Pulling Force Studies of Secretory Protein Translocation Into The

Home , Centriole, Golgi apparatus, SEC63, Secretory protein, TRAM2, Translocon

Theresa Kriegler Pulling Force Studies of Secretory Protein Translocation into the Endoplasmic Reticulum Pulling ForcePulling of Secretory Protein Studies Translocation into Endoplasmic Reticulum the Theresa Kriegler

Ribosome

Arrest peptide

Sec61 Signal sequence Sec63 Sec62 Cytosol TRAM ER lumen OST ISBN 978-91-7911-240-0 TRAP

Bip

Department of Biochemistry and Biophysics

Doctoral Thesis in Biochemistry at Stockholm University, Sweden 2020

Pulling Force Studies of Secretory Protein Translocation into the Endoplasmic Reticulum Theresa Kriegler Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Friday 18 September 2020 at 14.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.

Abstract More than 30% of human genes encode secretory or membrane proteins. Most secretory proteins are targeted to the Endoplasmic reticulum (ER) membrane via cleavable N-terminal signal sequences either in a co- or post-translational manner. They enter or cross the membrane using a protein translocating channel (translocon). Although the core of the translocon, formed by the Sec61 complex, was identified some time ago, the details of how signal sequences can facilitate channel opening and initiate protein translocation still remain unclear. Interestingly, the signal sequences of different proteins do not share any sequence homology—only general motifs have been described—but the precise sequence has been found to substantially affect the efficiency of translocation initiation. Many proteins require auxiliary components in order to enter the ER lumen. During ER stress conditions, these weakly gating proteins are prevented from entering, reducing the load of unfolded protein within the ER and protecting the cell. Consequently, it is tempting to hypothesize that the “inefficiencies” of signal sequences may actually provide a different message that works as a protective mechanism during ER stress conditions. Here, we employed a translational arrest peptide, which pauses the ribosome until a force—such as the interaction of the signal sequence with the translocon—acts on the nascent chain. We analyzed the different forces that are experienced by efficient and inefficient signal sequences during their biosynthesis in vitro. Our data shows that the efficient signal sequence of prolactin (Prl) experiences a strong biphasic pulling force while less efficient sequences, such as the ones from the Prion protein (PrP) or insulin, are pulled to a much lesser extent, indicating different modes of engagement with the translocon. The Prl signal sequence interacts first with a hydrophobic patch within the channel (the first pulling event), next it is inverted and intercalates into the lateral gate of the translocon, facilitating channel opening both laterally and axially. In the case of PrP or insulin, the initiation of translocation is delayed, suggesting that the opening of the channel might require auxiliary components. In order to explore this, we made use of semi-permeabilized cells (SPCs) prepared after siRNA knockdown of components of the translocation machinery and studied the effect on the observed pulling events and translocation efficiency. We found that the translocon-associated protein (TRAP) complex enhanced translocation of client proteins bearing weakly gating signal sequences that contained more glycine and proline residues. Additionally, we showed that TRAP plays a role in the translocation of intrinsically disordered domains with a high content of proline and glycine residues, and other regions of the mature protein enriched in positively charged amino acids. Chemical crosslinking revealed that TRAP contacts the insulin nascent chain before it enters the translocon channel suggesting that TRAP scans along the translocating protein and provides sequence-dependent assistance in facilitating channel opening and as a ratchet for challenging regions of the mature protein. Taken together, all of this data expands our understanding of the interplay between the signal sequence and the mature protein during translocation and protein folding and how the cell may take advantage of this to regulate translocation during ER stress.

Keywords: Co-translational translocation, Xbp1 arrest peptide, Translocon-associated protein (TRAP) complex, Prion protein, Insulin.

Stockholm 2020 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-183758

ISBN 978-91-7911-240-0 ISBN 978-91-7911-241-7

Department of Biochemistry and Biophysics

Stockholm University, 106 91 Stockholm

PULLING FORCE STUDIES OF SECRETORY PROTEIN TRANSLOCATION INTO THE ENDOPLASMIC RETICULUM

Theresa Kriegler

Pulling Force Studies of Secretory Protein Translocation into the Endoplasmic Reticulum

Theresa Kriegler ©Theresa Kriegler, Stockholm University 2020

ISBN print 978-91-7911-240-0 ISBN PDF 978-91-7911-241-7

Cover image, images and image modifications in comprehensive summary by Theresa Kriegler

Reprints in this thesis are in accordance to the author's rights for publications of the Cell Press and Elsevier publishing groups.

Printed in Sweden by Universitetsservice US-AB, Stockholm 2020 Look at this life - all mystery and magic.

- Harry Houdini

List of publications

I. Kriegler T., Magoulopoulou A., Amate Marchal R., Hessa T. (2018) Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest. Cell Chemical Biology, 25(7), 880-890.e3

II. Kriegler T., Lang S., Notari L., Hessa T. (2020) Prion Protein Translocation Mechanism Revealed by Pulling Force Studies. Journal of Molecular Biology, 432(16): 4447–4465

III. Kriegler T., Lang S., Notari L., Hessa T. (2020) Supporting data on Prion Protein Translocation Mechanism Revealed by Pulling Force Studies. Data in Brief, 105931.

IV. Kriegler T., Kiburg G., Hessa T. Translocon-associated protein complex (TRAP) is crucial for translocation of pre- proinsulin. (Submitted to Journal of Molecular Biology)

Contents

Introduction ...... 1 The route of secretory proteins in eukaryotic cells ...... 1 Overview of protein targeting routes to the ER membrane ...... 4 Post-translational targeting in eukaryotes ...... 4 Co-translational targeting and translocation in eukaryotes ...... 5 Co-translational targeting of proteins to the ER membrane ...... 8 The signal recognition particle and its receptor ...... 8 ER targeting signal sequences ...... 9 The Sec61 translocon ...... 12 Structural overview ...... 12 Signal recognition by the translocon and opening of the channel ...... 14 Translocation and insertion of membrane intercalating helices ...... 15 Assisted opening of the translocon ...... 17 Auxiliary components of the Sec61 translocon ...... 18 TRAM1 ...... 18 The translocon-associated protein (TRAP) complex ...... 19 OST ...... 22 Sec62/63 and Bip ...... 23 Additional auxiliary components of the translocon ...... 25 Auxiliary components of the translocon allow translocation regulation ...... 26 Co-translational protein folding on the ribosome ...... 28 Arrest peptides ...... 30 SecM ...... 30 Xbp1 ...... 31

ii Overview of the secretory proteins studied ...... 34 Prolactin -the “good” one ...... 34 Prion protein – the “bad” one ...... 35 Insulin – the “short” one ...... 36 Introduction to the methodology ...... 39 Arrest peptides as biochemical force sensors ...... 39 In vitro cell-free systems ...... 40 ER membrane donors – RMs and SPCs ...... 41 Chemical crosslinking and co-immunoprecipitation ...... 42 Additional biochemical methods used in these studies ...... 43 Conclusions and Speculations ...... 45 Populärvetenskaplig sammanfattning ...... 51 Populärwissenschaftliche Zusammenfassung ...... 53 Acknowledgements ...... 55 References ...... 59

iii

Introduction

The route of secretory proteins in eukaryotic cells

Cells are the smallest functional units of life. They are separated from their surroundings by the plasma membrane, formed by a hydrophobic lipid bilayer containing proteins. The smallest organisms, such as bacteria used to make yogurt or the yeast we use to bake bread, consist of single cells, while larger organisms such as humans contain several trillion cells of different types that vary in size, shape and function [1]. However, some principles are shared by all cells, including that cellular proteins are synthesized on molecular ma- chines known as ribosomes [2]. Ribosomes are large complexes of protein and RNA that “translate” the nucleotide sequence encoded on a mRNA-template into the correct amino acid sequence thereby leading to the creation of a protein. The amino acids are delivered to the peptidyl transferase center (PTC) bound to transfer RNA (tRNA). Only a tRNA that matches the mRNA-tram- plate can transfer its amino acids to the nascent chain, ensuring correct peptide synthesis. While growing, the protein chain is pushed from the PTC through the ~100 Å long ribosomal “exit tunnel” in order to leave the ribosome [3]. Protein synthesis is localized (with a few exceptions) to the aqueous cytosol of the cell, which then requires an advanced sorting and targeting mechanism to ensure that each protein reaches its correct final destination within the cell or is secreted to the environment. In eukaryotic cells proteins are sorted to the various different membrane-bound organelles by a number of machineries and internal protein signals [4–6]. My work has focused on proteins destined for secretion, which share their route with plasma membrane-bound proteins, ly- sosomal proteins, and the soluble proteins that remain in the luminal compart- ments of cellular organelles [7,8]. More than 35% of the ~20000 genes of the human genome encode secretory or membrane bound proteins and in some tissues, like the pancreas, secreted proteins represent more than 70% of the proteome [9]. Interestingly, about two thirds of all approved drug targets are membrane or secretory proteins, stress- ing the relevance of this class of proteins in medical treatments.

1 While membrane and secretory proteins in prokaryotic cells are targeted directly to the plasma membrane to be inserted or exported via a translocating channel (translocon) complex; eukaryotic cells use the endoplasmic reticulum (ER) as the entry gate for membrane and secretory proteins [10]. The ER is a continuous single-membrane network that is divided into three major mor- phologies: the nuclear envelope, peripheral “rough” sheets or cisternae and peripheral “smooth” tubules [11]. The nuclear envelope is formed by two flat lipid bilayers that surround the nucleus, forming the so-called perinuclear space, which is punctuated by nuclear pores [12]. The peripheral ER branches out from the outer layer of the nuclear envelope and all the way to the plasma membrane, creating a large network of sheets and tubules. The differentiation between the rough and smooth ER was first made by electron microscopy, where George Palade and his co-workers identified that the ER was forming large stacked sheets that were “dotted with small, dense granules” (later named ribosomes) and connected to each other via a tubular network [13]. Gen- erally, the cisternae are densely covered with ribosomes bound to the cytosolic side of the ER membrane giving them a “rough” surface, while tubules are often “ribosome-free” [14]. For this reason, it was assumed that the rough sheets are the site of membrane and secretory protein synthesis, while the tubules are the site of lipid synthesis and ER vesicle budding. However, it is likely that the functions are not so strictly divided, as the whole ER structure is very dynamic [12]. The fact that the ER is partially covered with bound ribosomes was an important finding that coupled the synthesis of proteins on the ribosome directly to their translocation into the ER (co-translational translocation) and will be discussed in more detail later on. Most secretory proteins entering the ER encode an N-terminal signal sequence which serves as a sorting signal for the cell and is cleaved off as soon as the protein reaches the ER lumen [15,16]. In contrast to the reducing environment of the cytoplasm, the ER lumen allows the formation of disulfide bonds between specific cysteine residues in nascent proteins, which contribute to the stability of the final mature structure [17,18]. This process, as well as protein folding in general, is often assisted by chaperones in order to ensure adoption of the correct structure and to prevent aggregation [19]. Another very common type of protein modification performed in the ER is the covalent attachment of sugar moieties (oligosaccharides or glycans) to specific asparagine residues (N) of the proteins. The N-linked-glycosylation is facilitated by the oligosaccharyltransferase (OST) complex and contributes to the final protein structure

2 and function [20,21]. Secretory proteins are transported from the ER to the Golgi complex in small coated vesicles, which fuse with the cis-cisternae of the “Golgi stack” and release the proteins into their lumen [22]. Proteins are further modified and matured while traversing the Golgi complex from the cis- to the medial to the trans-cisternae, finally being sorted by the trans-Golgi-network for transport to: other intracellular membranes such as the endosomes and lysosomes; the plasma membrane; or to be exported from the cell [8,23]. The jour- ney outlined above is schematically depicted in Figure 1.

extracellular space

cytosol 8 7 plasma Lysosomes, membrane Endodomes 6 trans-Golgi- network

Golgi 5 complex medial

cis side transport vesicles 4

3 Ribosomes

rough ER

2 Nuclear Nucleus envelope DNA -> mRNA Nuclear mRNA 1 pore complex

Figure 1: The route of secretory (red) and membrane proteins (blue) in euaryotic cells. (1) Genomic DNA is transcribed into mRNA in the nucleus. (2) The mRNA is transported into the cytosol via nuclear pore complexes. (3) Proteins are transcribed co-translationally on ribosomes bound to the ER membrane. (4) Folded and modified proteins are transported to the cis side of the Golgi complex. (5) Proteins are further modified as they traverse the Golgi complex. (6) Sorting of proteins to their final destination occurs mainly at the trans-Golgi- network. (7) Vesicles fuse with the plasma membrane to release secretory proteins into the extracellular space and deposit membrane proteins destined for the plasma membrane. (8) Proteins destined for endosomes or lysosomes local- ize via different vesicles. Figure created based on Bykov YS et al., 2017 [24] and Nelson DL and Cox MM, 2017 [25].

3 Overview of protein targeting routes to the ER membrane

Proteins can be delivered to the ER membrane either via the co-translational signal recognition particle (SRP)-dependent route or via SRP-independent post-translational routes. In the post-translational pathways, protein synthesis by the ribosome is completed before translocation across the ER membrane begins, while the co-translational pathway defines protein synthesis by membrane-bound ribosomes directly coupled to translocon channels, which allows the nascent protein to be translocated across the membrane while being synthesized [26]. In bacteria most secretory proteins are translocated via post-translational routes and the evolutionally conserved co-translational pathway is mainly used by membrane proteins [27]. In contrast, most proteins in higher eukaryotes are translocated co-translationally and only specialized protein groups employ post-translational pathways.

Post-translational targeting in eukaryotes

There are three major groups of post-translational substrates in eukaryotes: Tail-anchor proteins that carry no N-terminal signal but only a C-terminal transmembrane segment; very short secretory proteins; and proteins with mildly hydrophobic or structurally inadequate signal sequences [28]. The common feature in all of these cases is that they cannot be recognized by SRP as they either lack a proper N-terminal signal sequence or they complete synthesis before the signal is exposed. All post-translational targeting substrates must be prevented from folding or aggregating within the cytosol to ensure that they can still be transported across the ER membrane, since the translocon can only accommodate unfolded substrates. In particular, the hydrophobic sequences of targeting signals and transmembrane domains must be shielded from the aqueous environment [29]. Several chaperones that hold the precursors in an unfolded conformation have been identified for the different substrate classes. The main players are Bag6 for tail-anchor proteins [30,31]; and Hsp70/Hsp40 co-chaperones (heat shock proteins of 70/40 kDa) [28,32,33] or calmodulin [34] for weak signal sequences and small secretory proteins.

4 With the exception of the tail-anchor proteins, the central translocon pore formed by the Sec61 complex is used for both post-translational and co-translational translocation [28,35–37]. Additionally, the heterodimeric Sec62/Sec63 complex is recruited to the translocon during post-translational transport [38,39]. In yeast, Sec62/Sec63 is often used for signal sequences with lower hydrophobicity [40], while in mammals it is mostly employed as a “fail-safe” mechanism that ensures efficient secretion for very small secretory proteins, carrying an N-terminal signal sequence [41,42]. This is evidenced by the observation that proteins shorter than 100 amino acids cannot be efficiently tranlocated co- translationally, as they finish synthesis before efficient targeting to the ER membrane. These proteins depend on Sec62 for efficient translocation, which may act as the signal sequence receptor, while Sec63 is most likely involved in facilitating the opening of the translocon channel via its interaction with the ER luminal Hsp70 chaperone Bip [41–43]. Secretory proteins longer than 160 amino acids are commonly translocated co-translationally and proteins of intermediate lengths (100 to 160 amino acids) often employ both pathways [41].

Co-translational targeting and translocation in eukaryotes

The co-translational pathway is evolutionary conserved [44] and relies on a ribonucleoprotein complex known as the signal recognition particle (SRP) to recognize the N-terminal signal sequence or the first transmembrane domain of a protein as it emerges from the ribosomal exit tunnel [45,46]. In eukaryotes the interaction of SRP with the signal sequence induces translational stalling to ensure sufficient time for membrane targeting [47]. This stalling is facilitated by the Alu domain of eukaryotic SRP [48,49]. As bacterial SRPs are much sim- pler and lack an Alu domain, it is thought that translation does not slow upon SRP binding to the ribosome-nascent chain complex (RNC)[50]. In any case, SRP guides the RNC to the Sec translocon complex, where it interacts with its receptor (SR), hands the signal sequence over to the translocon, and translation resumes [51–53]. The RNC binds the Sec61 complex such that the ribosomal exit tunnel is aligned with the central translocon pore [54]. The interactions between the translating ribosome, the signal sequence, and the translocon complex can facilitate opening of the translocon pore towards the ER lumen and also laterally towards the lipid bilayer [55]. Hydrophobic domains, such as the signal sequence or transmembrane domains, are released through the lateral gate into the membrane, while soluble domains are translocated into the ER

5 lumen. During this co-translational process, protein folding and post-translational modifications are also possible. Once the chain is long enough, the signal sequence is cleaved off by the signal peptidase [56] and the transfer of N- linked glycans can be catalyzed by the OST complex at recognized sites [57].

Nascent chain SRP binding

Ribosome

Alu-domain signal mediates sequence (SS) elongational arrest SRP

Targeting to ER membrane

Cytosol SR

ER lumen Sec61 Interaction of Ribosome docking Y complex SRP with SR and translocon gating and handover Co-translational of SS to Sec61 Co-translational SS cleavage translocation axially Glycosylation Y and laterally Protein folding

Figure 2: Co-translational targeting of proteins to the ER membrane is facilitated by the signal recognition particle (SRP) and its receptor (SR).

Figure layout is based on Jiang Y et al., 2008 [58]. The outlines of the proteins are based on the 2D projection of the structures of 3D models (generated after Kobayashi K et al.,2018 [59] for SRP and SR, and after Lang S et al., 2017 [60] for the ribosome and Sec61 complex).

The coupling of translocation and protein synthesis of secreted and membrane proteins has two advantages compared to post-translational pathways [55]. The nascent chain can only pass through the narrow translocon channel before it is folded, which is ensured in co-translational transport removing the need for cytosolic chaperones. More importantly, co-translational translocation effectively shields the very aggregation prone hydrophobic transmembrane do-

6 mains or signal sequences from the aqueous cytosol allowing direct partitioning into the membrane as they emerge from the ribosome. These advantages have suggested that the co-translational pathway may have evolved after early post-translational routes, to offer a more sophisticated way to effectively de- liver membrane and secretory proteins while avoiding aggregationa and misfolding [29]. A schematic overview of co-translational targeting and translocation is shown in Figure 2. In the following chapters I will present in more detail all the steps involved in co-translational protein translocation in eukaryotes and describe the protein complexes responsible.

7 Co-translational targeting of proteins to the ER membrane

The signal recognition particle and its receptor

The first step of the co-translational route of proteins to the ER membrane, is interaction of SRP with the N-terminal signal sequence. The existence of SRP was proposed almost 10 years before it was identified in the early 1980’s in a mammalian system. SRP is a ribonucleoprotein particle consisting of six proteins and a roughly 300 nucleotide RNA [45,48,61,62]. It has two functional main domains, the Alu domain and the S domain [63]. The Alu domain mediates elongation arrest by contacting the ribomsome elongation factor binding site, blocking its accessibility, and thereby stalling translation [48,64]. The S-domain is responsible for initiating translocation by recognizing the signal sequence at the ribosomal exit tunnel, interacting with the ER membrane-bound signal receptor (SR), and handing over the signal sequence to the translocon complex in a GTP-dependent manner [63,65,66]. The two domains are arranged on opposite ends of the SRP 7S RNA, which provides the flexibility and reach needed for the interactions of the Alu and S domains to occur simultaneously [48]. The most evolutionarily conserved and functionally significant protein of the eukaryotic SRP is SRP54. This is exemplified by bacterial SRP, which contains only a homologue of this protein (Ffh) and a shorter RNA representing a “core” version of SRP that does not provide elongational arrest or any other regulatory features [67]. The SRP54 subunit contains three domains, the M, N and G domains. The M-domain provides a flexible hydrophobic groove that can accommodate various different signal sequences, the N domain interacts with the ribosome in the vicinity of the exit tunnel [48], and the G domain interacts with the receptor (together with the N-domain) [59,67,68]. Over time, it became obvious that SRP binds to translating ribosomes even before the exposure of a hydrophobic segment [69,70]. This so-called scanning mode is thought to sample the nascent chain and identify hydrophobic targeting sequences while they are still within the ribosomal exit tunnel, facilitating rapid and efficient SRP binding as soon as these sequences emerge into the cytoplasm [71]. In scanning mode the hydrophobic groove is occulded by a placeholder helix in SRP, preventing non-specific interactions with hydrophobic patches on other cytosolic proteins. How exactly the hydrophobic signal is sensed while still inside the ribosome exit tunnel and how this triggers the

8 conformational change in the M-domain necessary to accommodate the signal sequence is still not fully understood. Once the hydrophobic targeting sequence is bound to SRP, it targets the whole RNC to the ER membrane, where SRP interacts with its receptor, SR. The SR was identified in parallel by two different groups and consists of an evolutionarily conserved peripheral membrane protein SRα that interacts with SRP and a eukaryote-specific integral membrane protein subunit SRβ [51–53]. SRP and SRα both bind GTP, which is essential for their interaction [66,72]. The two GTP molecules are bound at the interface of SRP and SRα and it has been proposed that their hydrolysis only occurs after handover of the signal sequence from SRP to the translocon complex in order to ensure a sufficient time window for proper ribosome-translocon interaction [59,62]. Hydrolysis of GTP promotes their dissociation from one another, leaving them free to associate with a different translating ribosome (SRP) or an empty translocon (SR) [62,72]. How precisely the SR finds an empty translocon, how the handover of the signal sequence to the translocon is facilitated, and whether there may be a requirement for substrate-dependent auxiliary complexes still awaits further investigation.

ER targeting signal sequences

In the 1970s, Blobel and Dobberstein discovered that proteins secreted by cells carry a short N-terminal signal sequence, that is removable and labels the proteins for export [15,16]. This signal hypothesis was a major breakthrough in question of how proteins are transported across either the plasma membrane or internal membranes of cellular organelles such as the ER. It also shed light on the sorting of membrane bound proteins as many membrane proteins also contain a cleavable N-terminal signal sequence while, in some cases, the first transmembrane domain acts as an un-cleavable signal sequence [73]. The hypothesis originally assumed that all signal sequences would share a common recognizable motif that would facilitate protein targeting to the ER membrane. However, early comparative sequence analysis revealed that no common con- sensus motif could be identified in the primary sequence, but some general features shared by most signal sequences were noted [74]. Below I describe the general composition of signal sequences with some examples relevant to this work, highlighted in Figure 3. Signal sequences are typically 15-35 amino acids long (even though some exceptionally long sequences have also been found [75]) and are composed of three different domains: a basic (positively

9 charged) amino-terminal (N) domain, a 7-13 residue hydrophobic core (H) domain, and an often slightly polar cleavage (C) domain that habours the cleavage site [76].

cleavage site positively charged hydrophobic polar

+ H3 N N domain H domain C domain

Prolactin: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS- Prion (PrP): MANLSYWLLALFVAM WTDVGLC- Insulin: MALWMRLLPLLALLALWGPDPAAA-

Figure 3: Domain structure of ER signal sequences. Signal sequences have a net positive charge in the N-terminal domain (N domain) derived from the charged N-terminus itself + (H3N ) and charged amino acids (blue). The C-terminal part of the signal sequence often carries polar amino acids and contains the cleavage site (indicated by an arrow). The hydrophobic region (H domain, red) is the core of the signal sequence. Underneath example sequences relevant to this work are given in single letter amino acid code[77–79]. The figure design is based on Fekkes P and Driessen AJM, 1999 [80].

In addition to the lack of homology, the signal sequence of a protein is often interchangeable across evolutionary distant species [81]. In fact, as long as the hydrophobic core is not interrupted by charged residues, signal sequences seem to tolerate a large number of mutations while retaining their ability to target proteins for secretion. A study from the 1980s showed that about 20% of random sequences can facilitate secretion of yeast invertase [82]. As more protein sequences became available, it became obvious that many proteins carry a highly evolutionarily conserved signal sequence, not a simple degenerate and freely exchangeable sequence as first assumed [83]. Growing experimental evidence indicates that the exact primary sequence influences the targeting pathway, association with the translocon, and timing of signal sequence cleavage, which taken together affect the final folding of the mature protein [84]. Proteolysed fragments of cleaved signal sequences are also commonly presented as self-antigens on nucleated cells, an important aspect of the immune system. The major histocompatibility complex class 1 (MHC1) has an especially high affinity for antigenic peptides derived from signal sequences compared to other peptides from the same protein [85]. Peptides derived from intracellular proteins presented on MCH1 molecules are recognized by cytotoxic

10 T-cells as self-antigens, which inhibits their activity. Conversely, the absence of any antigen or the presentation of foreign antigens triggers a T-cell response leading to destruction of the cell. This demonstrates that the primary structure of signal sequences can have effects beyond targeting. However, for my work, the most relevant aspect of signal sequences is the fact that they differ in their translocation efficiency and their ability to initiate translocation [83,86]. Until 2006, it puzzled researchers why inefficient signal sequences would be preserved. That’s when Kang et al. demonstrated that subtle sequence differences provide the possibility to re-route substrates in a signal sequence-selective manner for degradation during ER stress conditions [87]. This so-called preemptive quality control protects the ER from accumulation of aggregated protein and allows only certain proteins (based on the efficiency of their signal sequence) to enter the ER lumen, providing an additional layer of regulation. Precisely what a signal sequence needs to efficiently open the translocon complex and what other factors are required to assist in gating of less efficient signal sequences is the subject of my thesis work and will be discussed in detail over the following chapters as well as a summary of my research papers.

11 The Sec61 translocon

Structural overview

The core of the protein translocating channel (the translocon) is commonly made up of three subunits. This central channel is evolutionarily conserved in all three domains of life - archaea, bacteria, and eukaryotes [88,89]. The typical Sec translocon (named for its role in “sec”retion [60,90]) is a heterotrimeric protein complex of the subunits named Sec61α, β and γ in mammals, SecY, E, and G in bacteria, and Sec61p, Sbh1p, and Sss1p in yeast [91–93]. The topology of the subunits is shown in Figure 4 with the binding sites relevant to my work indicated. Structural analysis revealed the arrangement of the individual subunits and helped to understand their functions [94].

N-terminal half C-terminal half N loop 6 loop 8 N N RBS ~70 aa RBS Sec61α CBS RBS ~30 aa cytosol C Sec61β Sec61γ Sec61α specially marked domains: Ribosome binding site (RBS)

ER lumen Lateral gate (H2+H3 and H7+H8) C plug TRAP Bip C BS BS loop1 loop 5 loop 7

Figure 4: Topology and relevant functional domains of the Sec61 heterotrimer. The membrane topology of each subunit of Sec61 is shown. Binding sites (BS) of Ca2+-calmodulin (CBS), the translocon-accociated protein (TRAP) complex, the ribosome (RBS, purple) and the ER luminal chaperone Bip are marked. The plug domain and the helices that form the lateral gate (dark blue) are indicated. Abbreviations: N, for N-terminus; C, for C-terminus; and aa, for amino acids. Information for this figure is taken from UniProtKB (P61619, Sec61α; P60468, Sec61β; and P60059, Sec61γ) and Lang et al., 2019 [95]

Sec61α has 10 transmembrane helices arranged into two”half-channels” (helices 1-5 and helices 6-10) that together form the central translocon pore (see Figure 4). In its closed or quiescent state, the channel takes on an hourglass shape, with the central constriction made up of hydrophobic side chains that form a “pore ring” that seals the channel along with the “plug” domain, a short

12 helix that reaches into the pore from the ER luminal side [55,60,94,95] (Figure 5). The channel can open both, towards the ER lumen and laterally towards the lipid bilayer [96,97]. The latter is accomplished by a “lateral gate” at the interface of helices 2 & 3, and helices 7 & 8 (Figure 4, dark blue, and Figure 5). The cytosolic and luminal loops provide interaction sites for the ribosome and various auxiliary components of the translocon machinery [95]. The cytosolic loops 6 and 8 provide the ribosomal docking site (Figure 4, RBS, purple)[98]. The single-spanning membrane proteins Sec61β and Sec61γ are located at the periphery of the Sec61α channel and while Sec61α and γ are very well conserved and essential for cell viability, some bacteria lack the β subunit [99,100]. In eukaryotes, Sec61β has been found to enhance co-translational transport interacting with the signal peptidase during translocation in vitro, probably assisting in its recruitment [101]. A recent study also suggested that Sec61β anchors the translocon complex to the cytoskeleton through interactions with microtubules [102]. Sec61γ is “clamped” around the two halves of the channel at the hinge region opposite the lateral gate, which is thought to contribute to the stability of the complex while it also contributes interactions with the ribosome via its ribosome binding site [94].

cross-section view front view

closed axially open closed laterally open

Pore ring Lateral gate

Plug-helix

Figure 5: Schematic representation of the Sec translocon in closed and open conformations. On the left a cross-section view cut through the middle of the pore perpendicular to the membrane plane showing: the closed conformation with its narrowed pore ring and the plug-helix sealing the translocon from the ER luminal side; and the axially open conformation, where the pore ring is widened and the plug shifts sideways, allowing protein tran- location. On the right, a side view of the translocon in the membrane plane showing the lateral gate (dark blue) in its closed and laterally open conformations that allows hydrophobic sequences to partition into the membrane. Figure based on Zhang B and Miller TF, 2010, [103] and Denks K et al., 2014 [39].

13 Signal recognition by the translocon and opening of the channel

In order to open, the translocon requires a ligand to interact with the cytosolic docking site formed by loop 6 and loop 8 of Sec61α. The best-studied ligand is the translating ribosome, but other effectors such as post-translational Sec63 in eukaryotes or SecA in bacteria can also interact with the same site to trigger conformational changes and channel opening [54,95,104–108]. Ribosome binding, mediated by ribosomal uL23 and eL29 proteins, causes conformational constraint in the docking site, which propagates through all the helices of Sec61α. This leads to a perturbation in the weak helical interactions at the lateral gate opening a small crack in the cytosolic half of the lateral gate between helices 2 and 8 [54]. In this ribosome-bound primed state, the overall structure of the translocon channel still resembles the closed conformation, but the slight opening of the lateral gate prepares the complex to re- ceive the signal sequence or a transmembrane segment of the nascent protein. The activation energy for the large conformational change required for complete opening of the channel is lowered by the primed state [95]. Specifically, conserved hydrogen bonding pairs between polar residues in helices 2 and 7 are destabilized and a hydrophobic patch in vicinity of the lateral gate is exposed [55,109,110]. It has been assumed that this patch provides the first point of interaction for the signal sequence H domain or the nascent transmembrane segment with the translocon channel. In this position, the exposure of the helical hydrophobic segment to the lipid bilayer drives the full intercalation of the targeting sequence in the lateral gate, displacing helix 2 of Sec61α [109]. Rigid body movement of the two half-channels of Sec61 induced by these interactions, results in an open channel conformation with the plug domain being displaced and the pore ring opening. The soluble parts of the nascent polypeptide can now be translocated through the channel into the ER lumen with subsequent hydrophobic domains exiting the translocon laterally through the open gate. Structural representations of the Sec translocon complex in the closed state and a signal sequence-intercalated open conformation are shown in Figure 6.

14 Closed state Translocating state (opened by a signal sequence)

cytosol

ER lumen loop 5

View from 90° 90° ER lumen

“plug” domain loop 5

Sec61α: Ribosome binding site Signal sequence of prolactin Lateral gate (H2+H3 and H7+H8) Sec61β Remaining helices of Sec61α Sec61γ

Figure 6: Models of the Sec61 complex in its closed and open conformations. Atomic model of the closed (left panel, PDB: 1RH5 [94]) and signal sequence-opened (right panel, PDB: 3JC2 [109]) conformations of the translocon either parallel to the membrane plane (top panel) or perpedicularily from the ER lumen (lower panel). The plug domain, indicated in the closed conformation (lower left panel), was not resolved in the structure of the open channel and is therefore absent in the right panels. Loop 5, an important interaction point for auxiliary proteins in the hinge region of the translocon, is indicated in the open structure.

Translocation and insertion of membrane intercalating helices

For secretory proteins, it is interesting to note that the structural data show that the signal sequence in the lateral gate has already acquired its final topology, with its N-terminus facing the cytosol and the C-terminal part of the signal

15 sequence is oriented towards the ER lumen, where the cleavage site is acces- sible to signal peptidase. It has been observed that the nascent chain forms a horseshoe-like loop when entering the translocon at this early stage of translocation [111]. This “hairpin-loop” insertion mode is likely driven by positively charged amino acid residues (in addition to the charged amino-terminus) in the N domain that prefer to remain in the cytosol according to the positive- inside rule [112]. Transmembrane helices of multi-spanning membrane proteins with the N-terminus in the cytosol are thought to insert the same fashion. In contrast, transmembrane domains with the N-terminus on the luminal side of the membrane use the “head-first” insertion mode, as these helices do not need to invert their orientation [113]. In Figure 7 the two modes of insertion for hydrophobic domains into the translocon pore are shown.

Hairpin-loop insertion Head-first insertion

N Cytosol N

ER lumen N N

Figure 7: Different modes of insertion of hydrophobic segments into the translocon channel. During co-translational translocation on translocon-bound ribososmes (gray), hydrophobic segments (red) such as signal sequences or transmembrane domains can be inserted into the Sec61 complex (blue) either in a hairpin-loop (left panel) or a head-first (right panel) fashion. See text for details.

Surprisingly, the head-first insertion is also favored by single-spanning membrane proteins whose final conformation has the N-terminus in the cytosol (type II proteins). The signal anchor domain starts its translocation like a cleavable signal sequence, i.e. as a “hairpin-loop”, which is inverted from its final orientation. As the growing polypeptide accumulates between the ribosome and the translocon, the transmembrane helix performs an energetically unfavorable “flip turn” of 180° to attain its final orientation [114]. During the translocation of soluble domains, including the loops of membrane proteins, into the ER lumen, the channel is open axially, but the lateral gate

16 remains partially closed, opening up only when a hydrophobic helix is accom- modated in the channel and needs to be released into the membrane [54,55,113].

Assisted opening of the translocon

An efficiently “gating” signal sequence results in productive hairpin-loop insertion following the strong interaction of the signal sequence with the hydrophobic patch of Sec61α and intercalation into the lateral gate. As mentioned before, the efficiency of signal sequences can vary a great deal and not all sequences can facilitate channel opening by themselves. It has been proposed that helix 2 of Sec61α acts as a selectivity filter allowing only sufficiently hydrophobic and helical sequences to efficiently displace it, therby completely opening the gate [55]. This model is supported by the previous suggestion of a “gating motif” in Sec61α, described as a conserved cluster of polar amino acids in the lateral gate. The cluster seems to set the hydrophobicity threshold to allow discrimination between bona fide transmembrane domains and hydrophobic stretches within soluble domains [110]. However, this could possibly hamper the membrane insertion of marginally hydrophobic domains. A possible solution to this problem was revealed by structural data, indicating that transmembrane helices can dock at a specific site immediately outside the lateral gate after partitioning into the membrane. It is attractive to speculate that a well-gating transmembrane segment might stabilize the laterally open conformation of the translocon to assist following marginally hydrophobic transmembrane regions during insertion into the lipid bilayer [115,116]. Interestingly, inefficient signal sequences do not primarily fail to be targeted to the ER membrane, but rather are unable to quickly and efficiently interact with the translocon, failing to initiate translocation and channel opening [83]. There is growing evidence that delayed translocation of these substrates triggers the recruitment of various substrate-specific auxiliary factors of the translocon machinery [78,117–121]. In the following chapter I will present our current understanding of the roles of the auxiliary components TRAM, TRAP, Sec62/Sec63, RAMP4, and ERj1, as well as the ER luminal chaperone Bip, and the OST complex, which performs co-translational N-linked glycosylation.

17 Auxiliary components of the Sec61 translocon

TRAM1

One of the first proteins found to provide substrate-specific translocation assistance was named translocating chain-associated membrane protein (TRAM) [37,122]. It was recently renamed TRAM1 due to the discovery of the homologs TRAM1L1 and TRAM2 [123,124]. Fluorescence resonance energy transfer (FRET) experiments showed that TRAM1 is permanently in close proximity to Sec61 complexes together with the TRAP complex [125]. TRAM1 contains eight transmembrane domains, but lacks any significant cytosolic domain [122,126]. The latter is the reason why TRAM1 could not be captured and identified with cryoelectron tomography (CET) in the vicinity of translocating translocons. It is assumed that a density, that is consistently found opposite of the lateral gate of an active translocon, represents TRAM, but this has not yet been confirmed [60,127]. Despite the finding that TRAM1 is displaced more eas- ily from the translocon compared to the TRAP and OST complexes (more below), indicating a weaker interaction, it is generally accepted that TRAM1 resides in proximity to both active and inactive translocon complexes [120,128]. TRAM1 is considered an essential component of the minimal in vitro reconstitution of translocation-competent proteoliposomes [37]. However, its contribution is substrate-dependent ranging from stimulatory to absolutely required for protein translocation (e.g. prolactin translocation is only mildly affected, while other substrates are essentially not translocated in reconstitutions lack- ing TRAM1 [37,129]). TRAM1 can be crosslinked to nascent chains very early in their translocation and was found to interact with the N-domain of the signal sequence [122,130]. Further analysis showed that TRAM1 is required for efficient lateral gate insertion of substrates carrying an especially short N-domain [118]. A recent proteomic analysis of TRAM depletion under physiologi- cal conditions confirmed these mild preferences, but also suggested that TRAM does not act as a receptor for signal sequences; rather, it plays a supportive role in translocation for various substrates [120]. In fact, it is very unlikely that TRAM plays a direct role assisting in channel opening. Live-cell calcium imaging has demonstrated that TRAM1 depletion does not result in altered calcium leakage, which was observed for components involved in gating [120]. In contrast, it was suggested that TRAM1 might affect the bilayer thickness and phospholipid structure close to lateral gate and

18 therby indirectly assist the lateral exit of signal sequences and transmembrane domains into the ER membrane [120,123].

The translocon-associated protein (TRAP) complex

The first subunit of the TRAP complex (TRAPα) was discovered in 1987 as a 35 kDa glycoprotein of the ER membrane that could be crosslinked to nascent chains of Prolactin [131]. It was originally termed signal sequence receptor (SSR), as it was suspected to act as such after targeting of the ribosome-nascent chain complex to ER membrane translocation complex. After the identification of the additional three subunits (TRAPβ, γ and δ), the name was changed to translocon-associated protein (TRAP) complex [132,133] and its involvement in later stages of translocation and not (only) as an initial receptor of the signal sequence became obvious [122,134].

TRAPα TRAPβ TRAPγ TRAPδ C

~60 aa ~60 aa RBS C N RBS C C cytosol

ER lumen ~130 aa ~200 aa ~120 aa Sec61 BS Sec61 BS OST BS

N N N

Figure 8: Topology and relevant functional domains of the four subunits of the TRAP complex. The membrane topology of each TRAP subunit is shown. Binding sites (BS) to Sec61, the OST complex and the ribosome (RBS) are indicated along with the approximate length in amino acids (aa) of the relevant domains. N, N-terminus; C, C-terminus. Figure is based on UniProtKB P43307 (TRAPα), P43308 (TRAP1β), Q9UNL2 (TRAPγ) and P51571 (TRAPδ); and based on Görlich et al., 1990[132]; Hartmann et al., 1993[133] and Peffer et al., 2017[135].

The heterotetrameric TRAP complex (Figure 8) was found to associate stably with the Sec61 translocon [128] and FRET experiments showed that TRAP is permanently in close proximity to Sec61 complexes, even in the absence of translocating polypeptides [125]. Indeed, TRAP seems to be a stoichiometric and permanent component of the otherwise very compositionally dynamic translocon complex [136]. Unlike TRAM, the TRAP complex contains soluble

19 domains, which allowed its location relative to other translocon components to be confirmed by 3D-reconstruction after CET [135–137]. Despite its proximity, TRAP is not required for all (albeit many) proteins translocated via the Sec61 pathway. Subtrates with signal sequences or N-terminal transmembrane domains that interact strongly and quickly with the translocon, and therefore have efficient gating potential, are transported inde- pendently of TRAP [78]. Additionally, TRAP has been identified to assist in the adoption of proper protein topology by modulating the “positive inside rule” and facilitating the translocation of positively charged residues across the membrane [138,139]. In order to identify common characteristics of TRAP clients, quantitative proteomics was used to analyze the changes in cellular protein abundance upon TRAP depletion [119]. This study identified a combination of lower hydrophobicity combined with a higher glycine and proline content in TRAP-dependent protein signal sequences. The amino acids glycine and proline are known as “helix-breaking” residues that decrease helical propensity. These two proper- ties are likely to affect the ability of the signal sequence to intercalate at the lateral gate of the Sec61 complex, displace helix 2, and open the channel [95]. These weakly gating signal sequences might be assisted by TRAP that facilitates Sec61-channel opening. This function is consistent with Ca2+ imaging data, that demonstrates that TRAP stimulates the open conformation and therefore increased Ca2+ leakage from the ER [119]. The structural picture of TRAP function was obtained by CET[135] (Figure 9). The fourth transmembrane subunit TRAPγ is positioned in the centre of the mammalian complex. Its substantial cytosolic domain contacts the translocon- bound ribosome at eL38 and the small rRNA, in close proximity to a recently discovered site that coordinates signal sequence binding to SRP in the bacterial homolog, [140] which suggests that TRAPγ could directly assist in the handover of the signal sequence from SRP to Sec61 [119]. This interaction could stabilize the signal sequence on the cytosolic side and facilitate insertion into Sec61 in a hairpin conformation. The subunits TRAPα/β are both single spanning type 1 membrane proteins, whose ER luminal domains form a heterodimer that contacts loop 5 of Sec61α in the hinge region. Via this interaction, TRAP could facilitate the opening of the channel and assist translocation initiation. Furthermore, the position of the dimer below the Sec61 channel also supports the possibility that TRAP interacts with the nascent polypeptide and could act as a molecular ratchet on the translocating chain.

20 The position of the last subunit of the complex—interacting with the OST complex—supports other findings that TRAP might also play a role in the coordination of N-linked glycosylation. This is supported by data showing that patients carrying certain mutations in TRAPδ have a congenital gycolsylation disorder [141,142]. Whether this is a direct or indirect effect remains to be eluci- dated.

Figure 9: The molecular organization of the mammalian TRAP complex. The positioning of the resolved regions of the TRAP subunits within the isolated density obtained by CET are indicated. The luminal TRAP α/β heterodimer, which contacts the Sec61 translocon and possibly translocated nascent chains (indicated by the dashed red line), is outlined in magenta. The cytosolic domain of TRAPγ, which interacts with the ribosome at rpL38 (purple) and rRNA (yellow), is outlined in red. The subunit TRAPδ, which interacts with the OST complex (shown in light red in the right panel) is outlined in blue. Scale bar, 10 nm. Figure modified with permission from Pfeffer et al., 2017[135].

It is worth noting that in the CET structure only the polytopic transmembrane domains and the cytosolic component of TRAPγ were resolved, while the single transmembrane domains of the other three subunits and the small cytosolic domain of TRAPα (58 amino acids) were not distinguishable from the membrane density. This could be due to a more flexible positioning of the other subunits around the more rigid TRAPγ, yet it also means that the precise position of substrates relative to TRAP is still not known in detail. The role of TRAP as an essential substrate-specific auxiliary component of the translocon complex posits that ER protein import can also be regulated at

21 this level. To this end, TRAP has been found to bind Ca2+ and be subject to phosphorylation [143,144]. It has been shown recently that TRAP interacts with calnexin in a redox-sensitive way and that disruption of their interaction during ER stress inhibits co-translational membrane insertion [145]. Furthermore, all TRAP subunits are upregulated during ER stress and it is thought TRAP might be involved in the endoplasmic reticulum associated degradation (ERAD) pathway [146] as it preferentially binds to misfolded proteins, acceler- ating the degradation of ERAD substrates. Taken together, the TRAP complex seems to be involved in several processes required in the translocation of specific substrates across the ER membrane and may have an important function in translocation regulation during ER- stress conditions.

OST

In eukaryotes, the OST complex is present as two multimeric paralogs, Stt3A and Stt3B. They are named after the catalytic core subunit contained in each of the different complexes [147]. In bacteria and archaea, OST exists as a mon- omer, a homolog of the core eukaryotic Stt3 subunits. Stt3 catalyzes the transfer of glycan units from a lipid carrier moiety onto asparagine residues present in the trimeric motif Asn-x-Ser/Thr, where x≠ Pro in the ER lumen [148–150]. In eukaryotes, the transferred N-linked glycan is always the tetradeca- saccharide Glc3Man9GlcNAc2 that comes from a dolichyl-diphosphate carrier, which is assembled in a process that is conserved from yeast to human. The mammalian Stt3A and Stt3B complexes contain an additional 7 or 8 subunits in addition to the core subunit of which 6 are common to both complexes (namely Ribophorin (Rpn) 1, Rpn2, DAD1, TMEM258, OST4 and OST48). Stt3A has the added subunits DC2 and Kcp2, while Stt3B contains either MagT1 or TUSC3 [95,151,152]. Our current understanding is that Stt3A acts co- translationally, while Stt3B can complement missed glycosylation sites post- translationally [57]. Glycosylation sites close to the C-terminus of the translocated protein are usually glycosylated by Stt3B, as protein synthesis completes before the sites can reach the catalytic center of Stt3A, which is located at a distance of 30-40 Å from the luminal surface of the ER membrane [153,154]. Stt3B is a stand-alone protein complex, while Stt3A is found in close proximity to translocating ribosome-translocon complexes. The ratio of OST complexes to Sec61 translocon complexes is tissue-dependent, probably reflecting the amount of N-linked glycosylated proteins in the respective cell types

22 [127,137]. Cryoelectron microscopy and tomography data have provided insights into the position of the co-translational Stt3A-OST complex relative to the Sec61 complex, the translocon-bound ribosome, and the TRAP complex [106,137]. The subunit DC2 binds selectively to Stt3A helices 10-13 and the C- terminal cytosolic loop. The Stt3B subunit cannot interact with DC2 due to sequence variations in these regions [95]. The luminal loop of DC2 interacts with N-terminal half of the Sec61 complex, as well as the short C-termini of Sec61 α and γ [106]. The interface with the ribosome is mediated by the Rpn1 C-terminal tetra-helix bundle. This 4-helix structure fits into a cavity formed by the ribosomal protein eL28, the rRNA segment ES7a and the rRNA helices H19 and H20, promoting stable ribosomal binding to the translocon complex [155]. Interestingly, the Stt3B subunit also interacts with Rpn1, but an extension in one of the cytosolic loops sterically hinders ribosome binding. Together, these data explain the specificity of the two paralogs for co- or post-translational glycosylation [106]. As mentioned above, the δ subunit of the TRAP complex interacts with the translocon-bound OST. This interaction is likely to coordinate translocation and co-translational glycosylation to ensure synergism between the complexes [135] (note the organization of the ribosome-translocon complex shown in Fig- ure 9, right panel).

Sec62/63 and Bip

As previously discussed, the heterodimeric Sec62/63 complex is recruited to Sec61 during post-translational transport [38,39]. In yeast, the complex requires additional two subunits, Sec71 and Sec72, that do not have mammalian homologs [38,156]. The ER luminal Hsp70 chaperone Bip (Kar2p in yeast) is also required to facilitate post-translational transport across the Sec61/62/63 complex in an ATP-dependent manner [157,158]. Bip is thought to act as a molecular ratchet that drives unidirectional transport across the translocon and prevents backsliding of the chain [158]. Its activity in the post-translational complex is stimulated by recruitment to the luminal J-domain of Sec63, which is an Hsp40 co-chaperone [159].

The recent structures of the yeast post-translational complex, obtained by cryo electron microscopy, determined that Sec63 is positioned opposite the Sec61 lateral gate [104][105]. In the structure it is interacting tightly with the hinge re-

23 gion and is most likely responsible for the unexpectedly wide open conformation of the translocon that was observed. Contacts are present in the cytosolic, luminal, and transmembrane domains, and with all three subunits of Sec61 involved. The universal docking site of Sec61α in loop 6 and 8 is blocked, indicating that concurrent binding of the ribosome and the Sec62/63 complex is unlikely [105]. Additionally, comparing the structures of the yeast post-translational complex and the mammalian co-translational complex presented above, indicates overlaps in the transmembrane domains of OST and Sec63 and steric clashes between the binding of luminal domain of Sec63 and the TRAP complex to Sec61α [160]. These structural observations strongly ar- gue that different post- and co-translational transport assemblies exist, which is supported by biochemical studies showing that Sec62 bound to Sec61 is displaced by SR binding to the translocon complex [161]. Sec62 was not well- resolved in the structures, but its cytosolic domain could be placed in the vicinity of the lateral gate, supporting the biochemical data that it plays a role in membrane protein insertion in yeast or as a signal sequence receptor for short secretory proteins in mammals [41–43,162].

Despite, the structural clashes that are observed in the superimposition of the known “co-translational translocon supercomplex” with the “post-translational translocon supercomplex”, there is growing biochemical evidence that the Sec62/63 heterodimer plays a supportive role in co-translational translocation of certain substrates in mammals [42,117,121,163]. Interestingly, mammalian Sec62 has gained a function during evolution compared to its yeast homolog. It is able to interact with the ribosome near the exit tunnel, supporting the idea that it could participate in co-translational translocation in mammals, but not in yeast [164]. As there is no structural data of the mammalian post-translational complex, one can only speculate about the possible combinations of auxiliary factors that could be recruited to the Sec61 channel during protein translocation. Depending on the gating efficiency of the signal sequence and other sequence-specific features coded in the mature part of the translocated protein, it is possible that sequential recruitments of SRP, followed later on by Sec62/63 could be required for certain substrates [161]. A very recent proteomics analysis under Sec62/63 depleted conditions revealed that longer but less hydrophobic stretches within protein signal sequences, together with downstream clusters of positively charged amino acids, are characteristics for Sec62/63-dependence during co-translational translocation [121]. For some of these substrates the recruitment of Bip has also been found to be essential. Thus, it has been suggested that in the case of this type of slowly gating signal

24 sequence, Sec62/63 facilitates the opening of the channel via the recruitment of Bip, which can assist gating by its interaction with loop 7 of Sec61α (see Figure 4) [43,163].

Additional auxiliary components of the translocon

In addition to the complexes described above, other resident ER membrane proteins have been identified to play a role in protein translocation or the processing of precursors. The first one was mentioned already. The signal peptidase cleaves off signal sequences from secretory proteins and membrane proteins with cleavable signals [56]. It can act on the nascent protein once the chain is elongated enough to expose the cleavage site. However, the exact timing of the cleavage is protein-dependent and can be delayed, for example, by the formation of secondary structure that occludes the cleavage site [84,165–167]. It is assumed that the signal peptidase complex is recruited to the translocon tran- siently via the Sec61β subunit [101]. After cleavage, the freed signal sequence can be further cleaved into smaller fragments by signal peptide peptidases, degraded completely, or go on to serve additional biological purposes [84,168]. For many of the other proteins identified to be somehow involved with translocon-bound ribosomes, a concrete function is not yet well understood. The ER-resident J-domain protein 1 (ERj1) binds to ribosomes near the exit tunnel and recruits Bip to the Sec61 complex via its J-domain [169,170]. This functionally overlaps with Sec63, which recruits Bip in a similar manner [171]. In its free form, ERj1 inhibits translation initiation of translocon-bound ribosomes, but not in its Bip-bound conformation. A possible interpretation is, that protein translocation is only initiated when Bip is available in the ER lumen to assist in translocation (as a ratchet) or in protein folding. The protein ribosome-associated membrane protein 4 (RAMP4; also known as SERP1, stress-associated endoplasmic reticulum protein 1) is recruited to the translocon when a transmembrane helix is within the exit tunnel. The ribosomal protein uL22 senses the transmembrane segment within the tunnel and transmits this signal to the translocon resulting in the recruitment of RAMP4 to the complex [172]. This way, the channel is primed for insertion of the nascent segment into the membrane. RAMP4 is also induced during ER stress and most likely promotes refolding of membrane proteins under these conditions [173].

25 There is a number of additional effectors that control calcium levels in the ER or regulate protein transport during ER stress, but here I will only mention calmodulin. This cytosolic calcium binding protein is involved in a number of second messenger systems via calcium-dependent binding. In our context, it limits calcium efflux during protein translocation through binding at the N- terminus of Sec61 (see Figure 4). This is crucial as the cell needs to maintain high calcium levels within the ER compared to the cytosol [174]. Interestingly, calmodulin has also been identified as a targeting factor for small secretory proteins to the Sec61 complex as mentioned above [34]. This shows once more how complex, interconnected and strickly regulated the protein network around the central translocon channel is and how many exciting questions still remain to be answered.

Auxiliary components of the translocon allow translocation regulation

The interplay of the various components that associate with the Sec61 complex do not only ensure the efficient protein translocation of various different substrates, but they also present an interesting layer of selective translocation regulation [175]. This possibility is supported by the previously mentioned observation that inefficiencies in protein signal sequences may be evolutionarily conserved for a functional reason, rather than being the product of sequence degeneration due to lack of specificity in SRP recognition. For efficient translocation initiation the signal sequence needs to interact with the translocon forming a hairpin loop (see Figure 7), and facilitate channel opening in the lateral and axial directions (see Figure 5). The auxiliary components discussed above can assist in one or both of these essential functions, if the signal sequence alone fails. In forming the hairpin loop: TRAM1 or TRAP might stabilize the loop conformation by interacting with the N-terminal part of the signal sequence on the cytosolic side [119,130]; interaction of ER luminal chaperones like Bip, recruited via Sec63, ERj1, or other factors, may stabilize the conformation on the luminal side, preventing backsliding and thereby topology inversion [171,176,177]. The roles that some of the components play in channel opening have been discussed above, such as for TRAP or for the Sec62/63 complex via Bip [43,135,163]. Many of the factors discussed, seem to have overlapping functions and substrate specificities. It is, therefore, likely that their precise interplay finetunes translocation efficiency for each substrate in different cell types and under

26 different cellular conditions [175,178]. During ER stress this layer of regulation would allow the cell to quickly lower the load of unfolded proteins in the ER lumen by prohibiting the entry for substrates requiring additional components of the translocation machinery and only allowing certain proteins to enter [87,179]. An additional role during ER-stress has been discussed for TRAP and RAMP4, indicating that they might “switch” their function from protein translocation assistance to contributing to the re-establishment of cellular protein homeostasis [145,146,173,180]. Phosphorylation sites have been identified on many of the auxiliary components, providing a potential mechanism to regulate their function under stress conditions [175]. However, precisely how translocation is regulated during ER stress is not very well understood and is the subject of further investigation.

27 Co-translational protein folding on the ribosome

Proteins must acquire their precise three-dimensional structure—a process re- ferred to as protein folding—in order to fulfill their biological functions. When this goes wrong and proteins misfold, it can lead to disease, including a number of neurodegenerative diseases (such as Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob disease), cystic fibrosis, and sickle cell anemia [181,182]. In most cases this is caused by mutations in a protein’s sequence, but in some cases it can be caused by the inability of cellular chaperones to assist the protein finding its proper fold, leading to aggregation. Many proteins begin the process of folding co-translationally during their synthesis on the ribosome [183]. Coupling protein synthesis and folding makes the maturation of a protein into a vectorial process, as only the protein that has been synthesized can undergo folding. This can allow the stepwise formation of protein domains, constrain the number of possible intermediate conformations that the nascent protein can acquire, and may direct the folding pathway to the correct final structure rather than an energetically stable aggregate [184]. Contributing to this, the precise mRNA template sequence affects the rate of translation as most amino acids are encoded by more than one codon, but not all codons can be paired with a cognate tRNA at the same speed. Codons that occur at a lower frequency are paired slower than common codons resulting in slower translation. These differences in translation speed have been found to be essential for the proper co-translational folding of some proteins and making synonymous codon changes can result in altered cell fitness [185– 187]. Interestingly, rare codons are often found ~30 amino acids downstream of autonomous folding domains, suggesting that by slowing down translation elongation the cell can guarantee enough time for the folding event to happen [188]. Additionally, clusters of rare codons positioned ~35-40 amino acids downstream of the signal sequence have been identified to promote SRP-binding by slowing translation, thus enlarging the time span for signal sequence recognition [189]. Therefore, the cell controls the rate of protein synthesis to help coordinate folding, targeting and downstream modifications such as N- linked glycosylation [190]. In addition to the mRNA sequence, the ribosome itself also affects co-translational protein folding. As mentioned above, the growing nascent protein chain travels from the ribosomal active site through a ~100 Å long exit tunnel. This tunnel was long thought to be passive, not interacting with the nascent chain and not involved in protein folding. Today it is established that the exit tunnel

28 does not only provide a constricted space that allows specific secondary structures and small protein domains to fold, but it can also affect translation rate via interactions with the nascent chain, as we will see in the following chapter about arrest peptides. The diameter of the exit tunnel ranges from 10 Å near the PTC to 20 Å at the vestibule, where it provides sufficient space for helix-formation, small protein domain folding, and the generation of other tertiary structures [191–194]. The formation of α-helices close to the PTC has been observed for transmembrane helices, but not soluble ones, suggesting that the ribosomal exit tunnel specifically stabilizes hydrophobic helices [191,195,196]. In this way, the ribosome could assist in SRP recognition and in membrane insertion as both require a helical conformation. Apart from interactions in the ribosomal exit tunnel, the surface of the ribosome also provides a specific interaction site for nascent chains. The local environment of the ribosome delays and restricts folding events, thereby preventing misfolding and aggregation by directing the folding pathway towards the native protein structure [190].

29 Arrest peptides

Specific interactions of certain nascent chain sequences with the ribosomal exit tunnel can induce translational stalling. These natural stalling sequences, termed arrest peptides, usually derive from regulatory proteins and have been found in a number of organisms. Although the different arrest peptides that have been identified so far do not share any homology, they all seem to induce translational stalling by forming interactions with the ribosomal exit tunnel, leading to a conformation at the PTC that does not allow peptide bond formation of the amino acid following the stalling sequence to the nascent chain [197,198]. Their biological functions are usually as cis-acting modulators of translation. Depending on the cellular situation, the stalling can be overcome by interaction with specific partners that exert “pulling” on the nascent chain, while in absence of this pulling force the arrest is maintained [199]. In both cases a specific biological response is triggered, often involving mRNA secondary structure formation or maturation, leading to downstream processes, such as regulation of target gene expression [200,201]. The stalling of many arrest peptides also requires the presence of a small molecule—like a metabolite—in the ribosomal exit tunnel (e.g. TnaC), allowing them to become active only under specific metabolic conditions [202]. Thus, arrest peptides naturally serve as specific sensors of cellular activities and allow the cell to react accordingly. Two important examples that do not require additional molecules within the exit tunnel are explained in more detail below.

SecM

One of the most well-studied examples of translation-stalling peptides is that of the secretory protein SecM. The 170 amino acid long bacterial protein “secretory monitor”, SecM, regulates the expression of SecA, which facilitates post-translational protein translocation in bacteria [203,204]. Both, SecM and SecA, are encoded on the same mRNA transcript with SecM located upstream of SecA [205]. Under cellular conditions, a mRNA loop structure forms in the intergenic region between the coding sequences, masking the translation initiation site of SecA and therefore preventing translation of SecA. When a ribosome translating the mRNA reaches the end of a 17 amino acid long arrest peptide sequence contained near the C-terminus of SecM, interactions between the arrest peptide and the ribosomal exit tunnel induce a conformational change at the PTC, slowing peptide-bond formation and tRNA translocation

30 ultimately resulting in translational stalling [206]. While stalled, the mRNA loop structure described above unwinds and translation of SecA begins [203]. How- ever, the stalled SecM arrest peptide interactions are overcome if sufficient SecA is present to “push” SecM through the translocon, restoring the conformation at the PTC and allowing translation to resume [199,207]. Therefore, SecA levels are controlled by this negative feedback loop mediated via SecM translational stalling. Excitingly, the arrest peptide induced stalling can be released not only by SecA “pulling”, but by any “pulling force” exerted on the nascent protein. This has led to the short stalling sequence of SecM to be widely used as a force sensor to study co-translational protein folding events (see above) and membrane insertion [192,208]. Additionally, variants with enhanced stalling capacity have been used to capture the structure of small folded protein domains within the ribosomal exit tunnel [192,193]. Thus, the use of the SecM arrest peptide as a tool has helped our current understanding of co-translational protein events in bacteria.

Xbp1

The work presented here is performed in a eukaryotic system and therefore cannot employ a bacterial arrest peptide. The mammalian arrest peptide, used instead, is derived from the X-box binding protein 1 (Xbp1), which is an essential component of the unfolded protein response (UPR). This response is triggered during ER stress conditions, when the cell’s protein folding capacity is overloaded and unfolded proteins accumulate in the ER lumen [209]. The UPR consists of three main branches, acting in parallel to ensure rapid return to protein homeostasis. One branch includes Xbp1 as a mediator to activate UPR-specific protein expression [210]. In brief, the Xbp1 mRNA is translated by ribosomes until the point of arrest peptide-induced stalling. The protein is co-translationally localized to the ER membrane, where it interacts with—but does not insert into—the membrane, which overcomes the stalling and pro- duces the final membrane associated product [211]. However, under ER stress conditions, the mRNA of the still arrested Xbp1 can be spliced by the stress- activated endonuclease IRE1. IRE1 is an ER resident membrane protein that is capable of removing an intronic sequence of Xbp1, leading to an alternative protein variant that acts as a transcription factor, upregulating the expression of a number of UPR genes [212]. The translational stalling of Xbp1 does not

31 only allow sufficient time for the splicing reaction, it is also required for proper targeting to the ER membrane, the site of its splicing [211].

Figure 10: Translational stalling induced by an Xbp1-derived arrest peptide. Interac- tions of the nascent chain with the ribosomal exit tunnel proteins uL22 and uL4 forces the chain into a looped conformation that does not allow subsequent peptide bond formation at the peptidyl transferase center. The rRNA base C4398 adopts a premature closed state, caused by the postioning of leucine residue 259 of the arrest peptide. This conformation clashes with the proper positioning of an A-site bound asparaginyl-tRNA, thereby blocking elongation. Residues of the arrest peptide are color coded according to their contribution for the strength of the translational arrest. Substitution of the strongly contributing residues (blue) reduce stalling, while certain mutations in the weaker contributing residues (red) can increase the stalling. [198]

The Xbp1 arrest peptide is 25 amino acid long and mediates stalling via several interactions with the ribosomal exit tunnel. These interactions impose a loop conformation on the nascent chain in the upper region of the exit tunnel,

32 compressing the last amino acids of the arrest peptide and preventing the transfer of the next residue in the Xbp1 sequence, an asparagine, onto the nascent chain [198] (Figure 10). The translational stalling can be overcome by an event that introduces tension in the nascent chain and breaks the interactions of the arrest peptide with the exit tunnel, which releases the unfavorable chain conformation so that translation can resume. The strength of the arrest peptide can be modulated by making point mutations within the arrest peptide sequence [198], allowing the capture of both strong and weak force generating events, depending on the investigation. In the method section, I describe the use of arrest peptides as force sensors for biomolecular interactions and in protein folding studies.

33 Overview of the secretory proteins studied

The research work presented in this thesis has focused on three different secretory proteins, their signal sequence efficiencies, modes of engagement with the translocon, and some of the auxiliary factors required for their translocation. In this chapter, a brief overview of the function and some relevant features of each of these three proteins—the “good”, the “bad”, and the “short”— is given.

Prolactin -the “good” one

One of the most well-studied proteins, in the context of protein secretion and translocation, is prolactin (Prl). It is translated as the precursor form, termed pre-prolactin, that carries an N-terminal signal sequence, which is cleaved off to release the mature prolactin into the ER lumen [213,214]. Prl is a peptide hormone of ~23 kDa that is secreted by lactotrophic cells in the pituitary gland into the blood. Additionally, Prl is secreted by various other tissues and can be found in a number of bodily fluids [215]. Experiments on rats showed that serum levels of Prl are important for immune function and survival and that they can be elevated by synthesis in non-pituitary tissues, if required [216]. In vertebrates, about 300 different actions for Prl have been reported, including water and electrolyte balance, metabolic functions, growth, development, and reproduction, as well as behavior and immune regulation [215]. The best-known function is induction and maintenance of milk production in mammals, where it is responsible for the synthesis of all the main components of the milk: lactose, milk protein, and lipids [215,217,218]. In biochemical studies, preprolactin has long been used to study secretory processes. It has turned out to be very efficient in translocon gating and it does not require any additional components for its translocation, at least in vitro, and can be targeted and translocated with the minimal cellular machinery, consisting of SRP, SR and the Sec61 complex [37,87]. Its very efficient signal sequence—with long hydrophobic and positively charged domains—has been

34 useful in identifying components of the secretory pathway. Its strong interaction with SRP helped to identify that the SRP54 subunit is responsible for signal sequence binding [219,220] and components such as the TRAP complex or TRAM1 via crosslinking reactivity to Prl [131,221]. Recently, the efficient intercalation of Prl at the lateral gate was used to obtain a structure of the mammalian Sec61 translocon channel opened by a signal sequence (see Figure 6). Prl has a long history as a star model protein in the field of protein translocation and is likely to serve as an exemplar of an efficiently secreted protein in many future studies.

Prion protein – the “bad” one

In contrast to Prl, which behaves well in biochemical studies and translocates efficiently, the Prion protein (PrP) is more of a “troublemaker”. It is expressed in various tissues, with the highest expression in the peripheral and central nervous systems [222]. Several functions have been suggested and discussed for PrP, including in memory and sleep, and neuroprotection [223]. However, no conclusive function for PrP has been identified so far. While its cellular function remains elusive, it is infamous for its involvement in the development of prion diseases. Misfolding and aggregation of PrP causes the transmissible neurodegenerative conditions, known as Bovine Spongiform Encephalopa- thy (BSE or mad cow disease) in cows, Scrapie in sheep and goats, or Creutz- feldt-Jakob disease in humans [224]. The normal form of PrP, a GPI anchored plasma membrane protein, can be converted to the infectious insoluble variant and propagate the conversion of other PrP molecules to the infectious form [225]. How the first infectious molecule forms is not well understood and only about 10-15% of cases of prion-related disease in humans can be traced to a mutation in the PrP gene (PRNP) [226]. Most cases (85-90%) are termed spo- radic Creutzfeldt-Jakob disease, where the reason for disease onset remains unclear. However, in some rare cases, the cause of disease can be tracked to contact with external infectious prions, as observed in the transmission of Kuru among the cannibalistic Fore people in Papua New Guinea, contact with infectious tissue during medical procedures (iatrogenic forms), or after the consumption of infected beef (variant forms) [226,227]. From a biochemical standpoint, the cases of genetically inherited prion disease are the most relevant ones. Interestingly, PrP contains a marginally hydrophobic segment that has been observed to drive a small portion of the protein population to partition into the membrane adopting two topological forms:

35 with the N-terminus inside the ER lumen (Ntm form), or with the N-terminus towards the cytosol (Ctm form) [228]. Certain mutations within this hydrophobic domain have been connected to the increased formation of the potentially neurotoxic Ctm form and propagation of neurodegenerative disease. Addition- ally, the inefficiency of the PrP signal sequence promotes formation of Ctm and the aggregation prone cytosolic form [86,229]. The PrP signal sequence has a fairly short hydrophobic domain (8 amino acids) and no additional charges in the N domain other than the naturally positively charged N-terminal amino group (see Figure 3). Studies have shown that about 10% of PrP does not reach the ER lumen in cultured cells, but remains in the cytosol due to inefficient gating of the PrP signal sequence [83,86]. During ER stress, the cell decreases inefficient translocation substrates preventing PrP from entering the ER lumen. In combination with the mutations discussed above, this could contribute to neurodegeneration [87,179,229]. In addition to the insufficient signal sequence, the marginally hydrophobic domain, two glycosylation sites, and the GPI-anchor signal, PrP also contains a cluster of positively charged amino acids that follows directly after the signal sequence and a downstream intrinsically disordered domain, including an oc- tapeptide repeat region, followed by another cluster of charges [230]. The combination of the weakly gating signal sequence and the first charged cluster have been found to be responsible for the recruitment of auxiliary components required for translocon gating [78,163]. This included the TRAP complex, but also the Sec62/63 complex [78,117], suggesting a potential co-translational role for Sec62/63 as discussed above. All of these features make PrP an interesting model protein to study the translocation and protein folding of a more complicated client.

Insulin – the “short” one

The short peptide hormone insulin is essential for the regulation of blood glucose levels by activating insulin-dependent glucose transporters, especially in muscles and adipocytes, to uptake glucose if glucose levels are high [231]. In- sulin is therefore a key player in providing energy to cells. Patients with diabetes mellitus suffer from absolute (as in type 1) or relative (as in type 2) insulin deficiency [232]. In type 1 diabetes, the insulin-secreting pancreatic β-cells are destroyed by an autoimmune reaction, in most cases leading to a complete loss of β-cells and absolute insulin deficiency. Patients suffering from type 2 diabetes usually develop insulin resistance, often as a consequence of obesity,

36 age, and inactive lifestyle [232]. These patients can usually still produce insulin, but as their cells have developed resistance, glucose cannot be taken up efficiently enough. Prolonged elevated blood glucose levels together with obesity can ultimately lead to β-cell dysfunction, further enhancing the problem [233]. Diabetes can also be triggered by genetic defects in either β-cell function or in insulin action [232]. In addition to autosomal-dominant inherited cases, pol- ymorphisms at various gene loci have been connected to an increased risk of developing type 2 diabetes, some of which might be mitigated by lifestyle changes and reducing environmental risks [234]. Many of the known patient mutations within the insulin gene that trigger diabetes have been characterized biochemically to identify the point in insulin synthesis, where the failure occurs. This does not only increase our understanding of the disease, but also the cellular control mechanisms that ensure only properly folded protein is secreted.

+ NH3

Disulfide bond formation C-peptide cleavage in the oxidizing environment by endopeptidases Signal sequence of the ER lumen in the Golgi apparatus

+ + NH3 NH3

C CH C C C CH HC C C C CH C

C CH C C HC C B-chain A-chain

C-peptide

Pre-proinsulin Proinsulin Insulin

Figure 11: The steps of insulin biosynthesis. Pre-proinsulin is targeted to the ER, where the signal sequence is cleaved and three disulfide bonds (orange) are formed to yield proinsulin. The bonds between the A and the B-chain of insulin stabilize the hormone after their cleavage from the C-peptide.

Insulin is synthetized as the 110 amino acid long precursor pre-proinsulin and processed to proinsulin in the ER lumen, where the signal sequence is cleaved off and three disulfide bonds are formed (see Figure 11). Proinsulin is trans-

37 ported through the Golgi apparatus and processed into mature insulin by re- moval of the C-peptide, connecting the A-chain and the B-chain. Finally, insulin and the C-peptide are stored in secretory granules of the β-cells [79]. Upon stimulation by increased glucose levels, the granules fuse with the plasma membrane and insulin and the C-peptide are released into the blood. For a long time it was thought that the C-peptide only served a biosynthetic purpose, but recently there is growing appreciation for its role in preventing osteoporosis and in maintaining healthy renal function [235,236]. The latter involves an evolutionarily conserved glycine-rich patch within the C-peptide that stimulates the activity of Na+ K+-ATPase in the renal tubule segments of rats. These results pose the question: are common complications that occur in patients with type 1 diabetes who depend on external insulin, due to the lack of C- peptide production and release? This and other questions about the role of the C-peptide will the the subject of further study. The precise components involved in either the co- or post-translational translocation of pre-proinsulin have not been studied in detail. It is commonly assumed that pre-proinsulin reaches the ER via the co-translational pathway, but growing evidence indicates that a post-translational pathway, via the Sec62/63 complex, might play an additional role to rescue precursors that fail to be efficiently targeted [41]. One patient mutation was studied where a positively charged arginine residue in the N domain of the signal sequence had been lost, resulting in decreased efficiency in the post-translational translocation of pre- proinsulin [237]. A recently identified polymorphism in the TRAP complex has been connected with higher susceptibility for type 2 diabetes, highlighting the contribution the translocation machinery makes in proper insulin homeostasis. Its role in the development of β-cell failure will need to be explored more carefully in the future [238].

38 Introduction to the methodology

Arrest peptides as biochemical force sensors

Arrest peptides can be engineered into any protein of choice at any position in the sequence to probe the pulling forces experienced by the nascent chain at any point in translation down to single amino acid resolution. Previous work using arrest peptides showed that the insertion of hydrophobic transmembrane segments into the ER membrane exerts a biphasic pulling force on the nascent chain, which was captured by engineering the arrest peptide into the model protein at various distances from the hydrophobic segment [208]. In my work, the Xbp1 arrest peptide system was employed to probe the pulling forces exerted by signal sequences or during the translocation of critical protein domains, such as intrinsically disordered regions. The arrest peptide was engineered into the protein of interest at a good distance from the sequence of interest (i.e. the putative force generator). When the arrest peptide was placed at the end of the protein, additional C-terminal amino acids were added to distinguish the arrested protein from the “pulled” full length (FL) protein population. From the basic construct, various shorter variants were created by truncation PCR to allow analysis of a complete force generating area. During translation, when the ribosome reaches the end of the arrest peptide and stalls, only the N-terminal amino acids, that are sufficiently distanced from the end of the arrest peptide, are exposed to the cytosol or potential interaction partners (~30-40 residues in human ribsomes) [239]. In our experiments, the Xbp1 arrest peptide is 25 amino acids long, thus sequences of > 10 residues in front of the arrest peptide should be exposed. If the exposed region is pulled strongly enough by an interaction or folding event, the tension this creates is transmitted along the nascent chain, breaking the interactions of the arrest peptide with the ribosome, allowing translation to resume and synthesis of the FL variant of the protein. In the absence of an event that creates sufficient tension to break the interactions of the arrest peptide, the ribosome remains in its arrested state, generating a shorter arrested protein variant (Figure 12). The two protein populations generated can be separated

39 by SDS-PAGE, allowing the calculation of the fraction of FL protein at each point in translation assayed. When the fraction of FL is plotted against the arrested chain length, the resulting force profile allows us to determine what protein sequences experience strong pulling forces, indicative of biomolecular interactions or folding events. Systematic mutagenesis within the sequences of interest helps us to understand substrate-specific events during co-translational translocation.

1 processed arrest peptide

signal pulling sequence 2 Full length cytosol no pulling (FL)

ER lumen 3 arrested Sec61

Figure 12: Schematic representation of the pulling force assay for signal sequence probing. Ribosomes stalled with an Xpb1-derived arrest peptide are targeted to the ER membrane via their exposed signal seqence. If the interaction of the signal sequence with the translocon generates a “pulling” event, (1) the arrest is overcome, the protein is translocated and processed (e.g. signal sequence cleavage and glycosylated as indicated by Y). If no pulling event occurs, the arrested protein population can be released from the ribosome by addition of RNAse (3). Spontaneous release or escaping from translocation can lead to a population of unprocessed full length protein in the cytosol (2). Figure modified with permission from Kriegler et al., 2020 [240].

In vitro cell-free systems

Many of the details we know today about the molecular machinery involved in the secretory pathway were discovered thanks to the development of reconstituted cell-free systems [241]. Initially, crude cell extracts were used, e.g. a bacterial crude cell extract was used to understand the genetic code [242]. The development of eukaryotic cell-free systems enabled researchers to identify the ER as the entry site for the secretory pathway and eventually the individual components essential for translocation [37,243]. Step by step, they reduced the components in the reconstituted system until a minimal system able to

40 transport proteins across an ER membrane (purified vesicles of rough ER from dog pancreas) was aquired. Even today, many basic research questions are tackled using similar in vitro systems, as they supply a way to reduce the number of variables present in the complicated processes of living cells and focus on understanding key details. The most well-established eukaryotic system is derived from rabbit reticulocyte lysate (RRL) [244,245]. Reticulocytes are pre-mature erythrocytes, which are packed with hemoglobin, have already lost their nucleus and genomic DNA, and contain only low levels of residual mRNA, which is usually destroyed by extract preparation, ensuring low background protein expression [246,247]. Today RRL is available as a robust and reproducible commercial product that yields good protein expression. Both, a coupled (transcription and translation are performed in one single reaction) and an un-coupled (transcription and translation are performed in two separate reactions) variant can be used depending on the precise experimental requirements. RRL can be combined with radiolabeling techniques by addition of radiolabeled amino acids, such as 35S-methionine, to the reaction, which are incor- porated to the newly synthesized protein, which can then be specifically and sensitively detected from within the complex lysate background. Additionally, the inclusion of a source of ER membranes enabled us to use the RRL system to study protein translocation.

ER membrane donors – RMs and SPCs

Canine pancreatic RMs played an important historical role in the discovery of protein translocation [243]. Pancreatic cells contain densely packed sheets of rough ER, as George Palade noted in his early electron micrographs [2,13]. Modern analysis of tissue-based protein expression confirms that pancreatic cells are highly specialized for protein secretion [9]. Consequently, they provide a good source of rough ER membranes that can be isolated and used in form of membrane vesicle for in vitro studies. They are equipped to carry out protein translocation, N-linked glycosylation, and other protein processing functions and have been used in countless studies [248]. While canine pancreatic RMs provide an excellent system, they cannot be ma- nipulated and researchers seeking a more in vivo-like situation during protein translocation have turned to the use of semi-permeabilized cells (SPCs) [249,250]. SPCs are created by taking the cell type of choice and subjecting it to

41 mild digitonin treatment, which permeabilizes the plasma membrane, but leaves the membrane-bound cellular organelles intact. The endogenous mRNAs are digested, the cytosol is washed out and replaced with RRL, and the SPCs provide an intact ER system for translocation. The preparation of SPCs and their experimental use are shown in Figure 13. Figure 2

A cytosol cytosol

ER lumen digitonin in vitro HeLa cells translation mix

B C D Figure 13: Schematic representation of the generation of SPCs. HeLa cells are harvested 1.0 and treated with digitonin to0.8 permeabilize the plasma membrane. The cytosol is releasedPrl AP (-SPC)and PrP AP (+SPC) Prl AP (+SPC) [240] replacedno membrane SPCswithRMs RRL to perform in vitro pulling force assays 0.8. f(CL) Prl AP 0.6 0.6

Gly 0.4 f(FL) FL 0.4 One of the main advantagesf(processed) of this approach is that it allows specific manip- 0.2 ulations of cellular proteins before the preparation of0.2 SPCs [42,163,240,250,251]. In arrest my thesis work, we used0.0 siRNA to knock-down components0.0 of the ER trans- 40 100 160 220 280 50 60 70 80 90 locationPrP AP machinery L126 , generating a toolLength to [aa] study the effect of individualLength [aa] components of the translocation machinery on the translocation of target proteins and identifying what precursor features require the engagement of auxiliary components during translocation.

Chemical crosslinking and co-immunoprecipitation

Protein-protein interactions are key to biological function. However, they are often transient and can be difficult to observe biochemically. Chemical crosslinkers can overcome this hurdle by physically linking a protein of interest to another protein when they come into close proximity. Chemical crosslinkers carry two chemically reactive groups, one on either end of a short spacer or linker molecule. Under the correct conditions (e.g pH), a covalent bond is formed between the reactive group of the crosslinker and a specific functional group on a protein [252]. Thus, two proteins, that spend sufficient time in close proximity to each other, can become covalently linked and this new complex

42 can be analyzed e.g. by sodium dodecyl sulfate–polyacrylamide gel electro- phoresis (SDS-PAGE) and co-immunoprecipitation to identify the interaction partner. Common reactive groups for chemical crosslinkers include N-Hydroxysuccin- imide (NHS) esters and maleimides [253]. NHS esters react with primary amine groups as can be found in lysine residues and at the N-terminus of proteins. Maleimides conjugate with sulfhydryl-groups such as the side chains of cysteine residues. Chemical crosslinkers can be either homobifunctional, bearing the same reactive group at both ends of the linker, or heterobifunctional, carrying two different reactive groups. The chemical crosslinkers used in the work presented here are: bismaleimidohexane (BMH), a homobifunctional maleimide crosslinker with a 13 Å long spacer; and N-[g-maleimidobutyr- yloxy] succinimide ester (GMBS), a heterobifunctional crosslinker with one maleimide and one NHS group connected by a 7.3 Å long spacer. Crosslinked protein complexes, that will selectively include the radiolabeled protein of interest (e. g. model proteins), together with interacting partners were visualized in the autoradiographic images. The likely interaction partners were predicted based on the size of crosslinking products and the biological context. The identity of interacting candidates were further confirmed by co- immunoprecipitation [254]. Briefly, specific antibodies raised against the interacting partner candidates were incubated with the crosslinking reactions that were co-immunoprecipitated by the addition of protein A- or G-coupled se- pharose beads and analyzed by SDS-PAGE and visualized by autoradiog- raphy.

Additional biochemical methods used in these studies

As protein targeting and translocation was my main area of interest during my doctoral thesis work, it was important to separate out only the properly targeted, i.e. membrane-bound, protein fraction from the untargeted protein of interest. This was achieved by membrane fractionation through a sucrose cushion. The sample was loaded on top of a 250 mM sucrose cushion and subjected to ultracentrifugation. The pellet fraction contained the ER membrane, and the luminally trapped and ER membrane-bound proteins, allowing this fraction to be analyzed separately. One important assay, that we performed on the membrane fraction, was protein digestion with proteinase K (PK). As PK cannot access proteins integrated

43 into or on the inside of the ER vesicles, only protein domains that are accessi- ble to the cytosolic side of the ER vesicles are digested. Additionally, any nascent chain-ribosome-translocon complexes can also shield the protein of interest from PK digestion. Protected protein fragments are analyzed by SDS- PAGE. Therefore, the PK digestion assay offers interesting insights into the translocation and topology state of the protein of interest at the point in translation assayed. In addition to the PK assay, we also used a glycosylation mapping technique to confirm the orientation of proteins. N-linked glycosylation sites are engineered into the protein of interest at various positions in the protein sequence, such as the loops between transmembrane domains, to determine the orientation of the protein in the ER membrane [255]. Glycosylation only occurs in the ER lumen, so the position of the engineered site can be determined based on whether the protein band shows an increase in size (i.e. that is has been glycosylated) or not. Hence, glycosylation mapping provides an additional method to the PK assay to determine a protein’s orientation in the ER membrane.

44 Conclusions and Speculations

In mammals, co-translational protein translocation across the ER membrane is facilitated by a dynamic super assembly of protein multicomplexes. Many steps of the procedure, such as interaction of SRP with the ribosome and the exposed signal sequence, the interaction of SRP with its receptor, and the general function of the Sec61 translocon channel, are fairly well understood. However, the detailed understanding on how signal sequences that have different levels of gating efficiency can trigger channel opening by the recruitment of auxiliary components to the complex is still a subject of intense investigation. Additionally, the involvement of auxiliary protein complexes that assist specific downstream domains (i.e. not the signal sequence) of a protein to be translocated via the Sec61 channel has recently become appreciated. In the work presented here, we have taken a novel approach to shed light onto the composition of the complete translocon complex at various specific points in translation for different signal sequences—and the mature proteins they traffic. Arrest peptides are short, naturally-occurring sequences that stall translation by preventing peptide-bond formation at the ribosome peptidyl transferase center by holding the nascent chain in an incomplatible conformation via nascent peptide-ribosomal exit tunnel interactions. For some arrest peptides, translational arrest can be overcome by events that exert tension on the nascent chain. This makes arrest peptides useful tools (“force sensors”) for the detection of biomolecular interactions or protein folding events that occur co-translationally. By sequentially engineering the arrest peptide into the model protein sequence, we can obtain a force profile for the translocation of the entire protein. We have made use of an eukaryotic arrest peptide to investigate the different forces experienced by efficient and less efficient signal sequences in an in vitro arrest peptide assay using rabbit reticulocyte lysate (Paper I). For the efficient signal sequence, in this case the model protein prolactin (Prl), a biphasic pulling force profile was observed. By making a number of mutations within the hydrophobic core and the N domain of the signal sequence, we gained further insight into the nature of the pulling events. The first, weaker

45 pulling event occurred at a chain length of 65 amino acids and was dependent on the hydrophobic core. Substitutions that increased the hydrophobicity, strongly increased the pulling force. This result, combined with our crosslinking data and observations in the literature, indicates that this event is the initial interaction of the signal sequence with the exposed hydrophobic patch of the primed ribosome-translocon complex. As the chain is elongated, a second, very strong pulling event was observed, which is linked to signal sequence intercalation into lateral gate and partitioning into the membrane. Mutations in the N domain revealed that the positive charges are essential to ensure correct inverted orientation that anchors the N-terminus in the cytosol and to observe the second pulling event.

Figure 14: Graphical abstract of the findings from Paper I. Using an arrest peptide-based assay, the differences between signal sequences were explored. Efficient signal sequences experience a strong pulling force when interacting with the translocon. They can efficiently interact and open the channel to be released into the lipid bilayer. Inefficient signal sequences experience only very weak pulling forces or none at all when interacting with the Sec61 complex. Translocation initiation is delayed and potentially needs assistance from additional factors.[256]

46 For less efficient signal sequences, such as the one from the prion protein (PrP), only a single weak and broad pulling event could be observed. The pulling could be enhanced slightly by increasing the number of hydrophobic amino acids, but did not convert the weakly pulled signal sequence into a biphasic strongly pulled sequence. The main findings of the first paper are sum- marized in the graphical abstract presented in Figure 14. In addition to the different pulling forces observed for efficient and inefficient signal sequences, the time point of signal sequence cleavage occurs also varies for different proteins. We compared the chain length in which we first observed signal sequence cleavage for the Prl (Paper I) and the PrP (Paper II) arrested constructs. We found that cleavage of Prl was almost complete at a length of 160 amino acids, while cleavage of PrP could not be observed until a length of 170 amino acids and only peaked at a length of 215 amino acids. To further investigate the pulling forces experienced by a protein carrying a weakly-gating signal sequence—as well as some additional interesting domains in the mature protein—we followed the translocation of full length PrP from start to finish by arrest peptide assay. It is important to note that we chose a weaker arrest peptide variant compared to the one used in the signal sequence study, allowing us to also capture weaker pulling events. In addition to the initial signal sequence-dependent pulling event, we observed a second pulling event at a chain length of 105-126 amino acids and a weak pulling towards the end of the protein (Paper II). Based on mutation studies, we hypothesize that the late weak pulling could be connected to the GPI anchor. The nature of the second pulling event could not be revealed by mutagenesis alone, therefore we took a new approach and performed the arrest peptide assay using semi-permeabilized cells (SPCs) instead of rough ER microsomes (method presented in Paper III). This allowed us to selectively knockdown different translocon-associated factors and investigate the effect on the observed pulling events. A siRNA-based knockdown was carried out on cultured mammalian cells, which were then used to prepare SPCs. The involvement of Sec62, Sec63, and the translocon-associated protein (TRAP) complex were tested and compared Sec61α depletion, which acted as a non-translocating control. Only mild effects were observed upon Sec62/63 depletion, but the second pulling event was reduced to baseline upon silencing of TRAPβ, which prevents formation of the TRAP complex. Returning to mutational analysis, we discovered that a cluster of positive charges located directly after the signal sequence, together with an intrinsically disordered glycine/proline (G/P)-rich

47 domain were responsible for the TRAP-dependent second pulling event. In- terestingly, G/P-rich signal sequences had been found to be a signature for TRAP-dependence by a genome-wide proteomics analysis [119]. Our data showed that this might also be true for mature domains of the protein. A graphical summary of the major pulling events experienced by PrP during translocation is shown in Figure 15. The fact that we could not observe any pulling for the insertion of the marginally hydrophobic segment within PrP, showed that, at least in our set up, there is no strong drive for it to partition into the membrane, as we would have expected for a real transmembrane domain. However, we were able to detect both of the transmembrane forms of PrP (Ntm and Ctm orientation), indicating that some of the protein partitioned into the membrane, probably in a slow and non-tension-generating manner.

Figure 15: Graphical abstract of the findings from Paper II and III. Pulling events during translocation of PrP were monitored using an arrest peptide-based assay. Two major events were captured, reflecting the initial interaction of the signals sequence with the translocon (left) and the TRAP-dependent translocation of a positively charged patch and an intrinsically disordered region within the protein (right).[251]

In our last project, we were able to gain more insights into the action of the TRAP complex in translocon gating and protein translocation. To our surprise, the pulling force analysis suggested that the signal sequence of insulin is inef- ficiently gating (Paper IV). The wild type produced barely any pulling force, but a single point mutation of a proline to leucine (P9L) in the signal sequence resulted in a strong pulling event. Together with crosslinking and co-immunoprecipitation experiments, we were able to show that TRAP was interacting with the translocating substrate in a signal sequence-dependent manner and

48 that the strongly-pulled P9L mutant interacted with TRAP to a lesser extent. Additionally, our siRNA-based knockdown of TRAPβ in SPCs revealed that the P9L mutant was less dependent on TRAP for efficient translocation. A number of additional mutants with a higher or lower G/P-content in the signal sequence confirmed the connection of G/P residues to the requirement of TRAP for translocation. Interestingly, a highly conserved poly-glycine patch in the C-peptide of insulin was found to contribute to the TRAP-dependence as well. This supports our previous findings that parts of the mature PrP protein require TRAP for efficient translocation. Furthermore, by probing the interactive environment of insulin arrested at various lengths, we could show that TRAPβ already interacts with the nascent chain on the cytosolic side of the translocon, before the chain enters the Sec61 channel (see the graphical summary in Figure 16). This interesting finding suggests a mechanism for the TRAP complex that has been suggested previously but had not been previously supported by biochemical data.

Figure 16: Graphical abstract of the findings from Paper IV. Crosslinking to insulin nascent chains stalled at different point during translocation show that the insulin nascent chain interacts first with the TRAP complex (green) via its β subunit, before it interacts with the Sec61 translocon channel (blue). This crosslinking pattern was observed for two native cysteines (Cys1 and 2, indicated as filled and empty stars, respectively) in the mature part of insulin directly following the signal sequence (compare the repeated pattern of Cys1 and Cys2 crosslinking intensities vs chain length on the right). The hypothesized positioning of the transmembrane segment and cytosolic part of TRAPβ is depicted in light green in the structural illustration (left panel). SS, signal sequence; ER, endoplasmic reticulum.

49 Based on our data, proteomic analysis [119], previous observations of an inverse correlation between the gating capacity of signal sequences and the requirement for TRAP [78], and cryoelectron tomography data [135], we propose the following mode of action for the TRAP complex. The TRAP complex as a permanent interaction partner of ribosome-bound translocons, interacting with both, the translating ribosome and the Sec61 channel. Electron tomography studies were unable to resolve the smaller cytosolic domains and the transmembrane segments of the α and β subunits, likely due to peptide chain flexibility and/or transient interactions that are av- eraged out in the final tomography density. Based on our crosslinking data, we propose that the short cytosolic tail of TRAPβ is positioned close to the nascent chain as it leaves the ribosomal exit tunnel, reaching into the gap between the ribosome and Sec61α. In this conformation the TRAP complex could potentially sense the sequence entering the translocon and provide assistance, when needed, via the large luminal heterodimer of TRAPα/β. In the case of a weakly gating signal sequence, delayed gating would lead to nascent chain accumulation in the space between the ribosome and the translocon, exactly where the cytosolic tail of TRAPβ is positioned. Sensing this, TRAP could trigger channel opening through the interaction of the α/β heterodimer with loop 5 of Sec61α on the luminal side. In the case of insulin specifically, the proline in the hydrophobic core of the insulin signal sequence would sterically prevent strong interaction with the exposed hydrophobic patch in the translocon, but could lead to a tighter interaction with TRAP. This interaction would signal a need for gating assistance from the cytosolic to the luminal side initiating channel opening. In the instances where TRAP assists with positively charged or G/P-rich protein domains outside of the signal sequence, we imagine a chaperone or ratchet-like function of the TRAP complex. However, it will require further investigation to clarify in what way TRAP mediates the efficient translocation of these domains.

50 Populärvetenskaplig sammanfattning

Cellerna är de minsta enheterna i livet. De separeras från sin omgivning av ett tunt membran, som består av fettmolekyler där proteiner är inbäddade. Många celler tillsammans bildar en vävnad, ett organ och slutligen en hel organism, till exempel en människa. För att cellerna och därmed hela organ ska kunna kommunicera med varandra, måste vissa proteiner kunna lämna cellen där de bildades. Till exempel reagerar vår kropp på sockerintag genom att släppa ut hormonet insulin från cellerna i bukspottkörteln. Under många år har forskare forskat på den väg som proteiner tar för att korsa cellmembranet för att kunna lämna cellen. I enkla bakterieceller kan proteiner transporteras direkt till cellmembranet, men i de mer komplicerade eukaryotacellerna så som växter, djur och människor, börjar resan vid det endoplasmatisk retikulumet (ER). ER är en så kallad cellorganell som är separerad med ett cellmembran i ett område i cellen. Proteiner som lämnar cellen måste transporteras över detta membran i ER. Porten bildas av en kanal, gjord av proteiner kända som translokon. Porten är väl stängd i inaktivt tillstånd för att förhindra att plasman av ER blandas med plasman i cellen. Proteiner produceras i cellplasman av molekylära ma- skiner, så kallade ribosomer, som består av många olika proteiner och RNA. Om ett protein görs som är avsett för att exporteras från cellen, bär det en så kallad signalsekvens i början. Denna signalsekvens produceras först och fun- gerar som en postadress då den känns igen av en "signaligenkännande partikel" (SRP). SRP flyttar ribosomen med det påstartade proteinet till membranet vid ER och translokonen. Där binds både, ribosomen och signalsekvensen, till ett translokon som öppnasså att det nya proteinet kan transporteras direkt in i ER medan det produceras. Denna process kallas co-translationell translokat- ion. Även om de flesta signalsekvenser kan föra sitt protein till ER, är det några som inte kan öppna translokonet utan hjälp. I detta arbete undersöktes de olika interaktionerna mellan effektiva och mindre effektiva signalsekvenser. I den första publikationen kunde vi visa att effektiva signalsekvenser dras in i translokonet, som öppnas och signalsekvensen släpps i sidled i membranet, medan resten av proteinet transporteras in i ER, varifrån det senare lämnar cellen. När vi testade en mindre effektiv signalsekvens, kunde vi inte obser- vera detta starka drag i transportkanalen, vilket visar att ytterligare faktorer

51 behövs för att öppna kanalen in till ER. I den andra och tredje publikationen utvecklade vi en metod som gjorde det möjligt för oss att undersöka i mer detalj vilka faktorer som är involverade i ett protein med en svag signalsekvens. Vi hittade överraskande nog att proteinkomplexet som kallas TRAP också är nödvändigt för transport av andra komplicerade områden i ett protein. Sådana komplicerade områden inkluderar regioner med positivt laddade ami- nosyror och / eller aminosyrorna glycin och prolin, som båda ger ostrukture- rade områden i proteiner. Dessa observationer bekräftades av data från den fjärde publikationen. Här kunde vi visa att TRAP-komplexet är nödvändigt för att transportera insulin prekursorn. Återigen identifierades glycin och prolin, som faktorer vilka gör både signalsekvensen och resten av proteinet bero- ende av hjälp från TRAP. Vi kunde också visa att TRAP tar kontakt med det ny-producerade proteinet på utsidan av ER membranet. Denna observation är särskilt anmärkningsvärd eftersom det bara finns ett mycket litet utrymme mellan ribosomen och translokonen där TRAP-komplexet kan agera. Där ver- kar komplexet känna om av om dess hjälp behövs för att öppna kanalen under transporten.

52 Populärwissenschaftliche Zusammenfassung

Zellen sind die kleinsten Einheiten des Lebens. Sie sind von ihrer Umgebung durch eine dünne Membran getrennt, welche aus Fettmolekülen besteht, in die Proteine eingebettet sind. Viele Zellen zusammen können ein Gewebe, ein Organ und schließlich einen ganzen Organismus bilden, wie zum Beispiel einen Menschen. Damit die Zellen und somit ganze Organe untereinander kom- munizieren können, müssen bestimmte Proteine die Zelle, in der sie gebildet wurden, verlassen können. Zum Beispiel reagiert unser Körper auf die Aufnahme von Zucker mit der Freisetzung des Hormons Insulin aus den Zellen der Bauchspeicheldrüse. Schon seit vielen Jahren erforschen Wissenschaftler den Weg, den Proteine nehmen müssen, um die Zellmembran zu überwinden um aus der Zelle freigesetzt zu werden. In einfachen Bakteri- enzellen, können Proteine direkt zur Zellmembran transportiert werden, doch in den komplizierteren Zellen von eukaryotischen Lebewesen wie Pflanzen, Tieren und Menschen, beginnt die Reise am Endoplasmatischen Retikulum (ER). Das ER ist ein sogenanntes Zellorganell und bildet einen durch eine Membran abgetrennten Bereich der Zelle. Diese Membran des ER müssen Proteine, die aus der Zelle hinaus transportiert werden, überwinden. Das Tor bildet dabei ein als Translokon bekannter Kanal aus Proteinen, der im inakti- ven Zustand gut verschlossen ist, um zu verhindern, dass sich das Innere des ER mit dem der sonstigen Zelle vermischt. Die Synthese von Proteinen erfolgt im Zellplasma an molekularen Maschinen, sogenannten Ribosomen, die aus vielen Proteinen und RNA bestehen. Wird ein Protein hergestellt, dass für den Export aus der Zelle bestimmt ist, so trägt es an dem zuerst synthetisierten Ende eine sogenannte Signalsequenz. Diese Sequenz fungiert wie eine Postanschrift und wird von einem „Signal erkennenden Partikel“ (SRP) erkannt. Das SRP bringt das Ribosom mit dem angefangenen Protein zur Membran des ER und dem Translocon. Dort binden sowohl das Ribosomen als auch die Signalsequenz an das Translokon und öffnen dieses, damit das neue Protein direkt in das Innere des ER transportiert werden kann, während es synthetisiert wird. Diesen Vorgang nennt man co-translationale Translokat- ion.

53 Obwohl die meisten Signalsequenzen ihr Protein erfolgreich zum ER bringen können, gibt es einige, die das Translokon nicht ohne Hilfe öffnen können. In dieser Arbeit wurden die unterschiedlichen Interaktionen von effizienten und weniger effizienten Signalsequenzen mit dem Translokon untersucht. In der ersten Publikation konnten wir zeigen, dass effiziente Signalesequenzen in das Translokon hineingezogen werden, wobei dieses geöffnet wird und die Sig- nalsequenz seitlich in die Membran entlässt, während der Rest des Proteins ins Innere des ER transportiert wird, von wo aus er später die Zelle verlassen kann. Im Fall einer weniger effizienten Signalsequenz, konnten wir keinen starken Zug in den Transportkanal beobachten, was zeigt, dass weitere Fak- toren benötigt werden um den Weg ins Innere des ER zu öffnen. Im zweiten und dritten Paper haben wir eine Methode entwickelt, die es uns ermöglicht hat, genauer zu untersuchen welche Faktoren bei einem solchen Protein mit schwachem Signal hinzugezogen werden. Überraschenderweise stellten wir fest, dass der TRAP genannte Proteinkomplex auch beim Transport anderer komplizierter Bereiche in einem Protein notwendig ist. Zu solchen komplizi- erten Bereichen zählen Abschnitte mit positiv geladenen Aminosäuren und/oder den Aminosäuren Glycin und Prolin, die beide für unstrukturierte Bereiche in Proteinen sorgen. Diese Beobachtungen wurden durch die Daten aus dem vierten Paper bestätigt. Hier konnten wir zeigen, dass der TRAP- Komplex notwendig ist, um den Insulinvorläufer zu transportieren. Erneut wurden insbesondere Glycin und Prolin identifiziert, welche sowohl die Sig- nalsequenz, als auch den Rest des Proteins von der Hilfe durch TRAP ab- hängig machen. Zusätzlich konnten wir zeigen, dass TRAP schon auf der Außenseite der Membran mit dem jungen Protein Kontakt aufnimmt. Diese Beobachtung ist insbesondere deshalb bemerkenswert, da zwischen dem Ri- bosom und dem Translokon nur ein sehr kleiner Spalt ist, indem der TRAP- Komplex agieren kann. Dort scheint er zu erfühlen ob seine Hilfe beim Öffnen des Kanals und beim Transport benötigt wird.

54 Acknowledgements

This work would not have been possible without the support of many great people around me that helped me in many different ways. I could not have done it without them. This is why I want to give some special thanks to some of them. First, I would like to thank my supervisor Tara for making this thesis possible in the first place by giving me the chance to work in her lab and on her projects. Your overflowing ideas were sometimes a bit overwhelming for such a small group, but I think we did our best, and together, we made a lot of it happen! Thank you for this opportunity.

My co-supervisor Dan, even though you were not directly involved in my projects, you always gave me good input after my presentations and talks. I appreciate that you always asked me how things are going and that you were always open to give me a good advice or to discuss about science, teaching, traveling, honey bees…

Gunnar, thank you for taking the role as my mentor, for all the good questions and suggestions during my lab seminars, and for your input on my manu- scripts.

Sven, meeting you at the EMBO meeting was inspiring and lead to a great collaboration. Thank you for welcoming me in Homburg, teaching me how to make SPCs and for always finding a good answer to my questions and thoughts.

I want to thank all the people that have been working with me in the Hessa lab for shorter or longer time. Anastasia, Emanuela, Martina, Andi, Sania and Mehwish thank you all for being with me for parts of this work! You all cheered me up when needed or celebrated with me if things were going well. Thank you for good times together in the lab! Also, I want to thank all the students that I had the honor to supervise during my PhD time and that contributed their share to the work: Rocio, Kevin,

55 Shuai, Zhe and Vivian. Special thanks to Génia, nicht nur hast du unglaub- lich fleißig an dem Insulinprojekt gearbeitet und alles versucht, es ans Laufen zu bringen, sondern du bist auch eine kluge und kreative Denkerin und ganz nebenbei eine gute Freundin! Danke für all deine Unterstützung auch über die Zeit in unserem Lab hinaus!

My wonderful current and former office mates Daphne, Ane, Hena, Rickard, Justin, Alice, Nir, Luigi and Renuka, thank you all for the good and creative atmosphere in the office. You were always happy to answer my questions, discuss problems and ideas and to give me any other support needed! Thank you all! Special thanks to Grant! Thank you for all the proofreading you have done for me during my PhD time, the time you took to listen to my problems, your help to find solutions for them and for cheering me up when my self- confidence and believe into scientific working was on the ground. Everyone from the GvH community, it was great to be part of this bigger group and get input from people working on so different topics. Your feedback during the lab seminars often inspired me and helped me a lot to develop my projects and to develop myself as a scientist. My fellow PhDs Felix, Rageia, Alex, Diana and James: All the best and good luck for the rest of you PhD time, it was great having you guys around! Thank you also to all the former members of the community: Patrick, José, Claudio, Kiavash, Thomas, Zhe,… Jan-Wilhelm, thank you for your questions during seminars and for interesting chats in the corridor or kitchen. The newest group that joint “our” corridor: Einar and Cecilia, you are a real asset to the group discussions with your different background and very different projects!

There is many other people within DBB that contribute to making it a great place to work, thanks everybody for all the good lunch conversations, chats in the corridor, the friendly smiles you gave me when passing by and small and big scientific help in form of chemicals, advice or equipment I could borrow. Among the colleagues, I had the honor to teach with, I would especially like to thank Ingrid for introducing me to everything in the biochemistry I course. And Alexandra and Olga, it was so fun to make the summer course in synthetic biology happen with you guys! Also, I would like to thank the members of the PhD council and the Pub com- mittee, we had so many good discussions and also fun times, thank you Joan, Linnea, Johan, Riccardo, Tobias, Sarah, Pascal, Frida, Eloy, Therese…

56 The department would fall apart if it did not have the great administrative crew it has. Special thanks to Maria and Alex for always being there and helping me with all the small and big paperwork and other issues! I also want to thank Matt for keeping the equipment in good shape and for being one of the most helpful people I have ever met!

Pia, you have been a great support for me, especially in the last phase of my PhD, thank you for always motivating me to hope for the best and for directing me through all the things that need to be completed before this thesis could be printed.

All the people in Homburg that took care of me during the weeks, I had the pleasure to work there. Moni, vielen Dank für deine Hilfe, besonders bei mei- nem ersten Besuch und vielen Dank für den super leckeren Kuchen und die netten Mittagspausen. Sarah und Mark, danke für die lustigen Abende mit und ohne Mölkky. Martin, dein Humor, die stetig gute Laune und dein Wis- sen sind eine großartige Kombination. Richard, vielen Dank, dass du mich in euren Laboren willkommen geheißen hast, ich habe viel gelernt und hatte eine gute Zeit bei euch!

Mama und Papa, dank euch habe ich geschafft, was ich geschafft habe. Ihr habt auch dann noch an mich geglaubt, wenn ich es nicht mehr getan habe und mich bei allem unterstützt, was ich tun wollte. Egal ob Zaubern, Tanzen, (schon wieder) Umziehen oder eben im Studium und bei der Promotion. Danke, dass ihr mir immer ermöglicht habt meinen Weg zu gehen und trotz- dem immer für mich da seid. Markus und Janina, es war so schön während meiner Zeit im Saarland mit euch immer ein Stück Familie in der Nähe zu haben. Die Zeit mit euch hat mit immer gutgetan! Johannes, du bist der Grund, warum ich es nie -egal was passiert ist- bereut habe meinen PhD in Stockholm gemacht zu haben. Wenn ich dich nicht ge- troffen hätte, würde ich vielleicht mit anderen Augen auf die stressige PhD- Zeit blicken. Aber allein das Wissen, dass du mich in den Arm nimmst, wenn gerade gar nichts gut läuft und mit mir feierst, wenn ich tolle Ergebnisse habe (auch wenn du sie eigentlich nicht richtig verstehst), hat mir die Kraft gegeben bis zum Ende durchzuhalten. Danke für Alles!

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