTS) are normally N-terminal (with exceptions (140)), have an overall positive charge and a propensity to form an am-phiphilic α-helix of variable length (10-80 residues). MTS are enriched in arginines that form part of the recognition site for the matrix processing pep-tidase (MPP). It has been suggested that more than 60% of all mitochondrial proteins are synthesized with N-terminal extensions (141). The non-cleavable signals can be found, e.g., in mitochondrial inner membrane carri-er proteins where they are localized in hydrophobic stretches or charged loops. Other internal targeting sequences are found in mitochondrial β-barrel proteins, which have a conserved motif (termed β-signal) within the last β-strand (142).

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Targeting to the organelle is mediated by soluble factors, chaperones like the Hsp70, 14-3-3 proteins and Hsp90, which can bind to different precursor proteins, keeping them in an unfolded import-competent state (143-145). It should be mentioned that protein import into mitochondria may not only be post-translational. This view has in fact been challenged even before its proposition in the late 70s (146,147). It has been established that approxi-mately half of the mRNA molecules encoding mitochondrial preproteins and a fraction of cytosolic ribosomes co-localize with the mitochondrial outer membrane receptors (148,149). Indeed, specific proteins like CoxIVp (150) and fumarase (151) have been shown to be co-translationally imported into the mitochondria. Additionally, proteins can be exchanged between organelles, e.g., from mi-tochondria to peroxisomes, by vesicular trafficking (152). Interestingly, a molecular tethering complex has recently been found to mediate physical contact between ER and mitochondria (mitochondria-associated membrane, MAM) (153). In yeast, the so-called ERMES (ER–mitochondria encounter structure) complex is composed of the outer membrane proteins Mdm10, Mdm34, Gem1 (a Ca2+ binding GTPase), the integral ER membrane protein Mmm1 and the adaptor protein Mdm12. These connection points are im-portant for exchange of lipids and Ca2+ between these two organelles, as well as for regulating mitochondrial membrane dynamics, morphology, and the ER redox conditions. It is however unknown if proteins are transported via these junctions (154,155).

4.2.2 Translocase of the outer membrane

Mitochondria are double-membranous organelles constituted by four compartments: the outer membrane (OM), the intermembrane space (IMS), the inner membrane (IM) and the matrix.

For correct mitochondrial sorting, virtually all the mitochondrial proteins have to engage in the translocase of the OM (TOM complex), Fig. 4. This 400 kDa complex consists of the central receptor Tom22, the small Tom5, Tom6, Tom7 (α-helical tail-anchored proteins important for TOM complex assembly and stability), the β-barrel pore-forming Tom40, and the two loosely associated “initial” receptors Tom20 and Tom70 that act on the cyto-

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solic side. The TOM complex has the ability to recognize and transport pre-cursor proteins of diverse topologies, including proteins with a β-barrel structure, single and multiple TM α-helices, and soluble proteins of the IMS and mitochondrial matrix (156,157). Structural analysis by electron micros-copy has revealed that the TOM complex seems to contain two or three pore-forming regions. This pore can accommodate translocation of substrates with sizes ranging from unfolded polypeptide chain, α-helical segments or a pre-protein in a loop (two α-helices) (158,159). Tom20 and Tom70 are anchored to the OM via an N-terminal TM segment, exposing hydrophilic domains to the cytosol. These receptors have different substrate preference: Tom70 binds to Hsp70/Hsp90 carrying unfolded sub-strates, and mainly interacts with proteins containing internal targeting in-formation, such as metabolite carriers of the IM. Most N-terminal prese-quence-containing preproteins are targeted to the Tom20 receptor that pref-erentially binds to the hydrophobic face of the targeting sequence. Tom22 (an α-helical tail-anchored protein) exposes a highly negatively charged N-terminal domain to the cytosol, interacting with the positively charged sur-face of the presequences via electrostatic forces (after recognition by Tom20). Subsequently the precursor is transferred through the Tom40 pore (160). Tom22 also provides a docking site for the Tom70 and Tom20 form-ing a receptor platform and has a role in the stability of the TOM complex (161). The driving force for protein translocation across the OM results from the increasing affinity of the precursors with the receptors and translocation pore along the import pathway - affinity chain (162). For presequence-containing proteins, the transport across the OM is tightly coupled to the transport across the IM via the TIM23 complex, see below. The mitochondrial OM contains both β-barrel and α-helical proteins. The insertion of the former into the OM relies on the SAM complex (sorting and assembly machinery). After passing through the Tom40 channel to the IMS, the precursors of β-barrel proteins bind to small Tim chaperones (poly-petides of 8 to 12 kDa, characterized by a central “twin Cx3C motif”), such as Tim9-Tim10 or Tim8-Tim13. These guide the β-barrel proteins to the SAM complex (the β-signal directs the precursor proteins to Sam35 and Sam50 the essential core components of SAM complex) (163,164). The α-helical OM proteins (single or multi-spanning) are handled by a large oligo-

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meric complex formed by Mim1, Fig. 4. These proteins are probably not transported via the Tom40 pore, however the Tom70 receptor interacts with Mim1 in the import of multi-spanning α-helical OM proteins (165). Interest-ingly, although independent, the SAM and Mim1 complexes can cooperate in the assembly of some OM proteins such as the TOM complex (166,167). Recently the ability of the TOM complex to release proteins laterally into the OM was analyzed and it was determined that the TOM pore could in fact, open laterally and release specific protein segments into the mitochondrial OM. This is a surprising result considering the β-barrel structure of the pore (168). The precursors for the small Toms were found to depend on Mim1 for mem-brane insertion (169), while Tom22 uses TOM receptors for targeting, fol-lowed by the SAM complex for insertion (166). Interestingly, certain C-tail anchored proteins may not require a proteinaceous machinery for OM inte-gration (170), e.g., Fis1. However, the lipid composition of the target mem-brane is in this case critical in regulating membrane insertion (171). Post-translational modifications of the OM translocases by cytosolic kinases can also regulate preprotein translocation. Phosphorylation of Tom22 and Mim1 by casein kinase (CK2) stimulates their biogenesis, whereas phos-phorylation of Tom70 by protein kinase A inhibits its receptor activity (143,172). This demonstrates that the environment outside the mitochondri-on plays a crucial role for the protein import activity of the mitochondrial translocases. Interestingly, it has been recently discovered that Mdm10, a protein known to be involved in mitochondrial distribution, morphology, and a constituent of the ERMES complex (see 4.2.1) (173), is also a component of the SAM complex and involved in the assembly of Tom40 with Tom22 (174), Fig. 4. These findings reveal an unexpected relation between mitochondrial protein import, mitochondrial morphology and mitochondrial-ER tethering proteins, hinting at the existence of a larger organizing system that coordinates protein transport and membrane architecture at the interface of the ER and mito-chondria (175).

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4.2.3 The intermembrane space

The majority of IMS proteins use the mitochondrial IMS assembly (MIA) pathway. Precursors such as the small TIMs are transported from the cytosol through the TOM complex to the IMS, where Mia40 recognizes re-duced cysteine motifs in the precursor, Fig. 4. A ternary complex composed of the substrate, Mia40 and Erv1 (176) promote oxidation of cysteines, re-sulting in disulfide bond formation, important for folding and complete translocation of the precursor protein. Electrons flow from the substrate to Mia40, Erv1 and finally to the respiratory chain via cytochrome c. The IMS environment is more oxidizing than the cytosol but less than the ER and periplasm (177,178).

There are a few IMS proteins that engage the TIM23 complex pathway, in which a hydrophobic segment following the N-terminal presequence is later-ally released from TIM23 into the IM and further cleaved by proteases. Smac/Diablo and cytochrome b2 are examples of such precursors that are cleaved by Imp1 after integration of the TM segment into the IM (179,180). Other proteins follow a more complex topogenic process, e.g., the Mgm1, a dynamin-related GTPase that will be object of study in this thesis. The N-terminal presequence of Mgm1 engages in the TIM23 complex and is then cleaved by the MPP. The first TM segment is laterally inserted into the IM, but only for 30-40% of the molecules, and remains integrated into the mem-brane, originating the l-Mgm1 isoform. For the rest of the molecules, the first TM segment is not arrested in the IM and translocates to the matrix side. The following second TM segment is laterally inserted into the IM via the TIM23 complex and, once in the membrane, is processed by the rhomboid protease, Pcp1, releasing the s-Mgm1 isoform in the IMS (181,182). The biogenesis Mgm1 isoforms is also known as “alternative topogenesis”, Fig. 5. These two isoforms are thought to form heterodimers or higher order as-semblies in the IMS required for mitochondrial fusion (183).

4.2.4 Translocase of the inner membrane

4.2.4.1 The TIM22 complex - the carrier import pathway

The TIM22 pathway is mainly used by members of the solute carrier family, such as the ADP/ATP carrier (AAC) or the phosphate carrier (PiC),

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and by multi-spanning IM proteins such as Tim22, Tim23 and Tim17. All known substrates are MPs with an even number of α-helical TM segments and expose their N and C termini to the IMS. They carry internal signals to which Hsp70 and Hsp90 bind in the cytosol, and are then transferred to Tom70 (184). After traversing the OM through the TOM complex in a loop structure, these substrates are bound by the small TIMs (Tim9-Tim10 or Tim8-Tim13) that prevent aggregation in the aqueous IMS and guide them to the TIM22 com-plex, Fig. 4. The TIM22 translocation channel is composed by Tim22, the pore forming subunit, Tim54 that provides the docking site for the small TIMs-substrate complex in the IMS, Tim18 that supports assembly of Tim54 and Tim22, and Sdh3 (185,186). After docking to the Tim54 receptor, the carrier is inserted in a loop fashion in the Tim22 pore (the Tim22 is thought to form two pores, each with a diameter of 18 Å in the fully open state) (187). This channel is activated by the internal targeting signals of the pre-cursor and by the Δψ that exerts an electrophoretic force on the positively charged loops of the substrate (188). The precursor release from the TIM22 complex, its IM insertion and assembly follows an yet unknown mechanism (139).

4.2.4.2 The TIM23 complex

The majority of TIM23 complex substrates have N-terminal posi-tively charged presequences (189,190). After interaction with Tom20 and Tom22 in the cytosol, while the C-terminal part is still spanning the TOM complex (191), the precursor associates with the IMS domain of Tom22 and is further handed on to the TIM23 complex. The TIM23 complex is the major preprotein translocase of the IM with 10 subunits so far identified: Tim50, Tim23, Tim17, Tim44, Pam18, Pam16, mHsp70, Mge, Tim21, and Pam17 (192), Fig. 4. Tim50 is a single spanning IM protein, exposes its large C-terminal domain to the IMS and functions as an import receptor for the incoming polypeptide (193). In addition it plays a role in regulating the permeability of the import channel (194,195).

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Tim23, the central component of the TIM23 complex, has four TM domains and exposes a 100 residues long N-terminal hydrophilic region to the IMS. There is evidence that the first 20 residues of this N-terminal region may insert into the OM, however this is still a matter of debate (168,196,197). The second part of this N-terminus contains an essential coiled-coil domain, which is critical for the dimerization of Tim23, interaction with Tim50 and substrate binding (198). Reconstitution experiments using recombinant Tim23 have shown that it is able to form a cation-selective channel with an inner diameter of 13 Å (199). The C-terminal part of Tim23 is inserted into the IM and interacts with Tim17. Tim23 and Tim17 together with Tim50 form the membrane-embedded core of the TIM23 complex, TIM23CORE. The Tim17 subunit, homologous to Tim23, also spans the IM four times but its function is not well understood and there are no reports of Tim17 forming a voltage-gated channel on its own (200). However, Tim17 is required for precursor transport, voltage-gating of the TIM23 complex (199), and both Tim23 and Tim17 have been crosslinked to a translocating (arrested) prepro-tein. It is, therefore possible that Tim17 plays a critical role in the formation and stabilization of the channel (201), as patch-clamp experiments using mitochondrial IM fused to liposomes revealed that the twin pore formed by Tim23 reorganized into a single pore upon Tim17 depletion (202). Tim44 is a peripheral IM protein facing the matrix side. Its N-terminal part interacts with the Tim17-Tim23 core (203), while its C-terminal domain interacts with cardiolipin (204). On the matrix side, Tim44 interacts with the chaperone mtHsp70, which is required for unfolding and vectorial move-ment of the translocating chain towards the matrix (Hsp70 cycles between ATP and ADP bound states that have low and high affinity, respectively, for the substrate). Tim44 also interacts with the Pam16-Pam18 subcomplex (205). Tim44 plays a key role in anchoring mtHsp70 to the import channel, which allows a precise coordination of mtHsp70 association/dissociation with the substrate and prevents premature release and backsliding. Mge1 is a soluble matrix protein that releases ADP from mtHsp70, so that new cycle can start (206,207). Pam18, a J protein, is integrated into the IM, exposes its N-terminal domain to the IMS, possibly mediating interactions with Tim17 (208). The J domain of Pam18 is exposed to the matrix side and stimulates the ATPase activity of mtHsp70, triggering binding to the translocating chain as it emerges in the matrix (209).

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Pam16 is a matrix protein peripherally attached to the IM by its hydrophobic N-terminal part. It carries a J-like domain and inhibits the stimulatory effect of Pam18 on mtHsp70 (210). Pam16 and Pam18 form a very stable complex through the J and J-like domains (211). Conformational changes between these two subunits may regulate interaction of Pam18 with mtHsp70, de-pending on translocating substrate availability (212). Tim44, Pam16, Pam18, Hsp70 and Mge1 are referred to as the presequence translocase associated motor (PAM) subunits (213). Tim21 is anchored to the IM by a single TM segment. It exposes its C-terminal domain to the IMS where it interacts with the IMS domain of Tom22 competing with the presequence binding. Thereby it promotes the onward transfer of the substrate protein to the TIM23 complex (214). Tim21 possibly plays a role in mediating the interaction of TIM23 complex with the respiratory chain supercomplex formed by the cytochrome bc1 and cyto-chrome c oxidase complexes, maximizing perhaps the electrochemical pro-ton gradient effect on preprotein translocation (215,216). Tim21 may also be involved in regulating the association of the import motor, PAM, with the membrane region of the TIM23 complex, which regulates the translocation of precursor proteins to the matrix (156). Tim21 and Pam17 are the only non-essential subunits of the TIM23 complex (217). Pam17 is anchored to the IM facing the matrix side. It promotes association of the Pam16-Pam18 complex with the Tim17-Tim23 core, and affects the stability of the former (218). Pam17 and Tim21 may have opposing roles in regulating translocation of the preprotein to the matrix by the TIM23 com-plex (156,217). At the gates of the TIM23 complex, Tim50, alone or together with Tim23, forms the receptor binding site for presequences (194). The Δψ drives trans-location of the presequence across the IM possibly by exerting an electro-phoretic force on the presequence or by stimulating the opening of the TIM23 complex channel (192,219). After the presequence reaches the ma-trix, Δψ is no longer required. If the preprotein is to proceed forward towards the matrix, after processing of the presequence, the translocating substrate is recognized by Tim44 and then bound by mtHsp70. These in a concerted action with the other PAM subunits (220) drive the vectorial translocation of

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the protein. Whether mtHsp70 works as either a stepping motor or actively pulls the translocating substrate to the matrix is still matter of debate (221,222). The TIM23 complex can function in both translocation and membrane inser-tion modes. It can recognize hydrophobic segments that contain “stop trans-fer” signals, arrest the precursor during the import at the level of IM, and insert them laterally into the lipid bilayer (219). Precursors with short seg-ments between the presequence and the “stop transfer” signal, and without tightly folded domains, do not require the PAM subunits for translocation and membrane insertion; in such cases the Δψ provides sufficient driving force (223,224). Currently there is an ongoing debate concerning two proposed models for the association of the TIM23 complex subunits, depending on the different translocating substrates: - the modular model suggests that the TIM23 complex exists in two forms. One form that is composed of the Tim23, Tim17, Tim50 and Tim21 subu-nits, denoted as TIM23SORT, and mediates presequence translocation to the matrix and lateral insertion of hydrophobic domains into the IM. The second form, TIM23-PAM, is free of Tim21 but is associated with the import motor, PAM, and handles translocation of proteins to the matrix. The TIM23 com-plex dynamically switches between these two forms. Tim21 and Pam17 play an important role in the interconversion between these two forms. Tim21 is involved in the association of TIM23SORT with the respiratory chain com-plexes and Pam17 leads to release of Tim21 from the TIM23SORT followed by recruitment of the PAM complex (156); - the single entity model proposes that all the components including the im-port motor are assembled (empty state, E) and will by default complete translocation of a presequence-containing precursor to the matrix (matrix translocating state, M). If a lateral sorting signal, in the substrate, is recog-nized, the translocation to the matrix stalls and the precursor protein is later-ally released to the IM (lateral insertion state, L) (217). A series of not so well defined conformational changes is in the basis of the altera-tion/conversion between the different states.

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Both models acknowledge the dynamic nature of the TIM23 complex and its flexibility in guiding differentially sorted substrates. Interestingly, the modu-lar model envisions the TIM23SORT form as the default state. The PAM com-ponents are recruited only for protein translocation to the matrix (225). The single entity model, however, considers the matrix translocation mode, M, as the default state, with specific stimulus/signals from the translocating protein triggering the formation of the lateral sorting state, L (201). The exact mechanism by which the TIM23 complex recognizes and inte-grates hydrophobic segments into the IM is not as well understood as it is for the Sec translocon (see section 3.1). Recent studies have shown that the TM helix 2 of Tim23, although not inherently amphipathic by sequence analysis, in the Tim23 channel, faces on one side a polar environment in the interior of the protein-conducting channel, and on the other side, a more nonpolar region. TM2 therefore seems to line an aqueous protein-conducting channel through the bilayer (226,227). Interestingly, the two TMs of the eukaryotic ER and bacterial Sec-type translocons that allow lateral gating, TM2B and TM7 (70) are similarly nonamphipathic. Understanding the TIM23 complex gating mechanism is still an ongoing quest. Although it has been determined that presequences promote channel opening, the involvement of other components such as Tim17 and Tim50 is not well understood. How the pore is maintained sealed, preserving mem-brane potential in the resting state or upon substrate translocation is also an open question (195,227,228).

4.2.4.3 The OXA translocase It is currently accepted that the TIM23 complex can only insert sin-

gle spanning MPs into the mitochondrial IM - a process termed the “stop-transfer pathway”. Typically, nuclear-encoded mitochondrial IM proteins that contain a presequence and multiple TM segments are handled by a third IM translocase, the OXA translocase (219). These substrates are post-translationally targeted to the mitochondria, engage the TIM23 complex and are translocated all the way into the matrix, whereupon they are inserted back into the IM by the Oxa1 – the “conservative sorting pathway” (229).

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The Oxa1 insertase contains a hydrophobic core of 5 TM domains with the N-terminus facing the IMS (96,230) and belongs to the YidC/Alb3/Oxa1 family. Interestingly, Oxa1 can replace YidC in bacteria, revealing a func-tional conservation and its bacterial origin (231). Oxa1 is also responsible for the co-translational integration of mitochondri-ally encoded proteins into the IM (232), Fig. 4. Oxa1 contacts with nascent polypetides very early on in their synthesis through the interaction of its C-terminal domain with the translating mitochondrial ribosomes (233). Other components such as Mba1 (a mitochondrial ribosome receptor) (234), Cox18 (homologous to Oxa1) (235) and Mdm38 (236) are involved in this process and in the subsequent co-and post-translational assembly events.

Recently, it has been proposed that Oxa1 may be involved in the biogenesis of mitochondrial IM carriers together with the TIM22 complex (237).

Mitochondrially encoded proteins as well as conservatively sorted proteins, follow the positive-inside rule (see section 3.1.2.2): the negatively charged flanking residues located in the loops or termini are more distributed towards the IMS side, while the positively charged residues tend to be on the matrix side (238,239). For the conservatively sorted substrates, the membrane po-tential is required for post-translation translocation of negatively charged domains across the IM during Oxa1-mediated insertion (240).

4.2.4.4 Stop-transfer vs. Conservative sorting The fate of N-terminally targeted mitochondrial IM proteins is de-

cided at the level of the TIM23 complex. Whether these will follow the stop-transfer pathway or will be conservatively sorted to the IM, depends on the determinants that the TIM23 complex uses to differentiate between domains that are “arrested” in the IM or “translocated” across to the matrix (219). Some of these determinants have been identified: laterally sorted preproteins tend to be somewhat more hydrophobic than those that are conservatively sorted (127,241); downstream charges of the TM segment are important for insertion into the IM (242); proline residues are typically absent in arrested TM domains, but present in conservatively sorted segments (241); the posi-tive-inside rule is only present in the proteins that follow the conservative

41

sorting pathway, which perhaps works as a differentiation signal used by the TIM23 complex (238). It is important to note that the stop-transfer and the conservative sorting pathways are not mutually exclusive and may cooperate in the membrane integration of proteins with complex topology, as observed by Bohnert et al. (243).

21

44

50

22705

667 20

40

3735

50 Mdm10 Mdm10

ERMESMim1

17 23

1618

70ATP

Mge1

18

Pam17 Mia40 Erv1

22 18 54Oxa1

OM

IM

IMS

Matrix

Δψ

+++

-SH-SH

9 9 91010 10

22

TIM22 complex

TIM23 complex

MIA-ERV system

OXA1 complex

SAMcomplex

813

8 813 13

TOMcomplex

Tims

Figure 4. Mitochondrial protein import machineries. The TOM complex is the entry gate for the majority of the mitochondrial proteins with the exception of α-helical outer membrane (OM) proteins that require Mim1 for membrane integration. β-barrel proteins are inserted into the OM with the concerted action of the TOM com-plex, small TIMs and the SAM complex. Mdm10, constituent of the ERMES is also a component of the SAM complex. Presequence-containing proteins are directed to the TIM23 complex and can either be translocated to the matrix or inserted into the inner membrane (IM) from the TIM23 complex (stop-transfer pathway). Polytopic IM proteins that lack presequence are inserted into the IM via the TIM22 complex. Small Tim proteins after reaching the intermembrane space (IMS) are trapped by the MIA-ERV disulfide relay system. Oxa1 inserts proteins into the IM from the matrix side. Its substrates may be encoded in the mitochondrial DNA, or arrive to the ma-trix through the TIM23 complex (conservative sorting pathway). Adapted from (244).

42

4.3 Mitochondrial protein import and the effect of lipids

Mitochondria are important sites for lipid synthesis and metabolism. While PA, PG and CL can be synthesized in the mitochondria, PC, PS and PC are synthesized in other organelles and transported into the mitochondria (11). Mitochondrial lipids have important functions on the import of proteins into the mitochondria.

Cardiolipin is a signature lipid for the mitochondrial IM, and has been impli-cated in the stability of the respiratory chain supercomplexes (245). It is therefore involved in maintaining the mitochondrial inner membrane poten-tial (246), regulating the ADP/ATP carrier activity (247), and the efficiency of MP integration by TIM23SORT (224). Co-translational insertion of mito-chondrially encoded proteins is also affected by the lipid environment, i.e., the transient binding of positively charged residues of a translating mito-chondrially encoded protein to negatively charged phospholipids, PG or CL, can prevent the transfer of those residues across the IM (248). The deletion of Tam41, a recently identified protein involved in the biosyn-thesis of PG and CL, also affects protein import into mitochondria (249). The mitochondrial OM, more fluid than the IM, is an important point of contact with the outside environment. Interestingly, subunits of the SAM complex have been implicated in the metabolism and in the transport of phospholipids together with constituents of the ERMES complex (155). Re-cently Ups1 and Mdm35 were proposed as the lipid transfer complex of phosphatidic acid from the mitochondrial OM to the IM (250) Surprisingly, CL has been detected in the OM, and CL mutants seem to af-fect stability of the TOM and SAM complexes, causing a slower assembly of β-barrels and α-helical OM proteins (251).

4.4 Mitochondrial protein import and the effect of MICOS/MINOS/MitOS

The mitochondrial IM is divided in two structurally and functionally

distinct subdomains: the inner boundary membrane and the cristae mem-

43

branes. The former aligns and faces the OM and is enriched in protein im-port systems. The cristae membranes that result from invaginations of the inner boundary membrane towards the matrix, form tubular protrusions and harbour the respiratory chain complexes. These two subdomains can connect forming very short tubules, termed cristae junctions (CJ). Additionally, it has been observed that the inner and the outer membranes can establish firm contact sites (CS) with little or no space in between. These contact sites are thought to be involved in mitochondrial fusion and fission, lipid transport and protein import (252).

How the mitochondrial morphology tunes the differential distribution of IM proteins, and organizes functionally distinct environments is poorly under-stood. Very recently, a protein complex involved in the formation of CJ and CS has been simultaneously reported in three independent publications (175,253,254). This complex, termed MICOS (mitochondrial contact site complex), MINOS (mitochondrial inner membrane organizing system) or MitOS (mitochondrial organizing structure), is composed of six proteins: mitofilin/Fcj1, Mio10/Mcs10/Mos1, Aim5/Mcs12, Aim13/Mcs19, Aim37/Mcs27 and Mio27/Mcs29/Mos2. The MICOS/MINOS/MitOS components reside in the inner boundary membrane and form physical and functional contacts be-tween the OM and IM (175,253,254). These components interact with the OM by binding to the SAM complex. Moreover, mitofilin/Fcj1 interacts with both Mia40 and Tom40 and affects the Mia40 pathway. It is of interest to understand the basis of these interac-tions and how this complex can regulate not only protein import from the cytosol, but also trafficking of proteins between the different IM subdomains (175,253,254).

44

5. Membrane protein maturation – focus on protein processing and membrane dislocation

The process of MP assembly is invariably connected with protein maturation events such as processing, modifications and assembly of pro-teins in complexes. Protein processing or dislocation from the membrane may alter protein sorting, condition its maturation and final location, which is especially important in the field of protein translocation. The cleavage of a targeting sequence (when present) is a process shared by protein targeting to the bacterial, ER and mitochondrial membranes. Cleava-ble signal peptides are processed by signal peptidases. In bacterial cells there are several signal peptidases but the most commonly used is the signal pep-tidase I, SPI (also known as leader peptidase, Lep), which is homologous to the catalytic subunit of the signal peptidase complex (SPC) in the ER. The E. coli SPI has two TM segments and a large periplasmic C-terminal domain containing the active site, which is predicted to be located at or near the exo-cytoplasmic membrane surface (255). In mitochondria the Imp1 and Imp2 enzymes, referred in section 4.2.3, are related to SPI (256). They are located in the IM, exposing the C-terminal active site to the IMS and are involved in the cleavage of hydrophobic sorting signals, releasing the mature form of the substrate to the IMS (257). In mitochondria, the N-terminal signal peptide MTS is in the majority of the cases removed by the mitochondrial presequence peptidase, MPP, a matrix-soluble heterodimer that consists of two homologous proteins, Mas1, which carries the catalytic domain, and Mas2. Interestingly, it has been demonstrat-ed that for some precursor proteins additional processing by other proteases can occur after MPP cleavage. Oct1 is a soluble matrix protease that cleaves an additional octapeptide from the precursor after MPP processing. Another example is the novel mitochondrial matrix aminopeptidase, responsible for cleaving a single amino acid (namely Phe, Tyr or Leu) from the precursor after MPP cleavage (257). These additional processing events generate ma-ture proteins with a more stable N-terminus and consequently with a longer half-life compared to the intermediate precursor generated by the MPP (141) – the so called N-end rule posits that certain amino acids present at the N-terminus are more or less destabilizing and therefore differentially affect protein half-life (258,259).

45

Rhomboid proteases are composed of six TM segments and catalyze prote-olysis of integral membrane proteins within the lipid bilayer. The crystal structure of a bacterial rhomboid protease, GlpG, revealed an aqueous chan-nel that leads to the active site located below the membrane surface (260). In the bacterial and ER membranes, rhomboid proteases are involved in protein quality control events, cleaving improperly assembled MPs (255). In the mitochondrial IM of yeast, the rhomboid protease Pcp1 has two known sub-strates, being involved in the biogenesis of Mgm1 isoforms (see section 4.2.3) and in the maturation of the cytochrome c oxidase, Ccp1 (261,262). The mammalian counterpart of Pcp1 is PARL and is involved in mitochon-drial morphology, stress response and the generation of apoptotic signals (263). AAA-ATPases (ATPases Associated with diverse cellular Activities) are especially important to the field of MP biogenesis due to their ability to dis-locate their substrates from the membrane environment. AAA-ATPases can be localized in the cytosol or at the bacterial or mitochondrial IM, among other organelles (255,264) (265). The mitochondrial IM harbors two types of AAA proteases with catalytic sites exposed to opposite membrane surfaces. The m-AAA protease is active in the matrix and is composed of the homolo-gous subunits Yta10 and Yta12 in yeast (266) and of paraplegin and AFG3L2 in humans (267). The i-AAA protease is composed of Yme1 subu-nits and exposes its catalytic sites to the IMS (268). The mitochondrial IM AAA proteases are involved in protein surveillance events, degrading unassembled subunits of the respiratory chain complexes or IM proteins that expose unfolded segments. The m-AAA protease acts on misfolded domains exposed to the matrix side, whereas the i-AAA protease on those exposed to the IMS side, however they also share overlapping sub-strate specificity (265). These proteases seem to recognize the folding state of a solvent-exposed domain of the substrate, and a 10-20 amino acid long segment protruding from the membrane is enough for the AAA protease to bind and trigger degradation (268). The crystal structure of the bacterial AAA protease FtsH (269) sheds light on the mechanism that allows extrac-tion of substrate from the membrane bilayer. The coordinated ATP-dependent conformational changes of the AAA protease subunits allow sub-strate threading through the central pore of the protease and translocation towards the proteolytic site. The substrates are completely degraded to oli-

46

gopeptides, which are subsequently either converted to amino acids by mito-chondrial proteases or released from the mitochondria (265,270). In addition to degrading substrates, the AAA proteases also have the ability to specifically cleave certain proteins. Examples are the yeast mitochondrial ROS scavenger Ccp1 and the mitochondrial ribosomal component MrpL32 (271,272). Ccp1 is nuclearly encoded and targeted to the mitochondria by a presequence that is cleaved off in a process that involves both the m-AAA and Pcp1 (261). In the model proposed for Ccp1 biogenesis, the first TM segment of Ccp1 partitions into the IM from the TIM23 complex, and is then dislocated from the membrane towards the matrix by the m-AAA protease in an ATP-dependent manner. This vectorial movement allows correct posi-tioning of a second TM segment of Ccp1 in the IM. The m-AAA protease cleaves Ccp1 once in the middle of its first TM segment, and the second TM (now in the IM) is processed by Pcp1. Finally, the mature form of Ccp1 is released to the IMS (262), Fig. 5. The m-AAA protease is responsible for removing the N-terminal targeting sequence of MrpL32 upon import into the matrix. In this case, the m-AAA does not recognize a specific sequence, but starts degrading the unstructured presequence until reaching the tightly folded mature portion of MrpL32. The processing of MrpL32 is necessary for its assembly into ribosomes (271,272). Inactivation of AAA proteases, specifically the m-AAA, results in pleiotropic cellular defects including loss of respiratory competence in yeast. This is mainly caused by the impaired processing of MrpL32 (273) rather than the deleterious effects that result from accumulation of non-native mi-tochondrial MPs. Recently, Bcs1, a mitochondrial IM AAA-ATPase that does not have proteo-lytic activity, was reported to translocate a folded substrate. The Rieske FeS protein (Rip1), carrying a 2Fe-2S cluster, is transported from the matrix side and is integrated into the cytochrome bc1 complex in the IM by Bcs1 in an ATP-dependent manner. Bcs1 forms an oligomeric structure with a central channel that might serve as a translocation pore (62,274). As discussed, AAA proteases are involved in protein dislocation and substrate unfolding prior to degradation/processing, while Bcs1 performs its function by an al-most contradictory/opposite mechanism. Homologues of Rip1 in bacteria and chloroplasts are transported via the Tat system (275,276) (see section 3.) across the bacterial IM and the thylakoid membrane, respectively. So far, a

47

mitochondrial translocase responsible for transport of folded substrate across the IM has not been found. It is possible that Bcs1 has taken over this func-tion of a long lost Tat system in mitochondria.

I87 M113 T160

Pcp1p

Mgm1

S117W128

K122A96

T160

Pcp1p

Cox5aT MFP

TIM23complex

Matrix

IMS

m-Ccp1

AE

S19 Y37 S67

Pcp1p

Ccp1

Pcp1

s-Mgm1

l-Mgm1

Pcp1

i-Ccp1

p-Ccp1Mgm1/p-Ccp1

IMB CF

D

m-AAA

Figure 5. Maturation pathways for Ccp1 (right) and Mgm1 (left). The first hydro-phobic segment (grey) in p-Ccp1 triggers insertion from the TIM23 translocon into the inner membrane (A). The membrane integrated form is dislocated from the membrane and concomitantly cleaved in the middle of the hydrophobic segment by the inner membrane (IM) m-AAA protease, generating the intermediate i-Ccp1 (B). Finally, the rhomboid cleavage region (green) in i-Ccp1, now in the IM, is cleaved by the rhomboid protease Pcp1 (C), releasing the mature form m-Ccp1 in the inter-membrane space (IMS) (261,262). In the alternative topogenesis model for import of Mgm1, the first hydrophobic segment (black) integrates into the membrane in 30-40% of the molecules (D), resulting in the membrane-anchored, long isoform, l-Mgm1. For the remaining molecules, the first hydrophobic segment translocates into the matrix (E) and the second hydrophobic segment (blue) integrates into the IM and is processed by Pcp1 (F), generating the short isoform, s-Mgm1 in the IMS (181).  

48

6. Quantitative studies of membrane partitioning As explained in the previous chapters, the integration of a protein in-

to a biological membrane such as the bacterial, ER and mitochondrial mem-branes requires several steps: targeting, interaction with receptors followed by engagement of the translocation machinery, partition into the lipid envi-ronment and folding/assembly of the MP.

I finalize this introductory part with a note on the study of how TM α helices come into thermodynamic equilibrium with the lipid environment.

6.1 Hydrophobicity scales

Translocation systems such as the SecYEG/61 or the TIM23 com-plexes can somehow recognize bona fide TM segments in translocating pro-teins. These will partition into the lipid environment while soluble domains will be completely translocated across the membrane. The mechanical work that this description suggests is, in fact, driven by a series of thermodynami-cally favorable reactions. In the case of TM domains, their free energy in a lipid environment is lower than in an aqueous medium. Hydrophobicity scales appeared from the analysis of the propensity of an amino acid-like molecule to partition into a solvent that mimics a membrane or into an actual membrane. Some representative examples of scales ranking the hydrophobicity of the each of the 20 natural amino acids are: - Kyte and Doolittle (277) developed a computer program that takes into account the hydrophilic and hydrophobic properties of each of the 20 amino acid side chains and determines the average hydropathy within a segment of predetermined length; - Radzicka and Wolfenden (278) created an early hydrophobicity scale based on partitioning of analogs of amino acid side chains, between water and cy-clohexane; - Wimley and White (279) measured the partitioning of model peptides be-tween aqueous and membrane-mimetic apolar phases. A single test amino acid was incorporated into the guest position in the host peptides with the

49

sequence Ace-WLm-X-LL where X is any of the 20 naturally occurring ami-no acids and m=1-6. The partition coefficients of the test residue were de-termined by evaluating the differential solvation in water versus octanol or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC). Octanol mim-icked the membrane environment whereas POPC represented the membrane interface region. The whole-residue hydrophobicity values (side chain plus backbone) showed that all charged residues were highly unfavorable in parti-tioning in the membrane interface, while aromatic residues were highly fa-vorable.

6.1.1 A “biological” hydrophobicity scale

The step towards a more realistic description was the development by von Heijne and co-workers of a “biological” hydrophobicity scale (280,281). This scale was derived from the quantitative measurements of the incorporation of model TM segments into the ER membrane by the mamma-lian Sec61 translocon. The model TM segments (H segments) were 19 resi-dues long, composed of Ala and Leu and flanked by two reporter glycosyla-tion sites, one on each side (282). Ala/Leu segments are known to form α-helices (283). An α-helix contains 3.6 amino acids per turn and each residue lengthens the helix by 1.5 Å, giving a total of ~ 30 Å for a 19 residue long segment, which corresponds to the thickness of the hydrophobic core of the membrane. Different H segments were engineered as an additional third TM domain in the E. coli protein Leader peptidase (Lep). Lep has two natural TM helices and inserts into the membrane in a well-defined orientation with both the N- and C-terminus in the lumen of the ER (284). The Lep variants were tested in vitro for membrane insertion of the H segments into the mammalian ER membrane, by assaying the modification status of the two glycosylation sites (282). The hydrophobicity requirement for membrane partitioning was ana-lyzed by varying the number of Ala vs. Leu in the H segment. The results demonstrated that the translocon–mediated insertion has the appearance of a thermodynamic equilibrium process and therefore the insertion of H seg-ments was used to derive an apparent free energy of membrane insertion (ΔGapp). The effect of each of the 20 natural amino acids on membrane inser-tion was assessed by placing a single test residue in different positions along the H segment (280,281).

50

The “biological” hydrophobicity scale (280,281) correlated surprisingly well with the Wimley and White octanol scale (279). The free energy profile of insertion for each residue along the TM span matched the physicochemical properties of the membrane, implying that both side-chain chemistry and specific position along the TM affect membrane insertion. Interestingly, the helical structure of a TM segment is a requirement for membrane insertion as indicated by the high free energy values required for placing a Pro residue in the middle of the membrane (see section 3.1.2.1). Membrane insertion of TM segments seems to be governed by a partitioning process of these TMs between the polar interior of the Sec61 channel and the apolar lipid surroundings (113). Although the exact mechanistic details of this process are not fully understood, it is possible that the translocon as a highly dynamic machine allows, during its open state, the translocating pol-ypeptide to rapidly sample the translocon-bilayer interface. Apparently, the Sec61 translocon not only provides an aqueous tunnel across the membrane, but also allows consecutive segments of the nascent polypeptide to make lateral excursions from the channel to probe the lipid environment (285,286). Similar experimental “biological” hydrophobicity scales have been obtained for a few other systems and show similar qualitative effects of the different residues on membrane insertion. Quantitative analysis showed, however, some differences. To achieve membrane insertion in the mammalian ER membrane mediated by Sec61, a segment with 16 Ala and 3 Leu is required in vitro (280,281). The same process in vivo in yeast requires 15 Ala and 4 Leu (287). For the YidC-mediated insertion into the bacterial IM, 17 Ala and 2 Leu are necessary (288). Based on the values obtained from the “biological” hydrophobicity scale of the mammalian Sec61 (280,281), a web server is now available at http://dgpred.cbr.su.se/. This server provides ΔGapp profiles of the membrane insertion of amino acid sequences. Applying this scale to natural protein sequences has revealed that the mammalian soluble proteins and single spanning MPs have very distinct free energies of membrane insertion with positive values for the soluble proteins and negative for the single spanning MPs. Interestingly, about 25% of the TM helices in multi-spanning MPs of known three-dimensional structure were predicted to have an unfavorable apparent free energy for membrane insertion. This suggests that membrane

51

insertion of TM helices in multi-spanning MPs may depend on interaction between neighboring TM helices and flanking loops (289) (see section 3.1.2.2). In order to develop predictors that are sensitive enough to correctly distinguish TM segments from non-TM segments in protein sequences from different organisms and different membrane environments, hydrophobicity scales have to be derived from systems that mimic as closely as possible the membrane of interest (290,291).

52

Projects

7. Aims The work carried out in this thesis had the general aim of under-

standing membrane protein assembly/biogenesis in the bacterial and mito-chondrial inner membranes. In the state of the heart of the protein translocation field, a comparative anal-ysis between the sequence determinants that governs protein insertion into different membranes is missing. This thesis had the aim of establishing ex-perimental systems that allow probing protein insertion into the bacterial and mitochondrial inner membranes by the SecYEG and TIM23 complexes, respectively. With these experimental set-ups, “biological” hydrophobicity scales for these two systems were determined and compared with the hydro-phobicity scale previously obtained for the Sec61-mediated insertion into the ER membrane (280,281). From these three systems, we aimed to understand in a greater detail the process of protein sorting mediated by the TIM23 complex. To this end, we investigated the role of charged flanking amino acids in regulating mem-brane insertion of a TM segment. In addition, we aimed to study the process of TM segments dislocation from the membrane by the m-AAA protease. A quantitative relation between the hydrophobicity of the TM segments that the TIM23 complex is capable of integrating, and that the m-AAA protease is able to dislocate into/from the mitochondrial IM the membrane, respec-tively, was established.

Finally, an in vivo experimental approach has been developed with the aim of assessing the sorting route taken by some of the TIM23 complex sub-strates, i.e., the conservative sorting vs. stop-transfer pathways.

53

8. Summary of the papers Papers I & II.

Certain protein translocation machineries are multifunctional, i.e., are able to distinguish soluble from membrane proteins and to transport pol-ypeptides between different environments. The Sec-type translocons and the TIM23 complex are examples of such machineries that can interconvert be-tween a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that by lateral opening inte-grates proteins into the membrane. The signals that trigger alternation between these two states are best under-stood for the Sec-type translocons. Hessa et al. tested insertion of designed TM segments into the ER membrane by the mammalian Sec61 translocon. These segments, termed H segments, were 19 amino acid long of Ala and Leu residues. In addition, the positional effects of specific amino acids along the H segment as well as the effect on membrane insertion of charged resi-dues immediately flanking the H segment were analyzed. As result, a “bio-logical” hydrophobicity scale giving the apparent free energy for membrane insertion of each of the 20 natural occurring amino acids was derived (280,281). Although the Sec61 complex shares with the SecYEG and the TIM23 com-plexes the functional ability to recognize TM segments in translocating pol-ypetides, the differences between these systems (summarized in Table 1) are sufficient to question the similarity of the molecular code for protein inser-tion into these different membranes. In papers I and II we developed two in vivo assays to probe the behavior of the same designed H segments in the E. coli SecYEG and in the yeast mito-chondrial TIM23 complexes, respectively. The results, summarized in Table 2, suggest that: - in all systems, the hydrophobicity of the H segments conditions membrane insertion, which implies that the interaction of the TM segments with the

54

lipid bilayer drives insertion into the membrane. However, the threshold hydrophobicity required for 50% insertion of the H segment of composition nLeu/ (19-n)Ala into the different membranes (n50%) differ. TM segments with a lower hydrophobicity, n50% ~ 2 Leu, are able to partition into the bac-terial IM, whereas the mitochondrial IM requires n50% ~ 3 Leu or n50% ~ 6 Leu (depending on the substrate), compared to n50% ~ 3/5 Leu in the ER membrane in the mammalian and yeast systems, respectively (280,281,287). This lower requirement of hydrophobicity for insertion into the E. coli IM is further confirmed by the fact that natural, marginally hydrophobic TM seg-ments insert better in this membrane than in the ER membrane and is also supported by the similar n50%= 1.5 obtained for the YidC-mediated insertion into the E. coli IM (288). The high hydrophobicity required for insertion into the mitochondrial IM when compared with the other membranes is surpris-ing, since mitochondrial IM proteins with an N-terminal presequence are known to be less hydrophobic than the bacterial or ER MPs (127) (however see Paper IV); Table 1. Comparison between the protein translocation properties of the Sec61, SecYEG and TIM23 translocons.

Translocon Properties Sec61 SecYEG TIM23 Co-/Post-

translation Both (yeast)/Mainly

Co- (mammals) Both Mainly Post-

Lipid composition

PC, PE PE, PG, CL PE, PG, CL

Protein/Lipid 50/50 70/30 70/30 Membrane potential

No Yes Yes

ATP requirement

No: protein synthesis drives co-translational

translocation Yes: SecA Yes: PAM complex

Positive-inside rule

Yes Yes Yes for mt-encoded and conservatively

sorted proteins

55

- comparing the “biological” hydrophobicity scales of these three systems further confirms that protein insertion follows the physicochemical proper-ties of the membrane where apolar residues are preferred in the middle of the H segment while polar ones are well tolerated outside the membrane hydro-phobic core; - aromatic residues (Trp, Tyr) are well tolerated at the extremes of the H segment, which is expected as seen by the stabilizing/positioning role of the aromatic belt (see section 3.1.2.1) for protein integration into membranes; - Pro residues when located in the middle of the H segment hinder insertion, which indicates that an α-helical structure of the H segment is an important determinant for translocon recognition and lateral partitioning. The Pro ef-fect is especially dramatic in the mitochondrial system; - charged residues strongly affect membrane insertion when positioned in the middle of the H segment, but are better tolerated when located near the ends for the three systems. Negatively charged residues (Asp, Glu), however, are only tolerated in the C-terminal part (IMS) of the H segment for insertion into the mitochondrial IM; - the effect of charged residues flanking the H segments is quite interesting in the mitochondrial IM system since positive charges (Arg, Lys) in either the N- or C-terminal part of the TM segment strongly promote insertion, whereas the Sec61 translocon, following the positive inside rule, promotes insertion of segments that have positive charges at the cytoplasmic end. The fact that the positive-inside rule is only found in mitochondrial IM proteins that are inserted from the matrix side is consistent with our findings (238). Negatively charged flanking residues (Asp, Glu) hinder insertion when lo-cated on the matrix side in the mitochondrial IM system and on the lumenal side of the ER membrane. Analyzing the distribution of charged flanking residues in MPs inserted by the TIM23 complex showed that negatively charged residues (Asp, Glu) are under-represented on the matrix side, whereas positive charges (Arg, Lys) are abundant both on the matrix and the IMS side. Whether specific properties of the TIM23 complex, the presence of membrane potential (negative in the matrix and positive in the IMS) or the negatively charged lipid, CL, in the mitochondrial IM underlie these effects of charged flanking residues is not clear.

56

Table 2. Comparison of the H segment recognition by the Sec61, SecYEG and TIM23 translocons.

H Segment Sec61 complex SecYEG complex TIM23 complex

Hydrophobicity n50%=3 (mammal) n50%=5 (yeast) n50%=1.9 n50%=5-6 or n50%=3

Proline Unfavorable in the middle

Same as in ER Same as in ER

Aromatic resi-dues

Favorable near the ends of H seg.

Same as in ER Same as in ER

Positively charged residues

Unfavorable in the middle of H seg.

Same as in ER Same as in ER

Negatively charged residues

Unfavorable in the middle of H seg.

Same as in ER

Unfavorable in the middle and N-term

part of H seg. Positively

charged flanking residues

Favorable in the cytoplasmic end of

H seg. --------- Favorable in both

ends of H seg.

Negatively charged flanking

residues

Unfavorable in the lumenal end of H

seg. ---------

Unfavorable in the middle and N-term

part of H seg. The reporter systems used for determining insertion vs. translocation of H segments in the ER, bacterial or mitochondrial IMs, are different and the two latter measure membrane insertion/translocation in an indirect way. There-fore, it cannot be excluded that the results obtained might be influenced to some extent by the nature of the system or model protein used. Although quantitative differences were found, the molecular codes for mem-brane insertion recognized by the Sec61, the SecYEG and the TIM23 trans-locons, correlate surprisingly well. Finally, the effect of the PAM motor in modulating membrane insertion into the mitochondrial IM was further tested by assessing insertion vs. transloca-tion of H segments in the yeast strain with impaired PAM motor function (pam16-3) (225). Interestingly, although the impairment of the “pull-ing”/trapping activity of the mitochondrial motor lowered the hydrophobi-city threshold from n50%=5-6 to n50% ~ 4, this effect was not as dramatic as the one seen for the control protein (Mgm1p wildtype) that led to virtually 100% partitioning into the membrane in the mutant strain at non-permissive

57

conditions, compared to the 50% membrane insertion observed in the iso-genic strain. Paper III.

The specific effects of charged flanking residues in controlling the insertion of H segments into the mitochondrial IM prompted us to analyze in closer detail the effects of naturally occurring charged residues flanking the TM segment of a mitochondrial IM protein. The protein selected was the model protein used in Paper II, the yeast mito-chondrial Mgm1p. The first TM segment of Mgm1, termed H1, is ineffi-ciently recognized by the TIM23 complex, and only 30-40% of the Mgm1 H1 molecules are inserted into the mitochondrial IM (181,182). Indeed, H1 is considerably less hydrophobic when compared with other TM segments of proteins that follow the stop-transfer pathway. It is of great interest to verify whether the charged residues flanking this H1 segment modulate membrane insertion and if the effects are comparable to the ones observed in Paper II. The charged amino acids that are localized upstream (within 14 residues), or downstream (within 10 residues) of the H1 segment, were systematically mutated to Ala or in some specific cases to another charged residue. The upstream N-terminal part of the H1 segment is rich in Arg and Lys resi-dues. Interestingly, single mutations of these residues to Ala did not affect membrane insertion, except for the 79R:A mutant that led to virtually com-plete translocation across the mitochondrial IM. 78R:D caused a similar phe-notype. Double or triple mutations including 79R:A or between 86K and 93R, also impaired membrane insertion. Additionally, the effect of a more hydro-phobic H1 variant, 101G102G103M:VVL, that partitions relatively well into the mitochondrial IM, is completely overruled by the 78R79R:AA mutations. For the C-terminally localized charged flanking residues, only the double mutation of 114E and 115E to Ala or Lys or the triple mutation, 112K114E115E: AAA, hindered membrane insertion of H1. Overall, we can conclude that the hydrophobicity of H1 is not the only de-terminant for membrane insertion, but charged flanking residues can indeed condition insertion of this segment. This effect is position-dependent and not

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additive. Specifically the effect of 79R:A mutation is so dramatic that even with an impaired PAM motor (pam16-3) (225), a considerable amount of molecules slip through the TIM23 complex. This is an interesting result since ATP levels, as a marker of the mitochondrial energetic state, are though to regulate insertion of H1 into the mitochondrial IM (181,182). Paper IV.

The m-AAA protease is a central component of the mitochondrial protein quality control system, and is able to extract and degrade misfolded polypeptides from the mitochondrial IM (265). Additionally, the m-AAA protease is involved in the biogenesis of specific mitochondrial proteins such as Ccp1. After targeting to the mitochondrion, the first TM segment in Ccp1 is recognized by the TIM23 complex and laterally released into the IM. This segment is then dislocated from the membrane by the m-AAA protease (261,262). In Paper IV we explored the relation between the process of TM segment insertion by the TIM23 complex into the mitochondrial IM and the disloca-tion of that TM segment from the membrane by the m-AAA protease in the yeast mitochondria. To this end, we replaced the first TM segment in Ccp1 with the same 19 amino acids long (nL/(19-n)A) H segments, used in Papers I & II, and analyzed the ability of the m-AAA protease in dislocating these segments from the membrane. We observed that the m-AAA protease could extract Ccp1 H segments with a hydrophobicity up to 5 Leu/15 Ala. Interestingly, in the absence of the m-AAA protease, we found that H segments with a hydrophobicity 1-2 Leu /18-17 Ala or higher could remain in the mitochondrial IM. These results not only establish a relation between the m-AAA protease dislocation activity and the hydrophobicity of the substrate but also indicate that the presence of the m-AAA protease increases the threshold hydrophobicity for retention of the engineered H segments in the IM. Testing the m-AAA dislocation activity on the previously analyzed Mgm1 H segment variants (Paper II) resulted in the same pattern observed for Ccp1 H segments. In the presence of the m-AAA protease, only Mgm1 H segments with a hydrophobicity corresponding to 5 Leu/14 Ala or higher remained in the IM. In contrast, in the absence of the m-AAA protease, Mgm1 H seg-

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ments with a hydrophobicity corresponding to 0-1 Leu/19-18 Ala or higher could be found in the IM. The wild-type Mgm1 is not a natural substrate of the m-AAA protease (182). Therefore, it is unclear why the replacement of the Mgm1 first TM segment with the nL/(19-n)A H segments somehow converts Mgm1 into a m-AAA protease substrate.      The m-AAA protease dislocation activity on the Mgm1 H segment variants explains the unexpectedly high threshold hydrophobicity observed in Paper II. These new results suggest a model where the threshold hydrophobicity for the TIM23-mediated insertion in the inner membrane is n ≈ 1-2. A con-siderably higher threshold of n ≈ 5-6 is required to withstand the force exert-ed by the m-AAA protease when it extracts protein segments from the mem-brane. This corrected threshold hydrophobicity for the TIM23-mediated insertion in the mitochondrial IM is in better agreement with the low hydrophobicity of the TM segments generally observed in N-terminally targeted mitochondrial IM proteins. Paper V.

The TIM23 complex sorts N-terminally targeted mitochondrial IM proteins using one of two pathways, i.e., it can laterally release TM segments into the mitochondrial IM (the stop-transfer pathway), or completely translo-cate the protein to the matrix side, whereupon it is inserted back into the IM by the Oxa1 or other protein complexes (the conservative sorting pathway). The determinants used by the TIM23 complex to differentiate between pro-teins that take the stop-transfer or the conservative sorting pathways are poorly understood. In Paper V we developed a method to assess which of these two sorting routes is taken by the TIM23 substrates in yeast. We fused a truncated or full length test protein of known sorting route to the C-terminal part of Mgm1 that contains the rhomboid cleavage region (RCR) and will function as a reporter. These fusions were termed Mgm1 fusion proteins, MFPs. If the test MFP takes the stop-transfer pathway a membrane-inserted MFP will be

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formed. Instead, if the test MFP takes the conservative sorting pathway, the RCR located downstream of the test sequence will be cleaved by the mito-chondrial IM protease Pcp1, yielding a processed form. We validated this method by testing various proteins with previously deter-mined sorting routes. The majority of the stop-transfer MFPs tested gave rise to the expected membrane-inserted form, while most of the conservatively sorted MFPs generated a cleaved form. Additionally, we extended this ex-perimental system to other mitochondrial IM proteins with unknown integra-tion mode. With this novel approach to determine the sorting pathway taken by some of the mitochondrial IM proteins, we then investigated whether certain growth conditions such as temperature and carbon source could affect the mode of protein trafficking. Surprisingly we found that the conservative sorting pathway is highly dependent on fully energized mitochondria, i.e., yeast growth on a fermentable carbon source caused some of the conservatively sorted MFPs to take a stop-transfer pathway and to be arrested at the level of the TIM23 complex. This was also verified when we analyzed protein sort-ing in mitochondria with an impaired PAM motor (pam16-3) (225), or in the absence of the m-AAA membrane dislocation activity. Overall, this paper not only offers a new experimental system to assess pro-tein sorting in vivo at the level of the TIM23 complex, but also establishes a relation between the metabolic state of the cell and the mitochondrial protein import, supporting previous studies (143,172). A detailed analysis of the hydrophobicity of the TM segments of mitochondrial IM proteins suggested that the TM segments of conservatively sorted proteins have a lower hydro-phobicity and are the most sensitive to the ATP levels in the mitochondria. Interestingly, most proteins that follow the conservative sorting pathway are directly or indirectly involved in the assembly of the respiratory chain com-plexes in the mitochondrial IM. The observation that their sorting mode is especially sensitive to the physiological environment may indicate that mito-chondrial functions are modulated depending on cellular demand.

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9. Conclusions Summary

Insertion of proteins into biological membrane through the action of multifunctional translocons is an intricate process. This thesis focuses on specific steps of this process:

- how the E.coli SecYEG and the yeast mitochondrial TIM23 complexes recognize TM segments. For each of these systems we determined a “biolog-ical” hydrophobicity scale. This scale describes the contribution of each of the 20 amino acids to the overall free energy of insertion of a single TM segment into the membrane. Additionally, important information concerning the threshold hydrophobicity for protein insertion into the bacterial and mi-tochondrial IMs, as well as specific effects of charged flanking residues in modulating TM segment insertion into the latter, were unraveled; - how the naturally occurring charged flanking residues modulate membrane insertion of the first TM segment of the mitochondrial IM protein, Mgm1. The results confirmed the determinant effects of charged flanking residues; - which are the hydrophobicity threshold values that permit protein insertion by the TIM23 complex and protein dislocation from the mitochondrial IM by the m-AAA protease; - how the TIM23 complex distinguishes between proteins that follow the conservative sorting or the stop-transfer pathways. We developed an exper-imental approach that allowed easy assessment of the pathway taken by a test protein. In addition, we could also establish a connection between the metabolic state of the cell and the import pathway. Significance & Hypothesis

The quantitative data presented in this thesis has for the fist time made it possible to compare different membrane insertion mechanisms side-by-side, suggesting that they are based on similar physicochemical princi-ples. Our results will hopefully contribute to the development of better pre-diction methods to determine TM segments in membrane protein sequences of different organisms.

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The study on protein insertion and dislocation into/from the mitochondrial IM suggests that membrane proteins that take the TIM23 pathway normally contain only marginally hydrophobic TM segments. This leads to the ques-tion of how these segments can partition into a membrane environment. I propose that charged residues flanking these segments are determinant sig-nals for the TIM23 complex sorting activity, and also have important roles in anchoring these segments in the mitochondrial IM. As the mitochondrial IM is enriched in proteins, it is possible that protein partitioning is not only driv-en by protein-lipid interactions, but that specific protein-protein interactions might play an important role in this process. Future perspectives

The studies carried out in this thesis have partially lifted the veil on how different proteins translocation systems handle membrane proteins, and made it easier to analyze insertion of proteins into membranes. In principle, the systems established here can be used or adapted to test membrane inser-tion of a wide range of other substrates, and can complement other currently used protocols.

The “biological” hydrophobicity scale for the SecYEG-mediated insertion into the E. coli IM can be used to optimize the existing ΔGapp predictor (http://dgpred.cbr.su.se/), to specifically distinguish TM segments of bacteri-al proteins. As the “biological” hydrophobicity scale determined for the TIM23-mediated insertion into the mitochondrial IM is affected by the dislocation activity of the m-AAA protease, prediction of transmembrane segments is more difficult in this case. The connection between membrane insertion pro-cess by the TIM23 complex and membrane dislocation by the m-AAA prote-ase could potentially be used to reengineer protein sorting in the mitochon-drial IM. Finally, the results here obtained may be applicable in the area of drug tar-geting. Understanding which properties allow molecules to cross membranes is of extreme importance, for example, in the development of drugs that have to cross the bacterial or the mitochondrial inner membranes.

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Sammanfattning på svenska

Vägen in och ut ur membraner

Cellen är den grundläggande byggstenen i alla levande organismer. Alla celler är omgivna av ett membran som möjligtgör en separation av cel-lens inre från den yttre miljön. Dessa membran består av lipider (fett) och proteiner. Proteiner i ett membran ansvarar för kommunikation med omvärl-den och möjliggör utbyte av substanser som näringsämnen eller andra ke-miska föreningar över membranet. Denna avhandling fokuserar på hur proteiner sätts in i cellens membran. Denna process har analyserats i både bakterier och bagerijäst. Även om båda är encelliga organismer, så har bakterier har endast två typer av membran medan jästcellen innehåller flera membranomslutna organeller med centrala funktioner för cellöverlevnad. Jag har särskilt studerat den organell som ansvarar för att producera energi, mitokondrien. Jag har undersökt hur två “insertionsmaskinerier”, det bakteriella SecYEG translokonet och det mitokondriella TIM23 komplexet, kan känna igen pro-teiner som är avsedda att sättas in i membranet. och har “dechiffrerat” de regler som styr insättningen i både bakteriella och mitokondriella membran. Likheten mellan de två systemen tyder på att de grundläggande principer är samma i båda fallen. Dessa arbeten bidrar till en bättre förståelse av hur proteiner sätts in i och extraheras ut ur membran och ger värdefull information för att förbättra bio-informatiska metoder som används för att förutsäga vilka proteiner integre-ras i ett membran.

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Acknowledgements

I would like to express my sincere gratitude to all the people that in one way or the other have contributed to the fantastic time I had during these 5 years at our beloved DBB! I have to start with my supervisor Gunnar! I have been so lucky and I feel so honored to be in your group as a PhD student. You have such a healthy atti-tude towards Science, and all your generosity allowed me to achieve many goals. Thank you for your kindness, guidance, advises, patience, for replying so fast to e-mails, and for always having your door open (even with the noisy ice machine) J! Joy, thank you so much for training me, letting me join your work and giv-ing me all the tools to do all these projects. You are such a caring and gener-ous person and I really appreciate all the support you gave me in the lab and outside. I feel so honored for being one of your first students and I am so proud of your new lab! Thank you for taking such good care of me! I cannot forget Stefan Nordlund, Elzbieta Glaser, Peter Brzezinski, Åke Wieslander and Joe Depierre for evaluating me and guiding me throughout the PhD studies. I must thank a lot my co-authors Marie Österberg, Karin Öjemalm, Koji Yamano and Kwangjin Park! Thank you for sharing your projects and knowledge, for teaching me, for all your hard work and dedication. You are an essential part of this thesis! J My sweet group!!! I adore you guys and I will miss you so much! I will nev-er find a lab like ours!! My office mates, Karin, Ola, Florian, Pati and Nina, we shared so many funny moments, I feel so comfortable in our office and that's thanks to all of you. Florian thank you for all the lunches together! Now you are not alone, Ola will hopefully control all the girlish topics! Ola and Florian, I wish you a great and successful time in our group!! My girls Karin, Nina, soon it will be four of you J. I´m looking forward for the ba-bies! Karin good luck for your thesis and thank you for helping me a lot!! Nina, you still have sometime so, enjoy it! Thank you for your kindness and friendship! My dear Pati..oh I will miss you! You are just too much fun! Thank you for being the best travel partner. I hope we will enjoy many more

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funny moments together. Good luck with your work and once your done, come and join me! J The “next office” people! Thanks for bearing with the noise from the ex-presso machine! Carmen, thank you for taking care of our lab and of all of us! Your organization and your kindness are really refreshing! Nurzianzita! You are a great lab bench mate! Thank you for being so kind, helpful, and generous and for listening to all my stories! Johannes we shared so much together! Thank you for always taking care of me, for being such a nice per-son and for always being there when I needed! Soon it will be your turn!! Rickard/Richie/Ricardo thank you for all our talks, I really enjoy your sense of humor and your sharp comments! Our lost members so far away in other offices, Bill and Patrick, Minttu!! You guys are so cool!! Keep it up!! Thank you to the past members, Susanna, Morten, Joanna, Kalle “Muscles”. Special thanks to Roger “out-of-control”!! All the GvH community, you are so many but you are all so nice that you deserve to be named: JWdeGier´s, thank you for being so nice, kind and helpful neighbors, Jan-Willem, Mirjam, Thomas, Anna, Susan, David thanks for my nickname and for being so cheerful; Dan´s, Dan, Isolde, Kalle, Jörg, Stephen; Rob´s, Rob, Diogo (representing the country!!!), Johan, Dan; Mar-tin´s, Martin and Jordi; Martin Ott´s, Martin, Manfred, Steffi, Kerstin, Mar-cus; David Drew; IngMarie, thank you for always treating me with such kindness and care and thank for always organizing great conferences. From neighboring labs I would like to give special thanks to Erika, my Big-exam study team Hans, Mousumi, Joachim! Silvia for great time in Greece!! My “unni” Young Ok you are so joyful and fun, I hope we will share more big lunches together!! Joy´s lab at SNU! Sungjun, Chewon, Hani, Hungsang, Kyungyun, Sungmin, Hayoung and Kwangjin! Thank you so much for all the great moments! It was a pleasure to meet your all! Special thanks to super duper Jake Kwang-jin! I was so fortunate to have met you! Thank you for all the hard work but also for being just who you are, the World has great plans for you!! My DBB “buddies” aka Portuguese Mafia, thank you so much for every-thing, you are such good friends!! Pedro and Tiago, from the very start un-til..forever I hope! Thank you for the care, friendship, for being there for me, always! Our adopted Portuguese/Polish sweet Bea, thank you for your smile and for being so nice! Changrong, you are great, Xié Xié!! Candan…my fortuneteller thank you for talks and dinners and coffees!!! Cata thank you for being our “Cata”, for taking care of me, for being always there for all of us! Pili, thank you for being there in the ups and downs, for all the support!!

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I will miss your presence always “in front of” me!!! J Helen, I miss you! Lunchtime was never the same after you left! Our “menino” Nuno the next generation of Portuguese at DBB!! Enjoy! Special thanks to Lotta, Ann, Maria for being so kind and helpful! Bogos, wherever you are, thank you for all your joyful greetings! My “famiglia”! How can I begin? You guys are my Friday and Saturday nights. PG, I think you have enough training as a dad! Good luck for the two big achievements! Marco, obrigada por seres um bom amigo e por todo o apoio! My gordita Eddie (Edurne) and my Sobrina (Ane), you girls are so sweet and generous...ooh I miss you so much! Uba, “keep on rocking in free world”! “Chilled” Alberto always with bono vino! Great Iman thank you for listening and being there for me! Euhana (Joni) and Dami you are so special to me J thank you for the breakfast and lunches, for taking care of me and listening to my endless speeches, Grande!! Jim, you are responsible for my best profile pic in FB (the hamburger one) you are great fun!! Burak, you are so sweet! Thank you for being so kind and treating me with such care! Olé Rafa! We had really funny moments together! Thank you for being there for me!! Irina so wild and so nice, thank you!! Nok thank you for throwing cra-zy parties and dinners!! Sandra, welcome to the family!!! Our MacGyver Nachito! The El Guapo Kizomba teacher António!!! Pablo, I miss you!!! Thank you for the cool times!! My girls Nita, Marta and Laia. We spent such wonderful times together! I miss you!! My neighbors at Lappis: Zahid, Shahzad, Hamid, Yasir, Moby, Shaban, the lovely couple Kenneth and Bing! Thank you for making me feel that I have a home! My chinese sister Wenzita, we always have a great time together!!! Pierre, thank you for being such good and sensitive friend! Linhua, my sweet “moonlight” you are so lovely, it was great meeting you! Germano! Thank you for all..time, love and care! Muito Obrigada aos meus queridos Pais!!!! I would like to thank Fundação para a Ciência e a Tecnologia, POPH-QREN, Portugal, for financial support (graduate student fellowship SFRH/BD/36107/2007).

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