Doctoral Thesis in Biochemistry, Stockholm University, Sweden
Membrane Protein Biogenesis in Saccharomyces cer evisiae
Johannes Reithinger
Membrane Protein Biogenesis in Saccharomyces cerevisiae
Johannes Reithinger
Cover: Adaptation of the historic Route 66 road sign against the background of Monument Valley in Utah, USA. Photo by Moritz Heupel.
© Johannes Reithinger, Stockholm University 2013
ISBN 978-91-7447-798-6, pages 1-72
Printed in Sweden by US-AB, Stockholm 2013 Distributor: Department of Biochemistry and Biophysics, Stockholm University
Meinen Eltern
List of publications
I. Hessa T, Reithinger JH, von Heijne G, Kim H. (2009) Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae. J Mol Biol. 386(5):1222-8.
II. Reithinger JH, Yim C, Kim S, Kim H. (2013) Structural and functional profiling of the Sec61 lateral gate. Manuscript
III. Reithinger JH, Kim JE, Kim H. (2013) Sec62 protein mediates membrane insertion and orientation of moderately hydrophobic signal anchor proteins in the endoplasmic reticulum (ER). J Biol Chem. 288(25):18058-67.
IV. Jung S, Kim JE, Reithinger JH, Kim H. (2013) The Sec62/Sec63 translocon facilitates membrane insertion and C- terminal translocation of multi-spanning membrane proteins. Submitted
V. Reithinger JH, Yim C, Park K, Björkholm P, von Heijne G, Kim H. (2013) A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER. FEBS lett. 587(21):3480-6.
vi Abstract
Membranes are hydrophobic barriers that define the outer boundaries and internal compartments of living cells. Membrane proteins are the gates in these barriers, and they perform vital functions in the highly regulated transport of matter and information across membranes. Membrane proteins destined for the endoplasmic reticulum are targeted either co- or post- translationally to the Sec61 translocon, the major translocation machinery in eukaryotic cells, which allows for lateral partitioning of hydrophobic segments into the lipid bilayer. This thesis aims to acquire insights into the mechanism of membrane protein insertion and the role of different translocon components in targeting, insertion and topogenesis, using the yeast Saccharomyces cerevisiae as a model organism.
By measuring the insertion efficiency of a set of model proteins, we studied the sequence requirements for Sec61-mediated insertion of an α-helical transmembrane segment and established a ‘biological hydrophobicity scale’ in yeast, which describes the individual contributions of the 20 amino acids to insertion. Systematic mutagenesis and photo-crosslinking of the Sec61 translocon revealed key residues in the lateral gate that modulate the threshold hydrophobicity for membrane insertion and transmembrane segment orientation. Further, my studies demonstrate that the translocon- associated Sec62 is important not only for post-translational targeting, but also for the insertion and topogenesis of moderately hydrophobic signal anchor proteins and the C-terminal translocation of multi-spanning membrane proteins. Finally, nuclearly encoded mitochondrial membrane proteins were found to evade mis-targeting to the endoplasmic reticulum by containing short C-terminal tails.
vii Contents
List of publications...... vi
Abstract...... vii
Contents ...... viii
Abbreviations...... x
1. Introduction ...... 11
2. Biological membranes...... 12 2.1 General features of biological membranes ...... 12 2.2 The endoplasmic reticulum ...... 15
3. Membrane protein architecture...... 18 3.1 β-barrel membrane proteins...... 20 3.2 α-helical membrane proteins ...... 20 3.3 Membrane protein topology ...... 22
4. Protein targeting in the eukaryotic cell...... 25 4.1 Signal sequences...... 25 4.2 Co-translational translocation...... 27 4.3 Post-translational translocation...... 30
5. Translocation machineries in the endoplasmic reticulum...... 32 5.1 The Sec61 translocon...... 32 5.2 The Sec62/63 complex ...... 36 5.3 Accessory proteins...... 38
viii 6. Insertion of α-helical membrane proteins into the endoplasmic reticulum membrane...... 39
7. Summary of papers ...... 42 7.1 A biological hydrophobicity scale in yeast (paper I)...... 42 7.2 Analysis of the Sec61 lateral gate profile (paper II)...... 43 7.3 Insertion and topogenesis of signal anchor proteins (paper III) ...... 45 7.4 Sec62 regulates membrane protein topogenesis (paper IV) ...... 46 7.5 C-terminal tail length affects membrane protein targeting (paper V) 47
8. Conclusions ...... 48
9. Populärvetenskaplig sammanfattning på svenska ...... 51
Acknowledgements...... 52
References ...... 55
ix Abbreviations
C-terminal Carboxyl-terminal ER Endoplasmic reticulum GFP Green fluorescent protein H-segment Hydrophobic segment Lep Leader peptidase N-terminal Amino-terminal OST Oligosaccharyltransferase PhoA Alkaline phosphatase RER Rough ER RNC Ribosome-nascent chain complex SER Smooth ER SP Signal peptidase SR SRP receptor SRP Signal recognition particle TA Tail-anchored TIM Translocase of the inner membrane TM Transmembrane TOM Translocase of the outer membrane
Amino acids
Full name 3-letter 1-letter Full name 3-letter 1-letter Alanine Ala A Leucine Leu L Arginine Arg R Lysine Lys K Asparagine Asn N Methionine Met M Aspartic acid Asp D Phenylalanine Phe F Cysteine Cys C Proline Pro P Glutamic acid Glu E Serine Ser S Glutamine Gln Q Threonine Thr T Glycine Gly G Tryptophan Trp W Histidine His H Tyrosine Tyr Y Isoleucine Ile I Valine Val V
x 1. Introduction
Biological membranes are key components of all living organisms. Membranes define the spatial dimensions of each cell and create a semi- permeable barrier to the outside of the cell, providing an enclosed space in which all cellular processes occur. Biological membranes are primarily composed of lipids and proteins.
Within eukaryotic cells, membranes form additional subcompartments called organelles, which perform specialized functions. The cell-enclosing plasma membrane and the different organellar membranes vary in both shape and composition, in accordance with their particular functional requirements.
Membrane proteins, serving e.g. as channels, receptors or pumps, are the gates and gatekeepers that conduct and regulate the traffic of molecules, such as nutrients, ions and signals, across the membrane. To perform their particular functions, membrane proteins must reach their designated compartment membrane, acquire a functionally active structure within the membrane and interact with other membrane proteins to form complexes.
This thesis includes five studies that explore different aspects of membrane protein targeting and subsequent membrane integration in eukaryotic cells, with a particular focus on the intrinsic signals that dictate the targeting route and destination of membrane proteins and on the major integration machinery that is present in the membrane of the endoplasmic reticulum. The yeast Saccharomyces cerevisiae served as a model organism for these studies.
11 2. Biological membranes
2.1 General features of biological membranes
Composed of a double layer of amphiphatic lipids, membranes have an average thickness of approximately 50 Å (Figure 1). The hydrophobic tails of the lipids of both layers are oriented towards the core of the membrane, while the hydrophilic head groups face the aqueous surroundings, forming polar interfaces on each side of the membrane.
The arsenal of lipids is vast, ranging from several hundred in bacteria to thousands in eukaryotes (Sud et al., 2007). Most lipids can be grouped into three classes: glycerophospholipids, sphingolipids and sterols. The lipid composition of membranes varies not only between different organisms but also within individual cells, according to compartment and function. For example, the well-studied Gram-negative bacterium Escherichia coli has outer and inner membranes, which contain predominantly phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL), but not phosphatidylcholine (PC) or sphingomyelin (SM) (Raetz, 1986; Raetz and Dowhan, 1990). However, in the plasma membrane of eukaryotic cells, PC and SM are the most abundant glycerophospholipid and sphingolipid, respectively (van Meer et al., 2008). In addition, PE, Phosphatidylserine (PS), phosphatidylinositol (PI) and cholesterol (ergosterol in yeast) are present in significant amounts (van Meer, 1989). The intracellular organelles of eukaryotic cells, such as the endoplasmic reticulum (ER), the Golgi apparatus, mitochondria, chloroplasts,
12 peroxisomes, lysosomes and the nucleus, contain distinct mixtures of lipids. For example, in contrast to the plasma membrane, the membrane of the endoplasmic reticulum contains only small amounts of sphingolipids and cholesterol. Instead, the major components are PC, PE and PI. In eukaryotic cells, the ER and the mitochondria are the main sites of lipid biosynthesis (van Meer et al., 2008). In addition to their membrane-forming role, lipids also perform important functions in energy storage and as messenger molecules in signal transduction.
Proteins account for another large portion of biological membranes (Figure 1). All proteins that are bound to a membrane are called membrane proteins and can be classified as either peripheral or integral membrane proteins (Singer, 1974). Peripheral membrane proteins interact with the polar headgroups of membrane lipids, primarily via hydrogen bonds, or/and with other membrane proteins. Due to their relatively weak interactions, these proteins can be separated from the membrane by carbonate treatment (Fujiki et al., 1982). Integral membrane proteins are deeply anchored within the lipid bilayer and often span the entire membrane (i.e., transmembrane). These proteins can therefore only be extracted by the application of organic solvents or detergents (Singer, 1974). For simplicity and due to the focus of this work, ‘membrane protein’ will henceforth denote integral transmembrane (TM) proteins. Depending on the organism and the cellular organelle, membrane protein content can vary greatly. For example, in the plasma membrane of E. coli and the mitochondrial membranes of eukaryotic cells, membrane proteins account for approximately 75% of the total mass, while the plasma membrane of yeast has a protein content of approximately 50%.
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Figure 1: Schematic view of a biological membrane composed of lipids and integral and peripheral membrane proteins.
Singer and Nicholson proposed the ‘fluid mosaic model’ that describes the membrane structure as a dynamic, two-dimensional viscous matrix formed by lipids, in which membrane proteins are embedded and float ad lib (Singer and Nicolson, 1972). Although the principal ideas of this model remain accurate, some aspects of the model have been refined. Biological membranes appear to be highly occupied by proteins. Specific interactions of proteins with lipid molecules, which may be essential for protein function, or with other proteins restrict their lateral mobility (Engelman, 2005). Additionally, the lipid bilayer can vary in thickness, likely due to interactions with membrane proteins (Mitra et al., 2004).
Each intracellular organelle possesses a characteristic and functionally relevant morphology. These shapes are generated by inducing bends and curvature into the otherwise flat lipid bilayer. Although asymmetric distribution of lipids in the two leaflets of the bilayer can contribute, membrane proteins appear to be the major players in inducing high membrane curvature (Zimmerberg and Kozlov, 2006). The two main mechanisms for introducing curvature are hydrophobic insertion (i.e., wedging) and scaffolding (Shibata et al., 2009). Wedging describes the insertion of a hydrophobic domain of a protein into one leaflet of the bilayer.
14 The resulting disturbance of lipid packing induces local membrane bending. In the second mechanism, scaffolding proteins acquire a rigid and intrinsically curved shape. Because the curved protein structure binds tightly to the membrane surface, the membrane is forced into the inherent shape of the scaffold (Shibata et al., 2009).
2.2 The endoplasmic reticulum
The ER is a continuous network of tubular or lamellar membrane structures with three main structurally and functionally distinct subdomains (Sitia and Meldolesi, 1992; Baumann and Walz, 2001): the nuclear envelope, the peripheral ER tubules and the peripheral ER sheets (English et al., 2009). The network encloses one common aqueous space: the ER lumen. In general, the diameter or thickness of the tubules or sheets is 60-100 nm (Shibata et al., 2006). Particularly in tubules, high membrane curvature is induced by the reticulon and DP1/Yop1 membrane protein families (De Craene et al., 2006; Voeltz et al., 2006).
Although the peripheral ER network extends throughout the cytoplasm, it must be able to constantly adjust to changing cellular conditions. This network is thus highly dynamic (Borgese et al., 2006). The ER is in contact with virtually all other organelles in the cell (Staehelin, 1997). As a major site of synthesis of membrane lipids, it seems plausible that these contacts are necessary for direct transfer of lipids from the ER to other organelles, such as mitochondria or peroxisomes (Voeltz et al., 2002; English et al., 2009). The relationship between the ER and the Golgi apparatus is of great importance in protein secretion. COPII membrane vesicles are formed at the ER membrane and transport proteins to the Golgi for secretion (i.e., anterograde transport) (Spang, 2009). Transport in the reverse direction is performed by COPI vesicles (i.e., retrograde transport). A subdomain of the
15 yeast ER is in close proximity to the plasma membrane (<50 nm), and these membranes may interact indirectly via protein complexes (Pichler et al., 2001).
In addition to lipid synthesis and protein secretion, the ER performs essential functions in drug metabolism, Ca2+ signaling and membrane protein biosynthesis. The tubules and sheets of the peripheral ER provide an example of the connection between the function of ER subdomains and the corresponding morphology. Electron microscopy revealed that the ER sheets, which are traditionally called the rough ER (RER), are predominantly covered with ribosomes, while the tubular ER membranes, which are called the smooth ER (SER), typically lack ribosomes or have low numbers of ribosomes (Figure 2) (Shibata et al., 2006). In yeast, the ER sheets carry up to 1100 ribosomes per µm2 (West et al., 2011). The RER is therefore the location of membrane and secretory protein biosynthesis and processing, and it is heavily enriched in secretory cells, such as rat liver cells (Vogel et al., 1990). The tubular SER is less involved in protein biosynthesis, but it is involved in protein packaging and transport to the Golgi apparatus and subsequently to the plasma membrane (Hobman et al., 1998; Voeltz et al., 2002).
16
Figure 2: Structural subdomains of the ER. (A) Electron micrograph of ribosome-covered RER sheets in secretory cells from silkworm silk glands (thin-section electron micorgraph) (Shibata et al., 2006). (B) Tubular structure of the SER from rat white skeletal muscle fibers (scanning electron micrograph) (Shibata et al., 2006). Both images are reprinted with permission.
17 3. Membrane protein architecture
Most membrane proteins inhabit three chemically distinct worlds simultaneously. One part of the protein is embedded in the hydrophobic core of the lipid bilayer, surrounded by the non-polar acyl chains of phospholipids, steroids, and other membrane proteins. The hydrophobic lipid tails in the membrane core present no opportunity for hydrogen bonding with the carbonyl oxygen and amide nitrogen atoms of the polypeptide backbone. As a result, intramolecular hydrogen bonds are a structural requirement for stable protein conformation (von Heijne, 1994; Chou and Elrod, 1999). Another part of the protein lies within the chemically diverse membrane interface formed by the phospholipid head groups, which offer possibilities for dipole-dipole interactions and hydrogen bonding (Killian and von Heijne, 2000). Finally, membrane proteins may also contain large domains in the water surrounding the membrane, either inside or outside of the cell or an organelle. To date, only two principle architectural types are known that are suitable for this anisotropic environment: α-helical bundles and β-barrels (Figure 3) (von Heijne, 1994; 1996).
Insights into these structures have been attained using electron microscopy and X-ray crystallography (Henderson and Unwin, 1975; Allen et al., 1988; Caffrey, 2003). However, high-resolution structure determination for membrane proteins remains a tedious and difficult task (Lacapère et al., 2007). The higher content of hydrophobic amino acids in membrane proteins and their associated insolubility in aqueous solution make crystal production a limiting step (Caffrey, 2003), as is the efficient expression and purification of native membrane proteins (Lacapère et al., 2007). Although membrane
18 proteins account for approximately 15% to 25% of all open reading frames (ORFs), they are dramatically underrepresented in the Protein Data Bank, accounting for less than 1% of all known structures (Wallin et al., 1997; Mitaku et al., 1999; Krogh et al., 2001). Crystallization in the meso- or cubic-phase and the use of stabilized recombinant proteins are examples of advances in this field that have led to an acceleration of structure determination (White, 2004; Bill et al., 2011; Cherezov, 2011).
Figure 3: Examples of the two principal types of membrane protein architecture. The voltage- gated potassium channel KcsA is an α-helical bundle (left) (PDB: 1BL8). OmpA is a β-barrel protein (right) (PDB: 1QJP). The grey lines indicate the approximate boundaries of the membrane.
The following sections will introduce the β-barrel and α-helical membrane protein structures in more detail and discuss the concept of membrane protein topology.
19 3.1 β-barrel membrane proteins
β-barrel membrane proteins are only found in the outer membrane of Gram- negative bacteria, such as E. coli, and in eukaryotic organelles originating from endosymbiosis, such as mitochondria and chloroplasts (Koebnik et al., 2000). Multiple anti-parallel β-strands assemble into a cylinder-like shape (Figure 3). In this conformation, all of the hydrogen bond donors and acceptors in the polypeptide backbone of the β-strands form intramolecular bonds with the neighboring strands. β-barrels are typically composed of an even number of β-strands, with 8 strands being the minimal number (Sansom and Kerr, 1995; Wimley, 2003). Each TM strand is composed of 9 to 11 amino acid residues.
β-barrel proteins are synthesized in the cytoplasm. After translocation of the unfolded peptide through the bacterial inner membrane via the Sec translocation machinery, the protein is assembled and integrated into the outer membrane by the Bam complex (Walther et al., 2009).
Functionally, β-barrels are involved in the transport of nucleosides and fatty acids, in signal transduction, in the permeation of host cell membranes and in a number of other processes (Wimley, 2003; Galdiero et al., 2007).
3.2 α-helical membrane proteins
The vast majority of integral membrane proteins (95%) are predicted to have the α-helical bundle architecture (Figure 3) (Wallin and von Heijne, 1998; Krogh et al., 2001). Although these proteins have various shapes and sizes, the basic unit is an α-helical TM segment consisting primarily of hydrophobic amino acids, such as leucine, isoleucine, valine, alanine and phenylalanine (von Heijne, 2007). The 14 to 36 amino acid segments, which have an average length of 26 amino acids, often traverse the entire
20 membrane bilayer, with a 21° tilt angle with respect to the bilayer (Bowie, 1997b; Ulmschneider et al., 2005). The H-bond potential of the polypeptide backbone is satisfied by bond formation within the α-helix (von Heijne, 1994).
α-helical membrane proteins can contain one (single-spanning) or more (multi-spanning) TM segments, with large soluble domains exposed to the aqueous surroundings. In multi-spanning membrane proteins, the helices are interconnected by soluble loops of varying length. Additionally, α-helical membrane proteins often interact with each other to form larger protein complexes, such as the 13-subunit cytochrome c oxidase in the inner mitochondrial membrane, which has a total of 28 TM α-helices (Tsukihara et al., 1996).
Biochemical studies and analyses of high-resolution structures of α-helical bundle proteins and complexes provided insights into the general features of helix-helix interactions within and between membrane proteins (Bowie, 1997a; Choma et al., 2000; Russ and Engelman, 2000; Zhou et al., 2001; Moore et al., 2008). Pairs of polar residues, such as asparagine, aspartic acid, threonine or serine, form intra-helical hydrogen bonds, which stabilize the membrane protein structure or drive complex formation between α-helical membrane proteins (Zhou et al., 2001; Hermansson and von Heijne, 2003; Meindl-Beinker et al., 2006). Tight helix association is also enabled by the GxxxG motif and GxxxG-like motifs. The two small glycines, which are separated by any three amino acids, allow close contact and promote van der Waals interactions between α-helices (Javadpour et al., 1999; Russ and Engelman, 2000).
Due to their functional diversity, the biological significance of α-helical membrane proteins cannot be overestimated. Prominent members of this class are G protein-coupled receptors (GPCRs), which are important for signal transduction in processes such as vision and olfaction, aquaporins,
21 which maintain the water homeostasis of cells, ATP synthase, and ion channels, which are essential for signaling and ion homeostasis (von Heijne, 2007).
The biosynthesis of α-helical membrane proteins will be discussed in detail in later chapters of this thesis.
3.3 Membrane protein topology
A topology map is a two-dimensional description of a membrane protein that contains information concerning the number of TM segments, their approximate location within the protein, and their orientation relative to the plane of the membrane (Figure 4) (von Heijne, 2006).
Single-spanning membrane proteins can insert in two different orientations, with their N-terminus facing either the extracytosolic/lumenal (Nout/Cin) or the cytosolic (Nin/Cout) side of the membrane; these proteins are termed type I or type II membrane proteins, respectively (Figure 4) (Shao and Hegde, 2011). Multi-spanning membrane proteins can be referred to as polytopic membrane proteins.
Figure 4: Different types of membrane protein topology.
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Topology maps of membrane proteins can be predicted using topology prediction algorithms (von Heijne, 1992; Punta et al., 2007) and using experimental approaches (Daley et al., 2005; Kim et al., 2006).
Topology prediction is simplified by the fact that the hydrophobic TM segments of α-helical membrane proteins can be easily recognized in the amino acid sequence. The ‘positive-inside rule’ is of high importance when determining the probable orientation of a membrane protein. This rule states that positively charged residues (i.e., lysine and arginine) are enriched in the loops that are exposed to the cytoplasmic side (i.e., the inside) of the membrane (von Heijne, 1986d; 1989). Prediction algorithms yield correct topologies for up to 70% of all proteins (Elofsson and von Heijne, 2007). Current widely used membrane protein topology prediction programs include SCAMPI (Bernsel et al., 2008), OCTOPUS (Viklund and Elofsson, 2008), ΔG-predictor (Hessa et al., 2007), and TOPCONS, which integrates results from a number of individual prediction algorithms into a consensus prediction (Bernsel et al., 2009).
Topology prediction is a valuable tool, because it gives first structural insights and can be applied on a genomic level. However, protein topology must ultimately be verified experimentally. A variety of strategies for topology assessment are available. Prominent examples of these strategies are described in the following section (Green et al., 2001).
With a protease protection assay, it is possible to assess which parts of a membrane protein are exposed to the aqueous environment on the in- or outside of a cell or a cellular organelle (Keeling et al., 1983; Wilkinson et al., 1996). Only water-accessible loops and domains are degraded by protease treatment, as the membrane domains are protected from proteolysis. The analysis of the proteolytic fragments, which may be performed using SDS-PAGE, allows for conclusions concerning which protein segments
23 reside in the lipid bilayer. By applying proteases to only one side of a membrane at a time, it is further possible to determine the cellular compartment in which the unprotected loops and domains are located.
Reporter fusions are other useful tools for mapping the parts of the protein that face the cytoplasm, the inside of cellular organelles or the cell exterior, and they have been successfully applied to the study of whole proteomes (Daley et al., 2005; Kim et al., 2006). These reporters, such as alkaline phosphatase (PhoA) or green fluorescent protein (GFP), typically fold and/or function correctly only on one side of the membrane, and their location can be assessed using specific assays (i.e., activity and fluorescence measurements for PhoA and GFP, respectively) (Horst and Lolkema, 2010).
N-linked glycosylation is of great importance for topology determination in eukaryotic cells. Glycosylation of proteins at the consensus sequence NXS/T, where X can be any amino acid except proline, is catalyzed by the eukaryotic oligosaccharyltransferase (OST) complex only in the ER lumen (Kornfeld and Kornfeld, 1985; Gavel and von Heijne, 1990; Dempski and Imperiali, 2002). By introducing N-linked glycosylation sites at different places in a membrane protein sequence, precise mapping of luminal loops is possible (Chang et al., 1994; Kim et al., 2006).
Combinations of different strategies, such as the glycosylatable GFP fusion reporter (Lee et al., 2012) and the fluorescence protease protection (FPP) assay (Lorenz et al., 2006) have also been applied successfully.
24 4. Protein targeting in the eukaryotic cell
The correct targeting of proteins to their designated cellular or subcellular destinations is a fundamental requirement for protein function. In eukaryotic cells, the vast majority of proteins are synthesized by cytoplasmic ribosomes and guided to their target locations by intrinsic signals (Blobel and Dobberstein, 1975; von Heijne, 1990; Rapoport, 2007; Cross et al., 2009). However, a small number of proteins are encoded in the mitochondrial and chloroplast genomes and synthesized by organellar translation machineries (von Heijne, 1986a). These proteins will not be discussed further in this thesis.
The following sections discuss the properties of signal sequences and the two main pathways of protein targeting: the co- and post-translational translocation pathways.
4.1 Signal sequences
Signal sequences act as address labels on peptide packages that must be delivered to the correct destination, such as the cell exterior, the ER membrane, mitochondria, etc. In this comparison, the mailbox is typically the membrane-embedded translocation machinery, which mediates either complete translocation across the membrane or insertion of hydrophobic segments into the membrane (Schatz and Dobberstein, 1996). The signal sequences are primarily located at the N-terminus of polypeptide chains, and the address information is encoded in the structural and chemical properties of the amino acid sequence. These properties, such as length, hydrophobicity
25 and charge distribution, are characteristic of different target locations and preferred targeting pathways (von Heijne, 1990; Ng et al., 1996; Reithinger et al., 2013).
Sequences that target proteins from the cytoplasm to the mitochondria, referred to as presequences, feature a strong tendency to form amphiphilic α- helices, with a high content of positively charged residues, and are usually 15 to 55 amino acids long (von Heijne, 1990; Dudek et al., 2013). For both soluble and membrane proteins, the TOM complex (translocase of the outer membrane) is the gateway through the outer mitochondrial membrane and into the intermembrane space. Presequence-containing proteins are subsequently transmitted into the matrix or integrated into the inner membrane by the TIM complex (translocase of the inner membrane) (Neupert and Herrmann, 2007). During sorting or matrix import, presequences are proteolytically removed by specific presequence peptidases (Mossmann et al., 2012).
ER targeting signals are more hydrophobic than mitochondrial presequences and often contain a positively charged N-terminal domain, a hydrophobic 7 to 13 residue long core with an affinity to form an α-helix, and a polar C- terminal domain (von Heijne, 1986c; 1990). All secretory proteins destined for cellular export, as well as membrane proteins of the ER, the Golgi and the plasma membrane, are initially targeted to the ER. Secretory proteins usually contain a cleavable signal sequence, with the cleavage site located downstream of the hydrophobic domain. The N-terminal charges help, in accordance with the ‘positive-inside rule’ (see above), to position the signal sequence in an Nin/Cout membrane topology. The luminal cleavage site is subsequently processed by the signal peptidase (Dalbey et al., 1997; Paetzel et al., 1998). In lieu of a cleavable signal sequence, membrane proteins are often targeted by the first hydrophobic TM segment, which functions as a
26 signal sequence and also anchors the protein in the ER membrane (Shao and Hegde, 2011).
Experimentally it was shown that the overall hydrophobicity of the signal sequence or anchor influences the pathway preference by which a protein is targeted; moderately and highly hydrophobic sequences from membrane proteins trigger co-translational translocation, while signal sequences with low hydrophobicity, which are often found in nuclearly encoded mitochondrial proteins and secretory proteins, enter the post-translational translocation pathway (Ng et al., 1996; Reithinger et al., 2013).
4.2 Co-translational translocation
The co-translational translocation pathway is dependent on the signal recognition particle (SRP) and its cognate ER membrane-associated receptor (SR). Both the SRP and SR are functionally highly conserved, and homologs are found in all three kingdoms (Keenan et al., 2001; Zimmermann et al., 2011). In higher eukaryotes, the SRP is a cytosolic ribonucleoprotein that consists of six polypeptides (seven in S. cerevisiae) and one RNA (Doudna and Batey, 2004). Functionally, the SRP is divided into two domains: the Alu domain and the S domain, which contains an integrated GTP binding site (Weichenrieder et al., 2000; Halic and Beckmann, 2005). SR is a heterodimer formed by two GTPases: the peripheral membrane protein SRα and the integral membrane protein SRβ. The latter protein anchors the complex to the ER membrane via an N-terminal TM helix (Tajima et al., 1986; Schwartz and Blobel, 2003).
The first step in the co-translational targeting of a newly synthesized secretory or membrane protein to the ER membrane is initiated by SRP binding, via the S domain, to the signal sequence or the first hydrophobic TM segment as it emerges from the ribosomal tunnel exit (Figure 5) (Wild et
27 al., 2004). Additional interaction of the SRP Alu domain with the elongation factor binding site of the ribosome decelerates polypeptide elongation rate (Walter and Blobel, 1981; Lipp et al., 1987; Halic and Beckmann, 2005). In a second step, the SRP-ribosome-nascent chain complex (SRP-RNC) is targeted to the ER membrane via the interaction of the SRP, via the S domain, with SRα (Figure 5). For stable SRP-SR complex formation, both SRP and SRα/SRβ must be in their GTP-bound state. Interestingly, the SRP- RNC complex can be targeted to the ER membrane even without the membrane anchor of SRβ, indicating that additional interactions support transient localization of the SR to the membrane (Ogg et al., 1998). In the third step, the signal sequence, together with the whole RNC, is transferred to the translocation machinery of the ER, the Sec61 translocon, where the translation of the nascent chain continues. The nascent chain is simultaneously translocated across or integrated into the ER membrane (Figure 5) (Keenan et al., 2001). In the fourth step, GTP-hydrolysis by SRP and SR induces the disassembly of the SRP-SR complex, resetting the components for a new round of the ‘SRP-cycle’ (Luirink and Sinning, 2004).
Co-translational translocation is the major mode of targeting for ER membrane proteins because it avoids exposure of hydrophobic TM segments to the aqueous environment of the cytosol, preventing protein aggregation and premature folding that would obstruct protein integration into the ER membrane.
In contrast to other eukaryotic organisms, the SRP was found to be non- essential in the yeast S. cerevisiae (Hann and Walter, 1991). Although cell growth is greatly slowed in the absence of the SRP, yeast can compensate for the loss of SRP function by reducing protein synthesis and inducing heat shock genes. As a result of these changes, the translocation machinery is unburdened and the additional heat shock proteins and chaperones, such as Hsp70, can keep newly synthesized proteins in an unfolded, translocation-
28 competent conformation (Arnold and Wittrup, 1994; Mutka and Walter, 2001). Under these conditions, secretory and membrane proteins are ultimately degraded in the cytoplasm or translocated via an alternative, SRP- independent pathway.
Figure 5: Schematic representation of the co-translational translocation of a polytopic membrane protein. In the initial step, the signal recognition particle (SRP) binds to the signal sequence of a nascent polypeptide chain as it emerges from the tunnel exit of a cytosolic ribosome and arrests the translation process. Via interaction with the SRP receptor, the SRP- ribosome-nascent chain complex is targeted to the endoplasmic reticulum (ER) membrane. Subsequently, both the ribosome and the nascent chain are transferred to the Sec61 translocon. During resumed translation, hydrophilic loops are translocated into the ER lumen, while hydrophobic transmembrane segments are released through the lateral gate of Sec61 into the lipid bilayer.
29 4.3 Post-translational translocation
In the post-translational translocation pathway, proteins are synthesized completely in the cytosol and only subsequently targeted to the translocation machineries of different cellular compartments (Figure 6) (Cross et al., 2009). Cytosolic heat shock proteins and chaperones, such as Hsp40 and Hsp70, keep the polypeptide chains in a translocation-competent state by interacting with the hydrophobic segments and facilitating targeting (Young et al., 2003; Stefanovic and Hegde, 2007; Johnson et al., 2012; Zimmermann et al., 2011). However, many aspects of post-translational targeting remain poorly understood.
Figure 6: Schematic illustration of the post-translational translocation of a eukaryotic secretory protein. The completely synthesized polypeptide is maintained in a translocation- competent state by cytosolic chaperones and targeted to the ‘post-translocon’ in the endoplasmic reticulum (ER) membrane, which consists of the Sec61 translocon and the Sec62/63 complex. The J-domain of Sec63 recruits and activates the luminal BiP (Kar2p in yeast), which acts as a molecular ratchet and facilitates the translocation of the polypeptide chain. After cleavage of the signal sequence by the signal peptidase (not shown), the polypeptide chain is released into the ER lumen.
30 While many secretory proteins destined for the Sec61 translocon in the ER and all nuclearly encoded proteins destined for the mitochondria, chloroplasts and peroxisomes are targeted post-translationally, ER membrane proteins typically utilize the SRP-dependent co-translational pathway. However, two types of ER membrane proteins are targeted to the membrane in an SRP-independent manner after complete synthesis in the cytosol: small single-spanning membrane proteins, such as the 3.6-kDa OST complex subunit Ost4p (Chi et al., 1996), and single-spanning tail-anchored (TA) proteins, which have their only TM segment within the last approximately 40 residues of the C-terminal end (Borgese et al., 2003; Shao and Hegde, 2011). Due to their short tails after the TM segment, both types are released from the ribosomal tunnel exit after complete translation and before the SRP can efficiently interact with the hydrophobic segment. The insertion of TA proteins into the membrane is not mediated by the Sec61 translocon (Steel et al., 2002), but instead occurs either without assistance or in an ATP- and chaperone-dependent manner (Borgese and Fasana, 2011). The Get3/TRC40 system (yeast/mammals) is the most prominent chaperone- mediated pathway for ER targeting (Denic, 2012; Johnson et al., 2012). After initial binding of the hydrophobic TM segment of a TA protein by the chaperone Sgt2, the protein is transferred to the homodimeric ATPase Get3. The Get3-TA protein complex is in turn targeted to the ER membrane by complex formation with the membrane-embedded Get3 receptor, which consists of Get1 and Get2. Subsequent ATP hydrolysis induces TA protein release at the membrane (Wang et al., 2011). The mechanism, however, by which TA proteins insert into the lipid bilayer remains elusive. Both spontaneous insertion and guided insertion by Get1/2 have been discussed (Denic, 2012).
31 5. Translocation machineries in the endoplasmic reticulum
The Sec61 translocon is the major facilitator of secretory protein translocation across and membrane protein insertion into the ER membrane. For efficient targeting and translocation of post-translational pathway substrates, the Sec61 translocon associates with an additional membrane protein complex, called the Sec62/63 complex (Figure 6).
In the following sections, the structure and function of both of these complexes will be discussed in detail.
5.1 The Sec61 translocon
The conserved eukaryotic Sec61 translocon is a heterotrimeric integral membrane protein complex, which is composed of Sec61α (Sec61p in yeast), Sec61β (Sbh1p) and Sec61γ (Sss1p) (Hartmann et al., 1994; Rapoport, 2007). It is functionally homologous to the trimeric SecY complexes in bacteria (i.e., SecYEG) and archaea (i.e., SecYEβ). Additionally, the α- and γ-subunits of the Sec61 translocon share a high sequence similarity with SecY and SecE, respectively (Van den Berg et al., 2004). The Sec61 and SecY complexes are both essential, but they reside in different locations in the cell; Sec61 resides in the ER membrane, and SecY resides in the plasma membrane.
Although early crosslinking studies identified the α-subunit as the translocation pore-forming component (Mothes et al., 1994), only the 3.2 Å
32 crystal structure of the Methanococcus jannaschii SecYEβ, which was solved by van den Berg et al., allowed for detailed insights into the channel structure (Figure 7) (Van den Berg et al., 2004). Several subsequent X-ray structures have since confirmed the SecY complex structure (Tsukazaki et al., 2008; Egea and Stroud, 2010).
Figure 7: Crystal structure of the trimeric SecYEβ translocon from M. jannaschii (PDB: 1RHZ), viewed from the side (left) and the cytosol (right). The channel, which is formed by the 10 TM segments of the α-subunit (SecY, beige), is blocked from the extracytosolic side by the plug domain (TM2a, yellow). TM helices 2b (orange) and 7 (blue) form the lateral gate that allows partitioning of hydrophobic segments into the lipid bilayer. The β- and γ-subunits (SecE) are depicted in grey. The grey bars indicate the approximate boundaries of the membrane.
The 10 TM segments of the α-subunit are divided into two bundles of five helices each (TM1-5 and TM6-10), which together form a membrane percolating hourglass-shaped pore, with the loop between TM5 and TM6 serving as the interconnecting ‘hinge’ (Van den Berg et al., 2004). The extracytoplasmic/lumenal funnel of the channel is sealed by a short α-helix (TM2a), referred to as the ‘plug domain’ (Figure 7). The narrow constriction in the midst of the channel, the ‘pore ring’, which is formed by six conserved hydrophobic residues, has a diameter of 5-8 Å and can harbor a translocating
33 polypeptide chain. The interplay of the ‘plug domain’, the ‘pore ring’ and extracytoplasmic factors, such as the luminal chaperone BiP (Kar2p in yeast), maintains the membrane barrier and impedes the unwanted flow of small molecules, such as ions or metabolites, across the membrane (Van den Berg et al., 2004; Park and Rapoport, 2011; Schäuble et al., 2012). In the course of polypeptide translocation, the ‘plug domain’ is pushed sideways into a cavity on the extracytoplasmic/luminal side of the translocon. In this state, the ‘pore ring’ serves as a seal around the translocating polypeptide chain and prevents the flux of small molecules (Park and Rapoport, 2011). Concerning the location of the ‘lateral gate’, which allows partitioning of hydrophobic TM segments from the channel into the lipid bilayer, both cross-linking and structural data point to the interface formed by TM2b and TM7, which is located opposite to the ‘hinge’ (Figure 7) (Plath et al., 1998; Heinrich et al., 2000; Tsukazaki et al., 2008; Egea and Stroud, 2010). For TM segment exposure to and subsequent integration into the hydrophobic environment of the lipid bilayer, the ‘lateral gate’ must undergo conformational changes and obtain an at least partially open state. However, the mechanism by which ‘lateral gate’ opening is triggered remains unclear. Molecular dynamics simulation suggests that hydrophobic TM segment binding within the translocon channel could stabilize an open conformation of the ‘lateral gate’ (Zhang and Miller, 2010).
Because the β-subunit of the translocon is non-essential and has only few interaction sites with the α-subunit, its function is currently unknown. The two TM helixes of the γ-subunit on the other hand, form a clamp-like structure on the ‘hinge’ side of the translocon (Van den Berg et al., 2004). Sss1p, the γ-subunit in yeast, was shown to be important for Sec61 stability, for co- and post-translational translocation, and for stable association of Sec61 with the Sec62/63 complex (Falcone et al., 2011). In addition, Sss1 was found to directly interact with a subunit of the OST complex and
34 coordinate efficient N-linked glycosylation of translocating polypeptides (Scheper et al., 2003).
During co-translational translocation, ribosomes tightly associate with the α- subunit of the Sec61 translocon at the rough ER (Kalies et al., 1994). This association was visualized using cryo-EM reconstitutions of 80S ribosomes in complex with Sec61 translocons (Beckmann et al., 2001; Ménétret et al., 2005). The ribosomal tunnel exit was found to align with the Sec61 channel, allowing direct transfer of the elongating polypeptide chain into the translocon. The cytosolic Sec61 loops 6 and 8 are the main interaction sites, reaching deep into the ribosomal tunnel exit (Becker et al., 2009). However, the previously proposal that tight ribosome binding inhibits ion flux through the Sec61 channel must be re-assessed, as the available structures show a large gap between the ribosome and the cytosolic surface of the Sec61 translocon (Becker et al., 2009; Zimmermann et al., 2011).
Although it is generally accepted that a single Sec61 molecule forms the functional translocation channel, it remains controversial whether the Sec61 translocon forms oligomers in the membrane (Park and Rapoport, 2012b; a).
The ER membrane of S. cerevisiae contains a protein of high sequence similarity to Sec61p (34% identity), named Ssh1p (Sec61 homolog one) (Finke et al., 1996). Together with Sbh2p and Sss1p, Ssh1p also forms a functional translocon. Because Ssh1p associates directly with the ribosome but does not interact with the Sec62/63 complex, it is believed to be exclusively involved in co-translational translocation (Finke et al., 1996; Wittke et al., 2002). Although Ssh1p is not essential, cells lacking components of this second translocation machinery exhibit reduced viability, defects in protein translocation, dislocation of unfolded proteins from the ER and a number of other defects (Wilkinson et al., 2001).
35 5.2 The Sec62/63 complex
The yeast Sec62/63 complex is composed of the essential subunits Sec62p and Sec63p, as well as the two non-essential subunits Sec71p and Sec72p (Figure 8) (Deshaies and Schekman, 1989; 1990; Green et al., 1992; Feldheim et al., 1993; Fang and Green, 1994). In mammalian cells, the complex contains only the two essential proteins Sec62 and Sec63, which are well-conserved in all eukaryotes (Meyer et al., 2000; Tyedmers et al., 2000; Müller et al., 2010).
Although no high-resolution structures are currently available, a combination of biochemical methods and sequence analysis provided insights into the structure of the Sec62/63 complex and its association with the Sec61 translocon (Deshaies and Schekman, 1990; Harada et al., 2011).
Figure 8: Schematic illustration of the yeast Sec62/63 complex components. Clusters of positively and negatively charged residues are indicated in the essential Sec62p and Sec63p proteins, respectively. The luminal J-domain is also indicated in Sec63p,
Sec62p is an integral membrane protein with two TM segments, and its N- and C-terminal domains are located in the cytosol (Figure 8) (Deshaies and Schekman, 1990). A cluster of positively charged residues in the N-terminal
36 domain of the protein was shown to be important for interaction with the negatively charged C-terminal domain of Sec63p (Willer et al., 2003; Wittke et al., 2000). Sec63p has three TM segments, with the N-terminus and the C- terminus residing in the ER lumen and the cytosol, respectively (Figure 8) (Rothblatt et al., 1989; Feldheim et al., 1992). In addition to the previously mentioned negatively charged C-terminal domain, Sec63p harbors a J- domain in the luminal loop that interconnects TM2 and TM3. The J-domain establishes the interaction with the luminal chaperone Kar2p (BiP in higher eukaryotes). Sec63p also contains a cytosolic Brl domain, which is located after the third TM segment (Feldheim et al., 1992; Ponting, 2000). The conserved Brl domain appears to be the site of interaction with the trimeric Sec61 translocon, most likely via the cytosolic loop 6 of the Sec61 α-subunit (Jermy et al., 2006; Harada et al., 2011). Sec71p is a single-spanning membrane protein, while Sec72p is only associated with the ER membrane and does not possess any TM segments (Figure 8) (Feldheim et al., 1993). Both proteins are integrated into the complex via interaction with Sec63p (Harada et al., 2011).
Although the Sec62/Sec63 complex was shown to be essential for efficient post-translational translocation (Figure 6) (Deshaies et al., 1991; Panzner et al., 1995; Lyman and Schekman, 1997), knowledge of the specific roles of this complex and the underlying molecular mechanisms remains limited. Photocrosslinking of secretory proteins to components of the Sec62/63 complex during an early stage of translocation suggested that the complex functions as a general signal sequence receptor in the post-translational translocation pathway (Müsch et al., 1992; Ng et al., 1996; Plath et al., 2004). In addition, cooperation between Sec63p and Kar2p was found to be required for translocation of co-translationally targeted substrates (Brodsky et al., 1995; Young et al., 2001; Lang et al., 2012). In this context, the luminal chaperone Kar2p functions as an ATP-driven molecular ratchet that facilitates the transfer of the polypeptide chain through the channel (Figure
37 6) (Matlack et al., 1999). The mammalian Sec62 associates with the ribosomal tunnel exit, further indicating a possible role in co-translational translocation (Müller et al., 2010). We recently showed that Sec62p functions not only as a facilitator for the translocation of moderately hydrophobic signal anchor proteins but also as a mediator of their topogenesis (Reithinger et al., 2013).
5.3 Accessory proteins
In addition to previously mentioned chaperones, such as BiP/Kar2p and the OST complex, a number of other factors modulate the polypeptide translocation process and are directly or indirectly associated with the Sec translocon (Shao and Hegde, 2011).
The signal peptidase (SP) is an ER membrane-anchored serine protease, with its active site located in the lumen (Dalbey et al., 1997; Paetzel et al., 1998; Auclair et al., 2012). SP is responsible for the processing of secretory or membrane proteins that are targeted to the Sec translocon via a cleavable N- terminal signal sequence. This processing is crucial for the subsequent release of secretory proteins into the lumen, as well as for the proper folding of membrane proteins.
In mammalian cells, TRAM (translocating-chain associated membrane protein) was found to be essential in early steps of translocation of a subgroup of proteins through the Sec61 translocon (Görlich and Rapoport, 1993; Voigt et al., 1996).
The TRAP (translocon-associated protein) complex is another factor required for efficient protein translocation into the mammalian ER (Fons et al., 2003). TRAP was recently shown to be involved in membrane protein topogenesis and may perform a role similar to that of Sec62p in yeast (Reithinger et al., 2013; Sommer et al., 2013).
38 6. Insertion of α-helical membrane proteins into the endoplasmic reticulum membrane
After successful co- or post-translational targeting of a membrane protein to the Sec61 translocon in the ER membrane, the TM segments must be recognized and correctly oriented in the channel before subsequent lateral integration into the lipid bilayer. The information required for insertion and correct topology is contained in the amino acid sequence.
The major factor that drives recognition of the TM segment by the channel and insertion into the membrane is the overall hydrophobicity of the segment (White and von Heijne, 2008). Comprehensive studies of the E.coli leader peptidase (Lep) in a variety of in vivo and in vitro systems led to the establishment of ‘biological scales,’ which contain information concerning position-specific contributions to the insertion efficiency of TM segments for all natural amino acids (Hessa et al., 2005; 2007; 2009; Lundin et al., 2008).
The apparent free energy of insertion (ΔGapp) is the quantitative term used to express the potential for membrane insertion (White and von Heijne, 2008).
The experimentally obtained ‘biological scales’ correlate well with data from biophysical partitioning experiments, in which insertion into the lipid bilayer was not mediated by a Sec translocon (Wimley and White, 1996). These findings, together with results from molecular dynamics simulations (Ulmschneider et al., 2011; Zhang and Miller, 2012), indicate that membrane integration is a thermodynamically driven equilibrium process, in which TM segments of sufficient hydrophobicity would partition from the hydrophilic channel interior into the hydrophobic environment. In this model, the Sec
39 translocon would only serve as a passive, continuously opening and closing conduit to the lipid environment (Heinrich et al., 2000; Osborne et al., 2005).
However, recent experimental data from yeast indicate that the Sec61 translocon might play a more active role in the partitioning process. Substitutions in the ‘pore ring’ and in the helices forming the ‘lateral gate’ altered the hydrophobicity threshold for TM segment integration (Junne et al., 2010; Trueman et al., 2012). Additionally, we found that substitutions that affected the translocation efficiency also changed the insertion rate of the TM segments (paper II). Substitutions in the gate and in the ‘plug domain’ of Sec61 also affected the topogenesis of membrane proteins (Junne et al., 2006; 2007).
Hydrophobicity and the distribution of charged residues are both sequence- intrinsic factors that direct TM segment topogenesis. Single-spanning membrane proteins of high hydrophobicity insert preferentially in an Nout/Cin orientation (Wahlberg and Spiess, 1997; Eusebio et al., 1998), while positively charged residues on one side of a TM segment favor cytosolic localization of that flanking sequence (i.e., the ‘positive-inside rule’) (von Heijne, 1986b; 1989).
Two models describe the possible mechanism of insertion of a single- spanning membrane protein after targeting to the translocon. In the first model, the signal anchor (TM segment) is initially oriented in an Nin/Cout membrane topology and the polypeptide enters the translocon channel as a loop, with the N-terminus being retained at the cytosolic surface of the translocon. In the case of a positively charged N-terminus, the protein would keep this orientation and depart from the translocon as a type II membrane protein (Shaw et al., 1988). Otherwise, the TM segment would undergo topological re-orientation in the channel and the protein would leave as a type I membrane protein (Goder et al., 1999). In the second model, the signal would insert ‘head-first’ in an Nout/Cin orientation and adjust to the final
40 topology in a second step, according to charge distribution (Goder and Spiess, 2003; Devaraneni et al., 2011). Both models are also applicable to secretory proteins, which are targeted by a cleavable signal sequence, as well as the first TM segment of multi-spanning membrane proteins.
For most multi-spanning membrane proteins, the insertion of the first TM segment determines the orientation of the following TM segments, which enter into the translocon channel in a sequential manner before partitioning into the membrane in an orientation opposite to that of the preceding TM segment (Sadlish and Skach, 2004). However, a basic sequential insertion model does not account for the complexity of multi-spanning membrane proteins (Shao and Hegde, 2011). Particularly in eukaryotic cells, many TM segments of polytopic membrane proteins are not sufficiently hydrophobic to insert into the membrane by themselves (Bernsel et al., 2008; Park and Helms, 2008). Helix-helix interactions with neighboring TM segments, such as hydrogen bonding, were shown to assist the insertion process of TM helices of moderate and low hydrophobicity (Meindl-Beinker et al., 2006; Enquist et al., 2009). The Sec61 translocon might provide the framework for the pre-insertion assembly of two or more helices, as it can harbor two translating polypeptide chains within the channel (Kida et al., 2010) and multiple chains in its direct vicinity (Sadlish and Skach, 2004; Ismail et al., 2008).
Although we have gained insight into the processes by which TM segments are recognized and the factors that contribute to their insertion and topogenesis, knowledge of the underlying molecular mechanisms is limited.
41 7. Summary of papers
7.1 A biological hydrophobicity scale in yeast (paper I)
After ER membrane targeting by either the co- or post-translational translocation pathway, hydrophobic segments of the newly synthesized membrane protein are released into the lipid bilayer through the lateral gate of the Sec61 translocon (Rapoport, 2007). The information that dictates how efficiently a segment is inserted into the membrane is encoded in its amino acid sequence. In previous studies, the ‘molecular code’ for the position- dependent contributions of individual amino acids, TM segment length, flanking charges and overall hydrophobicity to the membrane insertion efficiency, was deciphered in in vitro and in vivo systems, using dog pancreas rough microsomes and BHK cells, respectively (Hessa et al., 2005; 2007; Lundin et al., 2008). In this paper, we established comparable data in the yeast S. cerevisiae, using a modified version of the E. coli Lep and a glycosylation-based membrane insertion assay (Lundin et al., 2008). We measured the insertion efficiency of a set of 19-residue H-segments composed of varying numbers of Ala and Leu residues and found that the threshold hydrophobicity for membrane insertion is similar in yeast and mammalian cells. By placing each of the 20 natural amino acids in the middle of the Ala/Leu based H- segment individually, we could further determine the individual contribution of each amino acid to the apparent free energy of membrane insertion
(ΔGapp) and establish a biological hydrophobicity scale in yeast (Figure 9).
42
Figure 9: Biological hydrophobicity scale. The contributions of individual amino acids to membrane insertion were determined using Ala/Leu based H-segments, with the corresponding amino acid placed in the center (Hessa et al., 2009). Reprinted with permission.
Interestingly, although the yeast scale correlates well with the mammalian scale, we observed that the absolute contributions of the individual residues are decreased by a factor of approximately 2 in yeast. The reasons for this difference could include differences in translocon architecture and/or differences in lipid composition.
7.2 Analysis of the Sec61 lateral gate profile (paper II)
The conserved trimeric Sec61 translocon provides the main pathway across and into the ER membrane. The ten TM segments of the Sec61 α-subunit form an hourglass-shaped channel that facilitates the passage of secretory proteins into the ER lumen, while a ‘lateral gate’, formed by TM2b and TM7, allows partitioning of hydrophobic segments of membrane proteins from the channel interior into the lipid bilayer (Rapoport, 2007). Both co- and post-translationally targeted proteins are substrates for Sec61-mediated translocation. Although previous studies supported the hypothesis that TM segment insertion is a thermodynamically driven process in which a hydrophobic segment can partition freely between the hydrophilic channel
43 and the hydrophobic membrane environment, it remains unclear to what extent translocon itself modulates TM segment insertion and topogenesis (Hessa et al., 2005; Ulmschneider et al., 2011).
Here we investigated which residues of the lateral gating helices TM2b and TM7 are critical for modulating TM segment insertion using systematic alanine scanning and photo-crosslinking. We identified two groups of helix residues for which substitution with alanine resulted in an increase or decrease in insertion efficiency for the test H-segment in both single- and multi-spanning Lep-based model proteins. Interestingly, an increase or decrease in insertion coincided with an elevated or reduced relative amount of single-spanning model protein with an Nin/Cout membrane topology, respectively. Theses findings indicate that key residues in the ‘lateral gate’ modulate the conformational state of the gate; a more open conformation would reduce the threshold hydrophobicity for TM segment insertion and facilitate the reorientation of TM segments, while a closed conformation would induce the opposite effects.
To assign these key residues to specific positions within the profile of the gate helices, we performed in vivo site-specific photo-crosslinking experiments. We found residues in both TM2b and TM7 that are in close proximity to the other helix and are thus part of the gate profile. Mapping of these residues in the crystal structures of the homologue SecY (Van den Berg et al., 2004; Tsukazaki et al., 2008) indicates high structural similarity between archaeal/bacterial and eukaryotic translocons. Further, substitution of gate-forming residues with alanine was found to decrease the insertion efficiency of model proteins. This finding suggests that the key residues in the gate profile are necessary to maintain balance between the open and closed conformations.
44 7.3 Insertion and topogenesis of signal anchor proteins (paper III)
Proteins destined for the ER are targeted either co- or post-translationally by a cleavable or uncleavable signal sequence (i.e., signal anchor). It was previously demonstrated that highly hydrophobic signals are targeted co- translationally, while signals of low hydrophobicity are targeted by the post- translational pathway (Ng et al., 1996). The SRP plays an essential role in co-translational targeting, while the Sec62/63 complex is believed to function in post-translational translocation only. Subsequent translocation into the lumen or integration into the membrane is mediated by the Sec61 translocon (Rapoport, 2007).
In this paper, we used both model and cellular proteins with signal anchors of varying hydrophobicity to systematically evaluate the relationship between signal hydrophobicity and targeting pathway preference. The signal anchor proteins were expressed in temperature-sensitive S. cerevisiae strains, which were either deficient in SRP or Sec62p function, and tested for targeting and membrane insertion efficiency (Wilkinson et al., 2001). The SRP was shown to be important for the targeting and translocation of signal anchor proteins with a wide range of hydrophobicities, whereas the Sec62p defect only decreased the targeting and insertion efficiency of moderately hydrophobic signal anchors, indicating that the pathways might not be mutually exclusive. Additionally, we found that signal anchors preferentially acquired an Nout/Cin membrane topology when expressed in the yeast strain with defective Sec62p function, suggesting that the Sec62/63 complex plays a more general role in membrane protein biogenesis. This finding was investigated further in paper IV.
45 7.4 Sec62 regulates membrane protein topogenesis (paper IV)
To follow up on the results obtained in paper III, we investigated the potential expanded role of Sec62p in membrane protein topogenesis. In yeast, Sec62p is an essential subunit of the Sec62/63 complex, which also consists of Sec63p, Sec71p and Sec72p and combines with the trimeric Sec61 complex to form the heptameric ‘post-translocon’ (Harada et al., 2011). Sec62p has two TM segments that traverse the ER membrane and expose both the N- and C-termini to the cytosol. A cluster of positively charged residues in the N-terminal domain was shown to facilitate interaction with a negatively charged C-terminal domain in Sec63p (Wittke et al., 2000).
We first created a library of Sec62p variants and tested them for the ability to rescue a genomic sec62 deletion and form a stable complex with Sec63p in S. cerevisiae. Except for a triple-Ala substitution after the second TM segment, all variants complemented the deletion; however, the introduction of negative charges into the N-terminus impaired stable complex formation with Sec63p, affirming the N-terminal domain as the major site of interaction.
The Sec62p variants were then tested for their effects on the translocation efficiency of both natural and engineered model single- and multi-spanning membrane proteins. Substitutions in the Sec63p-interaction domain impaired the insertion of moderately hydrophobic TM segments into the ER membrane and the translocation of C-terminal domains into the ER lumen. Sec62p appears to function as a facilitator of C-terminal translocation for single- and multi-spanning membrane proteins by promoting an Nin/Cout TM segment topology.
46 7.5 C-terminal tail length affects membrane protein targeting (paper V)
Nuclearly encoded mitochondrial membrane proteins are targeted post- translationally to the mitochondrial import machineries TOM and TIM (Neupert and Herrmann, 2007). The pre-sequences and TM segments of these proteins are generally less hydrophobic than the signal sequences and TM segments of membrane proteins destined for the ER (von Heijne, 1986c). However, there are exceptions, such as the subunit of the mitochondrial succinate dehydrogenase Sdh3 and its homolog Shh3, which contain strongly hydrophobic TM segments at their C-termini and reside in the inner mitochondrial membrane (Szeto et al., 2012). Interestingly, Shh3 was mis-targeted to the ER when fused with a large C-terminal topology reporter domain (Kim et al., 2006).
Inspired by this observation, we investigated whether long C-terminal extensions could cause SRP-recognition and subsequent co-translational ER mis-targeting of hydrophobic mitochondrial membrane proteins. A set of Sdh3 and Shh3 fusion proteins with C-terminal extensions of different lengths was expressed in wild-type and SRP-deficient S. cerevisiae strains and tested for ER targeting using a glycosylation assay. We found that a C- terminal tail of 120 or more residues downstream from a highly hydrophobic TM segment is sufficient to trigger SRP recognition and at least partial targeting of a protein to the ER. Bioinformatics analysis further revealed that in yeast and mammalian cells, the vast majority of nuclearly encoded mitochondrial membrane proteins contain C-terminal tails that are shorter than 100 residues, thus suggesting that evading SRP recognition by having a short C-terminal tail may be a general strategy by which mitochondrial proteins avoid mis-targeting to the ER.
47 8. Conclusions
Although 40 years of research has revealed the main principles of membrane protein biosynthesis and the cellular components involved, knowledge concerning many aspects of the underlying molecular mechanisms remains limited. In this thesis, open questions were addressed using the yeast S. cerevisiae as a eukaryotic model organism.
The Sec61 translocon, the main translocation machinery in the ER membrane, allows for lateral partitioning of hydrophobic segments into the lipid bilayer and is a key component of membrane protein biosynthesis. Thus, an important question in the field of membrane protein biosynthesis is the degree to which translocons from different organisms are functionally and structurally similar. By establishing the sequence requirements for TM segment insertion into the membrane by the yeast Sec61 translocon and a ‘biological hydrophobicity scale’ for the individual contributions of natural amino acids to the apparent free energy of insertion (paper I) and comparing it with the results from previous, comprehensive studies performed in mammalian systems (Hessa et al., 2005; 2007; Lundin et al., 2008), we found that the ‘molecular code’ of the different translocons shows a high degree of similarity. Additionally, the identification of the residues involved in forming the profile of the ‘lateral gate’ in the yeast translocon and subsequent mapping of these residues onto the corresponding positions of the known SecY structures from bacteria and archaea (Van den Berg et al., 2004; Egea and Stroud, 2010) suggested a high degree of structural similarity among the translocon gates from different organisms (paper II). The profile of the gate appears to define the parameters of insertion, as
48 mutations within the gate helices 2b and 7 altered the threshold of hydrophobicity for TM segment insertion and membrane topology (paper II). It will be exciting to use these insights to gain an improved understanding of the mechanism of TM segment recognition and ‘lateral gate’ opening on a molecular level.
Although it was previously established that proteins are targeted co- or post- translationally to the ER membrane based on signal sequence hydrophobicity (Ng et al., 1996), we present the first systematic analysis of the relationship between hydrophobicity and targeting route preference (paper III). We found that for signal anchor proteins, the targeting routes are not mutually exclusive. In addition, we identified Sec62p, a part of the heptameric ‘post- translocon’ that was previously believed to be involved in signal-sequence recognition only, as a modulator of signal anchor topogenesis that promotes the insertion of these proteins as type II membrane proteins. Further analysis of Sec62p in single- and multi-spanning membrane protein topogenesis suggests that this protein plays a general role in C-terminal translocation (paper IV). This finding challenges the model, which posits that the Sec62/63 complex associates with the Sec61 translocon only transiently for translocation of post-translationally targeted substrates. However, future research is needed to establish the mechanism by which C-terminal translocation is facilitated by the Sec62/63 translocon.
The correct targeting of cytosolically translated membrane proteins to the designated organelle is a crucial task for eukaryotic cells. Nuclearly encoded proteins evade mis-targeting to the ER by containing TM segments of low hydrophobicity that are not recognized by the SRP. However, there are exceptions to this rule. We demonstrate that these exceptions sidestep SRP- recognition by having short C-terminal tails after strongly hydrophobic TM segments (paper V). These short tails likely cause the release of the
49 translated protein before the SRP can efficiently bind and mis-target the protein to the ER.
50 9. Populärvetenskaplig sammanfattning på svenska
Celler är de grundläggande byggstenarna i alla levande organismer. Medan de flesta bakterier och vissa svampar består av en enda cell, och kallas därmed för encelliga organismer, så består flercelliga organismer av två eller flera celler. Antalet celler i människokroppen uppgår exempelvis till biljoner.
Viktiga komponenter i alla celler är de biologiska membranen, som i huvudsak består av lipider och proteiner. Membranen omger cellerna och separerar deras inre från omgivningen, men kan även bilda små utrymmen inuti cellerna, så kallade organeller. De proteiner som finns i membranet (membranproteiner) uppfyller många olika och viktiga funktioner, såsom transport av näringsämnen eller signalering genom membranet.
Denna avhandling innehåller fem studier där vi försökt att förstå mer om hur membranproteiner syntetiseras i cellen, med hjälp av bagerijäst som modellorganism. Centralt för denna process är Sec61 translokonet, en molekylär maskin, som både kan transportera proteiner genom membranet och sätta in dem i membranet.
Vi kunde fastställa regler genom vilka Sec61 translokonen i jäst avgör huru- vida olika proteiner antingen transporteras genom eller in i membranet. Jäm- förelsen av våra resultat från jäst med data från bakterier och däggdjurscel- ler, visar att translokon från olika organismer fungerar på ett liknande sätt. Vidare fann vi att Sec62, en faktor associerad med translokonen, har hittills okända funktioner för korrekt och effektiv insättning av proteiner in i mem- branet.
51 Acknowledgements
So many people helped and supported me during the past years at Seoul National University and at Stockholm University and made me feel welcome and at home in both worlds. Thank you! It has been a pleasure working in such a positive and inspiring atmosphere! Here are some special thanks to people who made this thesis possible:
Joy, you have been a great supervisor and friend, and it has been fantastic and exciting to be part of the enterprise of starting your own group in South Korea! I am very grateful for all the things I learned from you on the way, not only concerning science, and will miss our discussions about the world and his brother. Thank you so much for taking good care of me and supporting me all the way throughout my thesis!
Gunnar, my supervisor, thank you for making this thesis possible in the first place and always being there when I needed help. It has been an honor to be part of your group! Your way of doing science and everything else is an inspiration!
Many thanks to Stefan, for taking care of all PhD-business and together with Elzbieta, Peter, Pia and Åke for guiding me through the various check points and helpful suggestions!
I owe a great deal to the members of Joy’s lab at SNU! Hunsang, thank you for being a good friend and fun lab mate, and for your patience and help
52 with all my Korean problems! Sungjun, Hani, and Chewon, it was great working with you! Thank you for many fun moments and interesting talks in and outside the lab! This thesis would have been impossible without you! Superduper Kwangjin, the spirit of the lab, thanks for a lot of fun! Kyungyeun, Sungmin, Ramla and Hayoung, thank you for the great working environment - Keep up the good work! You all helped to make me feel welcome and at home at SNU and I am very grateful for that!
Gyubum and Yeon Ji, from the Huh lab next door, thank you so much for showing me around Seoul! I enjoyed hanging out and talking about movies, the universe and everything!
Many thanks to all members, past and present, of the awesome GvH offices – it has been a frenzy! Thank you, Rickard and Bill, for all the laughs and fun discussions; Nurzian, my gym buddy, for taking care of everything and being so patient with us; Pilar, for all the support during learning and being a great office mate; Carmen, the center of peace in the office, for always being kind and understanding, and many great discussions; Salomé, for sharing so much during our time at DBB in countless good talks; Florian, for all the good Lantis times and help whenever needed; the next door ladies, Patricia, Nina, Karin and now Hena, as well as our new post-docs Annika and Ola, for a great working environment; our distant members Patrik and Minttu and former members Susanna, Roger, Joanna, Marie, Kalle and Morten, for sharing their knowledge and making the lab an inspiring place; Tara, for teaching me a lot and sharing your project with me.
I also thank all the members of the neighboring groups for the great atmosphere at DBB, especially: Dan and his group with Isolde, Stephen,
53 Kalle and Claudio; Jörg, for great fun, talks and distraction; IngMarie, for giving me a roof over my head and always being so kind and helpful; Catarina, Pedro, Beata and Erika in the EG lab; Jan-Willem, David, Susan, Thomas, Mirjam and Anna in the JWdG; Rob and his team, Diogo, Johan and Dan. Thanks also to Martin and David, and their groups upstairs; Manfred, for good times at the wall!
Many thanks to Ann, Maria, Malin, Lotta, Alex and Håkan for keeping things running and being always kind and helpful!
To all my friends, thank you so much for your support, although I have been away, so far and for so long! Special thanks to Moritz and Fabian for so many years of loyal friendship and uncountable great memories!
With all my heart, I thank my parents, my brother Martin with Alex and Paul, my sisters Susi and Lisa, as well as my grandmother for their unconditional love and support over the years! I am incredibly grateful for all the care and the possibilities my parents granted me, without which I could not have concluded this project!
Caroline, you are wonderful! Thank you so much for your love, support and always being there for me, even when half a world away! You enrich my life more than words can say!
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