Ludwig Institute for Cancer Research, Stockholm Branch and Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden

MEMBRANE CHAPERONES Protein folding in the ER membrane

Jhansi Kota

Stockholm 2007

Jhansi Kota

Previously published papers were reproduced with permission from the publishers. Published and printed by Larserics digital print AB Box 20082, SE-161 02 Bromma, Sweden  Jhansi Kota, 2007 ISBN 978-91-7357-102-9

~ To Johan and my family ~

Jhansi Kota

Abstract

Abstract The plasma membrane (PM), comprised largely of lipids and proteins, is a dynamic structure that establishes the integrity of cells. Newly synthesized PM proteins are initially inserted into the endoplasmic reticulum (ER) prior to being targeted to the PM via the secretory pathway. Many PM proteins are polytopic, i.e., they have multiple transmembrane segments (TMS) and domains located on both sides of the membrane. Polytopic proteins carry out many vital processes, including sensing environmental conditions, and facilitating metabolite transport in and out of cells. The mechanisms that control the functional expression of the PM proteins are not fully understood. The work documented in this thesis established that four highly specialized accessory proteins within the ER membrane of the yeast Saccharomyces cerevisiae function as membrane chaperones. These chaperones, Shr3, Gsf2, Pho86 and Chs7, are integral components of the ER that are individually required for the functional expression of discrete sets of polytopic PM proteins – their substrates. Although these novel chaperones do not share sequence or obvious structural similarity, they function analogously to prevent inappropriate molecular interactions between hydrophobic segments of their substrates as they insert in the ER membrane. Shr3 plays a critical role in enabling amino acid permeases (AAPs) to fold and attain proper structures required for functional expression at the PM. In the absence of Shr3, AAPs accumulate in the ER, where despite the correct insertion of their twelve TMS, they aggregate forming large molecular weight complexes. Shr3 prevents aggregation and facilitates the functional assembly of independently coexpressed split N- and C-terminal fragments of the general AAP Gap1. Shr3 interacts with the five TMS within the N-terminal fragment and maintains them in a conformation that can post-translationally assemble with the seven TMS in the Cterminal fragment. AAP aggregates that accumulate in shr3 mutants are redundantly targeted for ERassociated degradation (ERAD) by Doa10 and Hrd1 dependent pathways. In combination, doa10∆ hrd1∆ mutations stabilize AAP aggregates, and partially suppress amino acid uptake defects of shr3 mutants. Consequently, in cells with impaired ERAD, AAPs are able to attain functional conformations independently of Shr3. These findings illustrate that folding and degradation are tightly coupled processes during membrane protein biogenesis. A genetic approach identified SSH4, RCR1 and RCR2 as high-copy suppressors of shr3 null mutations. The overexpression of either of these genes increases steadystate AAP levels, whereas their genetic inactivation reduces steady-state AAP levels. Also, suppressor gene overexpression exerts a positive effect on phosphate and uracil uptake systems. Ssh4 and Rcr2 primarily localize to structures associated with the vacuole, however, Rcr2 also localizes to endosome-like vesicles. These findings are consistent with a model in which Ssh4, Rcr2, and presumably Rcr1, function within the endosomal-vacuolar trafficking pathway, where they affect sorting events that determine whether transport proteins are degraded or (re)routed to the plasma membrane.

Jhansi Kota

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Populärvetenskaplig sammanfattning

Populärvetenskaplig sammanfattning Allt liv – oavsett hur exotiskt och främmande det kan synas - består av celler. Det finns grovt räknat bara två sorters celler: prokaryota och eukaryota. Vi människor och de flesta livsformer vi är vana att se består av eukaryota celler. Bakterier är däremot prokaryota. Dessa är vanligen mycket mindre än eukaryoterna och saknar den cellkärna i vilken eukaryoterna förvarar sitt DNA. Med utgångspunkt från sitt DNA kan cellen tillverka livsnödvändiga proteiner. Ett protein består av en lång kedja av aminosyror och för att proteinet ska bli funktionellt (uppnå sin nativa form) måste kedjan av aminosyror så att säga ”slå knutar på sig själv” (veckas). Andra molekyler som finns i cellen kan lätt störa proteinveckningen och den lyckas inte alltid. Därför finns också speciella ”hjälpar-proteiner” (chaperoner) som ser till så att t.ex. inte proteinet trasslar in sig i sig själv eller andra proteiner (aggregeras) innan det hunnit veckas. Felveckade proteiner måste tas om hand och brytas ner igen av cellen. Den eukaryota cellen har, förutom cellkärnan, många specialiserade organeller. Dessa avgränsas alla av membraner som består av ett dubbelt lager av lipid- (dvs fett) molekyler och en stor mängd av olika membranproteiner. Eftersom lipiderna stöter bort vattenlösliga molekyler på samma sätt som fett stöter bort vattendroppar, hindrar de också de flesta molekyler från att ta sig genom membranen. I mitt arbete utspelar sig det mesta av intresse i cellens ytmembran (plasmamembranet), samt en organell som kallas endoplasmiskt reticulum (ER). ER ser ut som en stor skrynklig säck av veckat membran. Här syntetiseras många av cellens proteiner. ERs uppgift är också att i tillhandahålla utrymme där proteinerna kan veckas och eftermodifieras till sin slutliga form och felveckade och aggregerade proteiner kan tas om hand för nedbrytning. Därefter skickas korrekt veckade proteiner till rätt ställe i cellen med hjälp av cellens eget postväsende – the secretory pathway. Membranproteiner är som namnet antyder, proteiner som är associerade till cellmembranen. De kännetecknas av att de har fettlösliga sektioner (som begraver sig inne i lipidmembranet) och en eller flera vattenlösliga sektioner (som söker sig ut ur lipidmembranen). Därigenom kan de fungera som slussar, vilka selektivt överför information eller molekyler från en sida av membranet till en annan. Många moderna läkemedel riktar därför in sig mot just olika membranproteiner. Ett exempel är magsårsmedicinen ”Losec”, som minskar aktiviteten hos vissa ”protonpumpande” membranproteiner i några av magsäckens celler. Membranproteinernas betydelse för cellen (och läkemedelsbranchen) kan knappast överskattas. Tyvärr är membranproteiner besvärliga att studera, så vi har vetat förhållandevis lite om deras struktur, hur deras veckning och efterbearbetning går till och hur cellens mekanismer för att ta hand om felveckade membranproteiner fungerar. För de tre arbeten som denna doktorsavhandling bygger på användes jästceller, Saccharomyces cerevisiae, som försöksobjekt. Dessa är lätta att hantera och har en 7

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välutvecklad secretory pathway som är mycket lik den i t.ex. människan. Som modellproteiner användes en familj membranproteiner som transporterar aminosyror (Amino Acid Permeases = AAPs). AAPs tillverkas i ER och transporteras därefter av secretory pathway till cellytan, där de hjälper cellen att ta upp olika aminosyror från omgivningen. Avhandling handlar om hur cellens mekanismer för veckning och efterbearbetning av membranproteiner fungerar vid ER membranet samt vad som händer då veckningen misslyckas. Man har antagit att membranproteiner precis som många andra proteiner får hjälp av chaperone-molekyler under veckningsprocessen, men eftersom ingen direkt påvisat existensen av sådana membran-chaperoner kunde man inte veta säkert. I det första av mina tre arbeten visade vi för första gången att det existerar membran-chaperoner. Vi studerade ett protein som kallas Shr3 och lyckades experimentellt bevisa att dess funktion är att agera som en membran-chaperone åt AAPs. Det visade sig att vid avsaknad av Shr3 trasslar AAP-molekylerna ihop sig med varandra (aggregeras) i ER. Det andra arbetet fortsatte på samma spår som det första men försökte klarlägga mer i detalj hur membrane-chaperoner hjälper till vid veckningen av AAP. Vi undersökte också hur cellen tar hand om AAPs som aggregerat. Resultaten visar att cellen märker de felveckade och aggregerade AAP proteinerna och för de ”tillbaka” från ER till cytosolen för att bryta ner de. AAP-aggregaten brytas ner på två olika vägar av en redan känd mekanism som kallas ER-associerad nedbrytning. En oväntad upptäckt var att om nedbrytningsmekanismen i ER slås ut i jästceller som saknar Shr3 - och därför inte borde kunna vecka AAPs på rätt sätt - återfår ändå dessa celler förmågan att tillverka funktionella AAPs. Detta antyder att mekanismerna för membranproteiners veckning och nedbrytning i hög grad hänger samman med varandra genom processer som ännu inte är helt kända. I det tredje arbetet använde vi genetiska metoder för att undersökte vilka mekanismer som reglerar nivåerna av AAP när det väl befinner sig vid plasmamembranet - till exempel som respons på skiftande förhållanden i cellens omgivning. Under arbetets gång hittade vi tre membranproteiner (Ssh4, Rcr2, och Rcr1) som påverkar sorteringen av AAPs i secretory pathway, alltså var i cellen AAPs transporteras till. Våra resultat visar att Ssh4, Rcr2 och Rcr1 påverkar sorteringen av AAPs under den senare delen av secretory pathway, som leder till ökad koncentrationen av AAPs till plasmamembranet. Om jag får avsluta med en praktisk liknelse är cellen ett företag med en fabrik där proteiner tillverkas. Enligt denna liknelse har vi i våra tre artiklar avslöjat flera företagshemligheter kring hur membranproteiner färdigmonteras, paketeras och skickas ut till kunderna. Vi har också avslöjat flera knep som företaget använder i sin exemplariska kvalitetskontroll, samt vilken budfirma som anlitats för att sköta returnering av defekta exemplar.

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List of original publications

List of original publications This thesis is based on the following papers, which will be referred to in the text by their roman numerals:

I.

Kota, J. and P.O. Ljungdahl. Specialized membrane-localized chaperones prevent aggregation of polytopic protein in the ER. J Cell Biol. 168: 79-88, 2005

II. Kota, J., C.F. Gilstring and P.O. Ljungdahl. Membrane chaperone Shr3 assists in folding amino acid permeases preventing precocious ERAD. In press J Cell Biol. 2007 Reproduced with permission. Copyright 2007 Rockefeller University Press. III. Kota, J., M. Melin-Larsson, P.O. Ljungdahl and H. Forsberg. Ssh4, Rcr2 and Rcr1 Affect Plasma Membrane Transporter Activity in Saccharomyces cerevisiae. In press Genetics. 2007 Reproduced with permission. Copyright 2007 Genetics Society of America.

Additional publication not discussed in this thesis: Nyman T., J. Kota and P.O. Ljungdahl. Ancillary proteins in membrane targeting of transporters. In Topics in Current Genetics – Molecular Mechanisms Controlling Transmembrane Transport. E. Boles and R. Krämer, editors. Springer-Verlag, Heidelberg. 207-234. 2004

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Abbreviations AAP CFTR E1 E2 E3 ER ERAD Gap1 MVB PCC PM TMS TRAM UPR

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amino acid permease cystic fibrosis transmembrane conductance regulator Ubiquitin activating enzyme Ubiquitin conjugating enzyme Ubiquitin ligase endoplasmic reticulum ER-associated degradation general amino acid permease Multivesicular body protein-conducting channel plasma membrane transmembrane segment translocating chain associating membrane protein unfolded protein response

Table of contents

Table of contents Abstract

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Populärvetenskaplig sammanfattning

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List of original publications

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Abbreviations

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1. Introduction

13

2. Biogenesis of polytopic membrane proteins

17 17 19 19 20 22

2.1 The translocon 2.2 Targeting and translocation of membrane proteins 2.3 Membrane protein topology 2.4 Integration of polytopic membrane proteins 2.5 Membrane protein folding – Chaperones

3. Quality control and ERAD 3.1 Quality control in the ER – UPR 3.2 ERAD/Proteasome pathway 3.3 Substrate recognition - Folding or degradation

4. Targeting and downregulation of PM proteins 4.1 Protein trafficking in the late secretory and endosomalvacuolar pathways 4.2 Amino acid permeases (AAPs) as model cargo 4.3 Sorting of AAPs in the endosomal/MVB pathway

25 25 26 29 31 31 32 33

5. History of Shr3

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6. Results and discussion

37 37 39 40 41

6.1 Shr3 – an ER membrane chaperone (Paper I and II) 6.2 Novel class of membrane chaperones (paper I) 6.3 Folding and degradation of AAPs (Paper II) 6.4 Ssh4, Rcr2 and Rcr1- regulating transporters at the PM (Paper III)

7. Future prospects

43

8. Acknowledgments

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9. References

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Introduction

1. Introduction All forms of life – even those that appear utterly exotic or absolutely alien – consist of cells. Despite life’s manifested diversity, compare a human and jellyfish cell and you will find that they are astonishingly similar. All cells are enclosed by a semipermeable surface membrane, called the plasma membrane (PM). The PM is comprised of lipids and a multitude of proteins. The PM has several essential functions, perhaps the most important being that it enables cells to maintain an internal environment favorable for growth that is shielded from the extracellular milieu. To accomplish this barrier function, the PM contains various integral membrane proteins that either act passively as channels, or actively as transporters pumping molecules across the membrane. Other integral PM proteins function to transmit information across the cell membranes, acting as receptors that react to particular substances in the environment. Thus, the PM enables the selective uptake of required nutrients into the cell, the ability to maintain proper ionic composition and to transmit environmentally derived signals into the cell. Prokaryotes and eukaryotes, the two main categories of cells, differ in their intracellular architecture. Eukaryotes, unlike prokaryotes, separate various cell processes into sub-cellular compartments, or organelles, that are themselves enclosed by lipid membranes similar to the PM. One example of such a specialized membrane compartment is the endoplasmic reticulum (ER). Most integral PM proteins are synthesized on ribosomes associated with the ER membrane, and are cotranslationally inserted into the ER membrane. Subsequent to their insertion, membrane proteins must fold properly to attain their functional native conformations. After achieving their native structure, newly synthesized proteins are transported from the ER to their final destination. Several quality control mechanisms operate in the ER to ensure that unfolded, misfolded or partially assembled proteins are selectively retained and degraded. The ER membrane constitutes the first step of the intracellular transport system called the secretory pathway (Fig 1). Protein movement between the compartments of the secretory pathway occurs via small transport vesicles that form and bud from donor compartments, and target to and fuse with acceptor compartments. The process of directing newly made membrane proteins to particular intracellular destinations is critical to the organization and function of eukaryotic cells.

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Figure 1: The yeast secretory pathway. 1. Protein translocation/integration into the ER, 2. ER protein processing and maturation, 3. ER to Golgi (G) transport, 4. Intra-Golgi transport, 5. Golgi to PM transport, 6-9. Transport in the late secretory pathway

This thesis documents studies aimed at understanding how PM proteins enter the secretory pathway, with focus on the translocation and folding of polytopic membrane proteins (proteins comprised of multiple transmembrane segments) in the ER. The yeast Saccharomyces cerevisiae provides an attractive model organism for this purpose since yeast cells possess a secretory pathway, which in all essential aspects is identical to that found in cells of multicelluar organisms, such as humans. Additionally, yeast is a simple, single-cell organism that is readily amenable to both biochemical and genetic manipulation. In these studies, Amino Acid Permeases (AAPs) have been selected as model proteins, not only to address how polytopic membrane proteins insert and fold in the ER membrane, but also to investigate how the functional expression of PM proteins is regulated in response to environmental signals. AAPs constitute a family of around 20 highly homologous transport proteins that contain twelve transmembrane segments. AAPs facilitate proton gradient driven transport of amino acids into cells. Newly synthesized AAPs, like other PM proteins, are initially translocated into the ER membrane. After attaining native structures they exit the ER. In subsequent stages of the secretory pathway AAPs are subject to environmentally induced posttranslational modifications, including phosphorylation and ubiquitylation, which affects their functional expression at the PM. 14

Introduction

In the following two sections I will review the current understanding of the initial stages of the secretory pathway in the ER with a focus on translocation and folding of polytopic membrane proteins, and quality control mechanisms. In the third section I will briefly describe the general mechanisms governing the stability of PM proteins, and how environmental signals influence events in the late secretory pathway to regulate the functional expression of PM transport systems. Finally, I present a summary of my experimental contributions in a Results and discussion section.

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Biogenesis of polytopic membrane proteins

2. Biogenesis of polytopic membrane proteins Roughly one third of all proteins coded in genomes contain hydrophobic sequences that suggest that they are associated with membranes (Wallin and von Heijne, 1998). These proteins can be classified into three main groups according to how intimately they associate with membranes. Integral membrane proteins contain transmembrane segments (TMS) that completely transverse the membrane. Membrane-anchored proteins are partially embedded but do not transverse the membrane. The members of the third group, the peripheral membrane proteins, are only associated with membrane. These three classes can be readily distinguished based on the type of reagents required to extract them from the membrane. Integral membrane proteins, which include proteins such as transporters and ion channels, most often have a polytopic structure, i.e., they are comprised of multiple TMS. The biogenesis of polytopic membrane proteins remains poorly understood. Information from cross-linking studies examining translocation and integration of membrane proteins, the recently determined crystal structure of the proteinconducting channel, and the growing number of solved crystal structures of membrane proteins have provided important clues. Yet, these advances have only led to more detailed questions regarding the mechanisms enabling polytopic membrane proteins to properly integrate and fold in the membrane of ER.

2.1 The translocon As previously noted, the ER is the major site for translation and translocation of newly synthesized proteins in eukaryotic cells. During translocation, secretory proteins are transported across the ER membrane and enter the lumen of the ER, whereas the hydrophobic TMS of integral membrane proteins become inserted in the ER membrane. In either case, translocation proceeds through an aqueous channel in the ER membrane (Crowley et al., 1993; Simon and Blobel, 1991). This channel, i.e., the protein-conducting channel (PCC), is composed of a specific set of membrane proteins, termed the Sec61 complex in eukaryotes and the SecY complex in bacteria (Johnson and van Waes, 1999; Osborne et al., 2005). The PCC, often referred to as the translocon, is a highly conserved, heterotrimeric complex consisting of the Sec61α, Sec61β and Sec61γ subunits in mammals; the corresponding homologues are Sec61p, Sbh1p and Sss1p in yeast, and SecY, SecG and SecE in bacteria (Osborne et al., 2005). The crystal structure of the heterotrimeric SecY complex in Methanococcus jannaschii has recently been determined (van den Berg et al., 2004). This major accomplishment has laid down important pieces in the PCC structure-function puzzle. Contrary to previous models, the PCC is now thought to be monomeric, containing 17

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one copy of each of the three subunits (a single copy of the SecY complex) (Osborne et al., 2005; van den Berg et al., 2004). This new structure shows that the aqueous pore is located in the center of the complex formed by a single molecule of SecY (Sec61α in mammals), the largest subunit of the complex (van den Berg et al., 2004). This notion is supported by studies demonstrating cross-linking between a translocation substrate and SecY. The data are consistent with the movement of the polypeptide substrate through the center of the SecY molecule (Cannon et al., 2005). The other subunits, SecG and SecE, are located at the periphery of the pore complex. The location of SecG and SecE and the N- and C-terminal domains of SecY enable one side of the PCC to open laterally towards the lipid phase. The ability to open allows TMS of membrane proteins to exit the PCC and partition into the lipid bilayer (Osborne et al., 2005; van den Berg et al., 2004). Several additional components required for the efficient translocation/integration of membrane proteins have been found associated with the translocon (Johnson and van Waes, 1999). These include oligosaccharyltransferase, signal peptidase, TRAP (translocon-associated protein) and TRAM (translocating chain associating membrane protein). It has been suggested that TRAM, a multi-spanning membrane protein, functions as a chaperone during the translocation and integration process (see below). To maintain the integrity of the ER membrane and prevent leakage of small molecules, e.g., ions, from the ER into the cytosol, the PCC must act similarly to a sluice gate, or lock. As proteins are being translocated across, or become integrated into the membrane, the translocon must alternatively maintain a seal at the cytoplasmic or luminal face of the ER membrane. The recent structure suggests that a small helical segment, referred to as the plug, blocks the pore of the channel (van den Berg et al., 2004). It has been postulated that when the channel opens, the plug moves towards the inner pore wall of the complex to allow passage of a translocating polypeptide. However, recent studies in yeast question the role of the plug; a mutant form of Sec61 lacking the plug segment is functional (Junne et al., 2006). In an alternative model, the seal for small molecules on the cytosolic face is provided by the binding of the ribosome to the translocon, and on the lumenal side by the binding of BiP (the luminal Hsp70 chaperone) (Haigh and Johnson, 2002; Hamman et al., 1998). There is an active debate regarding these models, and the most recent cryo-EM structure of the PCC bound to a translating ribosome (Mitra et al., 2005) has raised additional and new questions. The cryo-EM structure suggests that two PCC functionally associate with their lateral openings facing each other. Clearly, additional experimental advances will be required to obtain a satisfactory understanding of the actual structure and function of the PCC.

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Biogenesis of polytopic membrane proteins

2.2 Targeting and translocation of membrane proteins Translocation through the Sec61 PCC occurs either in a co-translational or posttranslational manner (Rapoport et al., 1996). In the post-translational pathway, the polypeptide is completely synthesized and released from the ribosome in the cytoplasm before being translocated. In the co-translational pathway, proteins are translated by the ribosome at the same time as they are translocated through the PCC. Most membrane proteins in eukaryotic cells are co-translationally inserted into the ER membrane. Proteins are targeted to the translocon by signal sequences, comprised of stretches of hydrophobic amino acids (Blobel and Dobberstein, 1975; von Heijne, 1990). Two key components in this process are the signal-recognition particle (SRP) and the SRP receptor, an integral component of the ER membrane (Walter and Johnson, 1994). Targeting to the ER starts as soon as signal sequence from the growing nascent polypeptide chain emerges from a translating ribosome and is recognized by the SRP. Binding of the SRP causes a pause in the translation. Next, the ribosome-nascent chain-SRP complex is targeted to the ER by binding to the membrane bound SRP receptor in a GTP-dependent manner (Walter and Johnson, 1994). This latter interaction results in the release of the SRP from the nascent chain-ribosome complex, and concomitantly, the ribosome is transferred to the translocon. The release of the SRP allows translation to continue, and the newly synthesized polypeptide is translocated across or integrated into the ER membrane.

2.3 Membrane protein topology The orientation or topology of an integral membrane protein in the ER membrane depends on topogenic sequences (signal sequences, stop-transfer sequences and signal-anchor sequences) within the hydrophobic segments (Blobel, 1980; von Heijne and Gavel, 1988). Single-spanning membrane proteins can be classified into three types based on the orientation of the transmembrane segment (TMS) and whether the signal sequence is cleaved off or retained as a TMS (Spiess, 1995). Type I proteins have a cleavable N-terminal signal sequence that initiates insertion and a subsequent hydrophobic stop-transfer sequence that anchors the protein in the membrane with an Nlum/Ccyt topology. Membrane insertion of Type II and type III is directed by an uncleaved signal sequence, the signal-anchor sequence, located at the N-terminus that is responsible for both insertion and anchoring of the protein. The signal-anchor sequence in type II translocates the C-terminus into the lumen generating an Ncyt/Clum topology. In contrast, type III proteins insert with an Nlum/Ccyt topology. Although hydrophobicity is of great importance, it is not the only topogenic signal determining the topology of a membrane protein. An important feature that affects the orientation of a TMS is the distribution of charged residues flanking a 19

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TMS. Positive charges, arginine and lysines, usually remain on the cytosolic side of the membrane and thus affect the topology of the protein, referred to as the positiveinside rule (Gafvelin et al., 1997; von Heijne, 1986). For polytopic membrane proteins that span the membrane multiple times, the first hydrophobic segment, which is either a cleavable signal sequence or the first TMS, normally defines its own orientation and dictates the orientation of subsequent TMS. However, the integration of polytopic proteins is more complex since they have multiple topogenic signals. The topogenic information in a downstream TMS may affect the topology of upstream TMS. Also other factors such as the positive-inside rule, length of the TMS or the connecting loop, and interaction between the TMS, influence the overall topology of polytopic membrane proteins (Turner, 2003; von Heijne, 2006; Wahlberg and Spiess, 1997)

Figure 2. Topology of single membrane spanning proteins with different topogenic sequences.

2.4 Integration of polytopic membrane proteins As mentioned previously, a clear majority of polytopic membrane proteins are translocated and integrated into the ER co-translationally. During the synthesis of a membrane protein, the PCC facilitates the insertion of TMS into the ER membrane. As a TMS arrives inside the PCC, it is recognized and released into the lipid bilayer, a process that occurs laterally through an opening in the channel wall (Osborne et al., 2005; van den Berg et al., 2004). During the synthesis of polytopic membrane proteins, the ‘lateral gate’ of the channel must open and close several times in order to allow the partitioning of each TMS into the membrane. Recent structural studies regarding the PCC show that the gate may undergo ‘breathing,’ i.e., continuous opening and closing (Osborne et al., 2005). TMSs differ widely in sequence and it is therefore unlikely that they are specifically recognized based on protein-protein interactions alone. Rather, recent work has revealed the critical importance of protein20

Biogenesis of polytopic membrane proteins

lipid interactions during TMS recognition and membrane insertion (Hessa et al., 2005). To fully understand the integration of polytopic membrane proteins it is necessary to know at what moment during their synthesis that the TMS partition away from the aqueous interior of the channel into the lipid phase. Experiments attempting to analyze the environment surrounding the nascent TMS-chain of membrane proteins during translocation and integration have resulted in many models and proposed mechanisms. At the extremes are the “en masse” and the “sequential” models (Lecomte et al., 2003). According to the “en masse” model, all TMSs remain in the ER translocon until protein synthesis has been completed, after which they are released together into the lipid phase (Borel and Simon, 1996). In the “sequential” model, TMSs are sequentially released into the lipid phase during translation. The “en masse” model is not consistent with the recently determined crystal structure of the PCC, which indicates that the channel is too small to accommodate multiple TMSs; at most two TMSs can be present in the channel (Osborne et al., 2005; van den Berg et al., 2004). Data from numerous cross-linking studies strongly favor the “sequential” model (Heinrich et al., 2000; Ismail et al., 2006; Meacock et al., 2002; Mothes et al., 1997; Sadlish et al., 2005). Thus, based on the current understanding of polytopic membrane protein insertion it is likely that the TMSs exit the channel one by one or in pairs. The rate at which individual TMSs integrate into the lipid bilayer differs between proteins. Hydrophobic TMS partition rapidly into the membrane. Less hydrophobic TMS containing charged or polar residues, partition into the membrane less readily and are retained in close proximity to the translocon, or translocon associated proteins, i.e., TRAMs, which may chaperone their partitioning into the membrane (Heinrich et al., 2000). Also the partitioning and integration of a less hydrophobic TMS from the channel can be facilitated by interaction with the previously integrated TMS (Heinrich and Rapoport, 2003). Individual TMSs in a polypeptide also exhibit different requirements for their integration into the lipid phase. TMSs in some proteins are observed to move through specific molecular environments adjacent to the channel prior to entering the lipid bilayer (Ismail et al., 2006; Meacock et al., 2002; Sadlish et al., 2005). The requirements of a TMS to be released into the lipid bilayer can vary within the same polypeptide (Ismail et al., 2006). In other crosslinking experiments, it was found that TMSs in some proteins remain at the PCC channel and/or exhibit prolonged associations with translocon-associated proteins until translation terminates, at which point the TMS release into the membrane (Do et al., 1996; McCormick et al., 2003). In summary, the integration of TMS into the lipid bilayer is a complex process influenced by the lipid-protein interactions of the partitioning TMS, the translocon, and the presence of accessory proteins. The variation of the results obtained in different cross-linking approaches suggest that different membrane proteins might rely on alternative ways to integrate into the lipid bilayer. 21

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2.5 Membrane protein folding – Chaperones One of the key questions about the biogenesis of polytopic membrane proteins is how and in what order individual TMS assemble and fold following their integration into the lipid phase of the membrane. In comparison to soluble proteins, the starting point of membrane protein folding is likely to be more constrained because much of the secondary structure and topology is defined by the process of TMS insertion. Once in the membrane, the TMSs must correctly orient themselves to enable the proper folding of hydrophilic regions – a process that likely depends heavily on interactions between specific TMSs.

Figure 3: The two stages of membrane protein folding according to Popot and Engelman. Stage 1: The insertion of TMS into the lipid bilayer. Stage 2: Folding and assembly of the inserted TMSs

To describe the folding of integral membrane proteins, a two stage model was originally proposed by Popet and Engelmann (Fig. 3) (Engelman et al., 2003; Popot and Engelman, 1990). In the first stage of this process, membrane insertion of independently stable helices is achieved. In the second stage, the helices interact with one another to form the tertiary structure of the polypeptide. This second stage involves the assembly and reorientation of TMSs in order to attain native structures. However, this process raises questions regarding how TMSs of partially integrated polytopic membrane proteins avoid inappropriate interactions and/or misassemble prior to the complete translocation of remaining TMSs present in full-length proteins. For example, the crystal structures of bacterial PM transporters LacY and GlpT, each comprised of 12 TMSs, clearly show that interactions between TMS within the lipid phase of the membrane are not limited to immediately flanking TMS (Abramson et al., 2003; Huang et al., 2003). Thus, these proteins cannot complete folding until all TMSs are inserted in the membrane. Consequently, in order to fold properly, Nterminally localized TMSs that partition into the membrane of partially translated proteins must await the insertion of C-terminal TMS. To avoid non-productive 22

Biogenesis of polytopic membrane proteins

interactions with flanking TMS, or with other ER components prior to folding, it has been suggested that membrane chaperones exist to prevent such interactions from occurring, and to facilitate the folding and/or assembly of hydrophobic TMS (Alder and Johnson, 2004; Lecomte et al., 2003; Rapoport et al., 2004). Recent results regarding the membrane insertion of aquaporin-4 suggest that the Sec61 translocon possess intrinsic chaperone-like activity to facilitate correct folding of some polytopic membrane proteins (Sadlish et al., 2005). After individually passing through a single entry site in Sec61, several TMSs of aquaporin-4 were found to interact with secondary peripheral sites on Sec61. These results suggest that the translocon may transiently retain certain TMS to facilitate early folding events and to control their release into the membrane. TRAM, the translocon associated protein, has been proposed to function as a membrane chaperone in mammalian cells (Gorlich et al., 1992; Rapoport et al., 2004). TRAM is a membrane protein with six TMS that is found adjacent to the Sec61 channel. During insertion and integration of membrane proteins several TMS have been shown to associate with TRAM (Do et al., 1996; High et al., 1993; Mothes et al., 1998). In the case of TMS with charged or polar residues, TRAM might function to shield and chaperone their integration, and participate in guiding folding events (Heinrich et al., 2000). TRAM may also organize the assembly of several TMS before they release into the membrane as partially assembled proteins (Alder and Johnson, 2004; Do et al., 1996) In bacteria the membrane protein YidC, which has a similar topology to TRAM, is required for integration and folding of some membrane proteins (Dalbey and Kuhn, 2004). The direct involvement of YidC in the folding process has been demonstrated in studies analyzing the folding of the lactose permease, LacY. Using conformation dependent antibodies, it was shown that YidC is important for the inserted LacY to attain its correct tertiary structure (Nagamori et al., 2004). Direct evidence of membrane chaperones in eukaryotes was found in our research on Shr3 (Paper I and Paper II). Our results demonstrate that Shr3 prevents aggregation and facilitates the proper folding of a group of polytopic membrane proteins, the amino acid permeases in yeast. The existence of three other substrate specific membrane chaperones was demonstrated in work documented in Paper I. These findings are described in detail in the Results and discussion section of this thesis. Neither Shr3 nor YidC are required for the membrane integration and topology of their cognate substrates, apparently the translocon is capable of correctly interpreting topogenic signals in the absence of these chaperones (Gilstring and Ljungdahl, 2000; Nagamori et al., 2004). Nevertheless, their polytopic proteins substrates cannot assemble correctly to obtain native conformations without their assistance. This is consistent with the idea that membrane protein folding occurs essentially in two discrete stages, i.e., membrane insertion and folding (Bowie, 2005; Engelman et al., 2003; Popot and Engelman, 1990). 23

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In addition to the factors mentioned above, lipids also play a major role in membrane protein biogenesis. For instance, the lipid composition of the bilayer has been shown to affect the topology of membrane proteins, assembly of oligomeric membrane proteins, and its stability (Schneiter and Toulmay, 2007; van Dalen and de Kruijff, 2004). In conclusion, the biosynthesis of a functional membrane protein depends on a series of interactions between the nascent chain, ribosome, translocon, lipids, and several accessory ER proteins. This process is sensitive to the individual requirements of different membrane proteins for correct integration and assembly. Our current understanding is based on studies of only a few substrates and is still fragmented, which allows for several alternative interpretations. Thus, many structural and mechanistic aspects remain to be discovered before a more rigorous model can be proposed.

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Quality control and ERAD

3. Quality control and ERAD The microenvironment of the ER is optimized for proper folding and delivery of newly synthesized proteins to the secretory pathway. Despite this, not all proteins fold properly, and sometimes misfolding occurs at alarming rates. Several human disorders have been attributed to inefficient protein folding (Kopito, 1999; Zhao and Ackerman, 2006). All polypeptides that pass through the ER are subject to quality control, including soluble and membrane proteins (Ellgaard and Helenius, 2003). Quality control mechanisms discriminate between properly folded and terminally misfolded states, and monitor the assembly of multi-subunit proteins. Protein folding and quality control are intimately linked processes, both rely on chaperones to assist and monitor the folding process. Proteins that fail quality control are generally retained in the ER and ultimately targeted for degradation. Two distinct quality control mechanisms respond to the presence of misfolded proteins in the ER. The first is an ER-dedicated stress response termed the unfolded protein response (UPR). The UPR is sensitive to disturbances in the efficiency of protein folding, and when activated, induces changes in the pattern of gene expression in a manner that increases the folding capacity of the ER (Kostova and Wolf, 2003). The second, termed ER-associated degradation (ERAD) specifically recognizes misfolded proteins and retrotranslocates them across the ER membrane into the cytosol, where they are degraded by the ubiquitin proteasome degradation machinery (Meusser et al., 2005).

3.1 Quality control in the ER - UPR In yeast, the essential components of the UPR are: the Hsp70 molecular chaperone Kar2 (Bip in metazoans); the ER transmembrane protein kinase Ire1; and the transcription factor Hac1 (XBP1 in metazoans). Kar2 resides in the ER lumen where it plays an essential role during the translocation process, protein folding and assembly (Johnson and van Waes, 1999; Kostova and Wolf, 2003). Kar2 recognizes and binds with high-affinity to hydrophobic sequence motifs that are transiently exposed during these processes. Importantly, Kar2 also binds to the lumenal domain of Ire1, however with less avidity. Kar2 binding to Ire1 prevents homodimerization. Ire1 dimerization is a requisite for kinase and HAC1 mRNA splicing activity. In the absence of stress, the levels of free Kar2 in the ER are sufficiently high so that Ire1 dimerization is prevented. However under conditions of ER stress, Kar2 preferentially binds to misfolded proteins enabling Ire1 to dimerize and become active. The active Ire1 dimers promote HAC1 mRNA splicing, resulting in the synthesis of the Hac1 transcription factor. This factor enters the nucleus where it 25

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induces transcription of UPR target genes encoding proteins required for folding, quality control, and ERAD (Bukau et al., 2006; Hirsch et al., 2006). During conditions of stress, the UPR also enables cells to selectively reduce the new synthesis of proteins, which allows cells to decrease the load on the ER folding machinery. Recent studies on ER stressed cells show that cells can degrade discrete subsets of mRNAs to reduce the volume of newly synthesized proteins entering the ER. In metazoans, Ire1 mediates this selective degradation of mRNAs (Hollien and Weissman, 2006). Additionally, under conditions of stress, cells appear to be able to initiate the degradation of newly synthesized polypeptides that normally would target to the ER. Intriguingly, this degradation occurs prior to their entry into the ER (Kang et al., 2006; Oyadomari et al., 2006). This latter mechanism, designated preemptive quality control (pQC), recognizes information encoded within the signal sequence of proteins and prevents translocation into the ER. The untranslocated polypeptide is presumably degraded by the proteasome. The UPR target gene P58IPK is suggested to be involved in pQC (Kang et al., 2006). P58IPK is a cytosolic cochaperone that associates with the translocon, and was recently shown to be a key player in the cotranslocational ER degradation of stalled or compromised translocating polypeptides during ER stress (Oyadomari et al., 2006).

3.2 ERAD/Proteasome pathway Proteins that fail to attain native structures are generally degraded by ERAD, which is considered to be an essential component of quality control mechanisms (Meusser et al., 2005; Romisch, 2005). Both luminal and integral membrane proteins are subject to ERAD. ERAD is dependent on the cytoplasmic ubiquitin-proteasome pathway (Hiller et al., 1996; Sommer and Wolf, 1997). Consequently, luminal and integral membrane proteins selected for degradation must be transported back to the cytosol, a process known as retrotranslocation or protein dislocation, after which they become degraded by the 26S proteasome. The identity of the dislocation channel is still a matter of debate. Conclusive evidence is lacking, however, the Sec61 translocon is currently considered to be the best candidate to function as the dislocation channel. Experimental evidence from mammalian and yeast cells shows that Sec61 is required for proteasome degradation of misfolded proteins (Plemper et al., 1997; Romisch, 2005; Wiertz et al., 1996). Recently, it has been suggested that the membrane protein Derlin-1 (Der1 in yeast) forms the dislocation channel (Lilley and Ploegh, 2004; Ye et al., 2004). However, Derlin-1/Der1 are required for the degradation of only a subset of ERAD substrates, thus if they do indeed function to form a dislocation channel, other channel forming proteins must exist. Other candidates that have been implicated in forming the channel are the integral membrane E3-ubiquitin ligases Hrd1/Der3 and Doa10 (Meusser et al., 2005). 26

Quality control and ERAD

As ERAD substrates are retrotranslocated through the ER membrane, they become ubiquitylated (Jarosch et al., 2003). Ubiquitin, a 76 amino acid polypeptide expressed in all eukaryotic cells, functions as a tag for targeting proteins to the proteasome for degradation. The conjugation of ubiquitin to polypeptides takes place in the cytosol and requires the sequential activities of ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3) (Fig. 4). This ATP-dependent activation cascade leads to the covalent binding of ubiquitin onto lysine residues of target proteins. Proteins can be polyubiquitylated by the successive addition of ubiquitin molecules to lysine residues of previously attached ubiquitin.

Figure 4: The ubiquitin-proteasome system The ubiquitin-activating enzyme (E1) hydrolyses ATP and forms a high energy thioester linkage between its active site and the carboxyl terminus of ubiquitin. Next, the activated ubiquitin is transferred to a member of the ubiquitin-conjugated enzymes (E2). Substrate recognition is then mediated by a member of the ubiquitin protein ligase family (E3) which usually binds directly to the substrate. The E2 together with E3 then transfers ubiquitin to lysine residues of the substrate. A polyubiquitin chain is formed by successive addition of ubiquitin molecules to the ubiquitin molecules previously attached to the substrate. The polyubiquitylated substrate is recognized by the 26S proteasome and degraded.

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Ubiquitylation of ERAD substrates in yeast is mediated by the ER membrane localized E3 ligases Hrd1/Der3 and Doa10. Both of these E3 ligases are polytopic membrane proteins with cytosolic RING finger domain (Bays et al., 2001; Deak and Wolf, 2001; Meusser et al., 2005; Swanson et al., 2001). The Hrd1/Der3 ligase primarily functions with the E2 enzyme Ubc7, which is anchored to the ER membrane via Cue1 (Biederer et al., 1997). The Hrd1/Der3 ligase ubiquitylates a wide range of ERAD substrates (Meusser et al., 2005). Doa10 also functions with Ubc7, but can also function with another E2 enzyme, Ubc6 (Sommer and Jentsch, 1993). Doa10 ligase is thought to primarily ubiquitylate integral membrane proteins with large cytosolic domains (Meusser et al., 2005). The cytosolic AAA-ATPase Cdc48 has a crucial role in the dislocation of proteins from the ER (Jarosch et al., 2003; Ye et al., 2001). Cdc48 forms a complex with Ufd1 and Npl4, two factors that bind ubiquitylated proteins. The Cdc48 complex recognizes and promotes the dislocation of polyubiquitylated ERAD substrates. Mutations affecting the genes encoding the Cdc48 complex significantly impair the rate of ERAD substrate degradation; mutant cells accumulate polyubiquitylated ERAD substrates in association with the ER membranes. Thus, polyubiquitylation, in addition to serving as a targeting and recognition signal for proteasomal degradation, is important to ensure proper dislocation of misfolded proteins (Meusser et al., 2005). Considering the diversity of proteins that enter and fold in the ER, one might imagine that different surveillance mechanisms are required to identify the full spectrum of misfolded proteins. Recent studies examining ERAD substrates with defined folded and misfolded domains placed in different topological contexts have provided insights into this matter (Huyer et al., 2004; Ismail and Ng, 2006; Ravid et al., 2006; Vashist and Ng, 2004). These studies revealed that multiple surveillance pathways do exist (Vashist and Ng, 2004). Two different surveillance mechanisms that were proposed are the ERAD-L and ERAD-C pathways. Additional studies have revealed that these pathways are comprised of multimeric protein complexes organized around the two membrane localized E3 ubiquitin ligases, i.e., Doa10 and Hrd1/Der3 (Carvalho et al., 2006; Denic et al., 2006). Also, a new pathway was proposed in these studies, designated ERAD-M. Although notable exceptions exist, soluble secretory proteins and membrane proteins with misfolded lumenal domains are substrates of the Hrd1/Der3 dependent ERAD-L pathway. Membrane proteins with misfolded TMS are also targeted for degradation in a Hrd1/Der3 dependent pathway, but through the ERAD-M pathway (Carvalho et al., 2006). Consistent with the ability to handle an altered spectrum of folding perturbations, the composition of the ERAD-M Hrd1/Der3 complex differs from that of the ERAD-L complex. Finally, misfolded cytoplasmic domains of membrane proteins are targeted for degradation by the Doa10 dependent ERAD-C pathway. Thus, the topological location of misfolding regions within ERAD

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Quality control and ERAD

substrates determines which ERAD pathway targets it for degradation (Ahner and Brodsky, 2004; Huyer et al., 2004; Vashist and Ng, 2004).

3.3 Substrate recognition - Folding or degradation The initial step in quality control is to sense the presence of a misfolded proteins, and importantly, to differentiate them from folding intermediates and fully folded proteins. How cells distinguish between the vast arrays of possible protein conformations and place them into one of these three broad categories is unclear. Current data indicate that both lumenal and cytosolic chaperones play a significant role in this process. In addition, proteins involved in the glycosylation process are also important in determining the folding state of ER proteins. A common modification of proteins that enter in the ER is the covalent attachment of N-linked glycans (Helenius and Aebi, 2004). This well-characterized process provides important information regarding the folding status of glycoproteins and prevents both premature exit out of the ER and targeting of folding intermediates for degradation by ERAD (Helenius and Aebi, 2004). However, not all proteins are glycosylated. The ER luminal Hsp70 chaperone Kar2 (Bip) has been shown to have a critical role in determining the folding state of non-glycosylated proteins (Jarosch et al., 2003; Romisch, 2005). Also, calnexin, a chaperone that participates in the glycosylation process, appears to contribute to the quality control of some non-glycosylated membrane proteins (Swanton et al., 2003). There is extensive evidence demonstrating that cytosolic Hsp70 and Hsp90 chaperones are important in monitoring the folding status of membrane proteins with cytosolic domains (Romisch, 2005). With respect to folding of native polytopic membrane proteins in vivo, perhaps the most extensively studied protein is the PM Cl- ion channel of epithelial cells, designated CFTR. The ∆F508 mutation in CFTR results in the most common form of cystic fibrosis (Kopito, 1999). The ∆F508 mutation affects the folding efficiency of CFTR and the mutant protein accumulates in the ER and is targeted for degradation by ERAD (Kopito, 1999; Younger et al., 2006). Based on several criteria, the CFTR∆F508 mutant protein is not terminally misfolded, but rather appears to be in a kinetically trapped folding intermediate. In cells grown in the presence of chemical chaperones, e.g., glycerol, or at decreased temperatures (30 oC), CFTR∆F508 is able to achieve a folded conformation, exit the ER, and restore chloride transport (Brown et al., 1996). Recently it was shown that Hsp90, together with cochaperones FKBP8, p23 and Aha1, participate in maintaining the kinetically trapped state, and contribute to the efficient targeting of the folding intermediate for degradation (Wang et al., 2006). Remarkably, reducing Aha1 levels enabled CFTR∆F508 to exit the kinetically trapped state, fold and become functionally expressed at the PM. Clearly, lumenal and cytosolic chaperones participate in monitoring the folding of integral membrane

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proteins with exposed hydrophilic domains, however, it is not known whether these chaperones participate in the quality control of predominantly hydrophobic proteins. It is known that cells possess the means to monitor physical properties of TMSs that are embedded in the membrane. However the mechanisms remain undefined. The introduction of charged residues into TMS is known to be highly destabilizing, and modified proteins are efficiently targeted for degradation by ERAD (Bonifacino et al., 1991). This latter finding is very important for understanding the assembly of multi-subunit protein complexes, and the folding of polytopic proteins with TMS possessing charged amino acid residues. Unassembled TMS of α-subunits of the Tcell antigen receptor are retained and rapidly degraded by ERAD (Bonifacino et al., 1990). It was shown that the retention and degradation is due to two basic amino acid residues in the α-subunits of T-cell receptor. In yeast, the ER membrane proteins Nsg1 and Nsg2 associate with Hmg2 (HMGCoA reductase). Nsg1 and Nsg2 appear to have chaperone-like functions that affect the stability of Hmg2. When sterol levels are low, these chaperones interact with the sterol-sensing transmembrane domain of Hmg2. The interactions promote Hmg2 folding and prevent the targeting of Hmg2 for degradation by Hrd1/Der3 dependent ERAD (Flury et al., 2005). Similarly, the ER membrane chaperone Shr3 facilitates folding of AAPs, and also prevents premature degradation of partially folded AAPs (Paper II). In the absence of Shr3, AAPs accumulate as aggregates in the ER membrane and are subsequently targeted to ERAD. Mutations that impair both Hrd1/Der3 and Doa10 dependent ERAD pathways enhance functional expression of AAPs at the PM, even in the absence of Shr3. These findings highlight the intimate link between folding and degradation.

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Targeting and down-regulation of PM proteins

4. Targeting and down-regulation of PM proteins PM proteins that have been correctly processed in the ER are transported to the cell surface via the secretory pathway. The capacity of cells to control and remodel their repertoire of surface proteins in response to changes in the environment is essential for adaptation and survival. The presence of nutrients and other molecules in the extra-cellular milieu greatly influence the targeting and turnover of PM proteins. In addition to transcriptional regulation, the functional expression of PM proteins is controlled at the level of membrane trafficking in the late secretory pathway. The ability to control the trafficking of PM proteins enables cells to more rapidly respond to changing conditions. Several control mechanisms have been described that affect the membrane trafficking of metabolite transporters. Transporters can be down-regulated and eliminated from the PM by endocytosis. Subsequent sorting events within the endosomal pathway determine whether transport proteins are targeted to the vacuole for degradation, or rerouted back to the PM. The trans-Golgi compartment also provides a major sorting point in the secretory pathway where newly synthesized PM proteins are sorted and targeted either to the cell surface or to endosomal and vacuolar compartments. Finally, some transporters are stored in intracellular compartments. In response to environmental signals these transporters can be quickly mobilized to the PM. Signal mediated secretion enables cells to rapidly increase transport activity.

4.1 Protein trafficking in the late secretory and endosomal-vacuolar pathways The late secretory pathway in yeast is schematically presented in Figure 5. Newly synthesized PM proteins are generally targeted to the cell surface from the transGolgi compartment (Step 1). However, under certain conditions, proteins may instead be targeted to the endosomal membrane system (Step 2). This step represents one of the entry points of the well-studied En/MVB (endosomal/multivesicular body) pathway (Bryant and Stevens, 1998; Katzmann et al., 2002). The downregulation of PM proteins involves their endocytic removal from the cell surface (Step3). After endocytosis, transporters are moved by vesicular traffic through endocytic intermediates known as early and late endosomes. In late endosomal compartments, protein cargo destined for vacuolar degradation is sorted into membrane regions that invaginate, leading to the formation of lumenal vesicles (Bryant and Stevens, 1998; Katzmann et al., 2002). The resulting vesicular compartments, called multi-vesicular bodies (MVBs), fuse with vacuoles (Step 4). Consequently, the lumenal vesicles including their cargo and lipids are degraded. Proteins that reside in the limiting membrane of the MVBs end up in the vacuolar membrane. Membrane proteins, 31

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internalized from the cell surface and targeted to the endosomal pathway can also be sorted and routed to other cellular compartments and thereby escape vacuole/lysosome degradation (Lemmon and Traub, 2000). Sorting and trafficking in the endosomal-MVB pathway enables storage and recycling of proteins that may have been miss-targeted to the vacuole for degradation.

Figure 5: Protein trafficking in the late secretory pathway 1. Trans-Golgi to PM transport. 2. Trafficking between trans-Golgi and endosomes/MVB pathway 3. Trafficking between PM and endosomes/MVB pathway. 4. vacuolar targeting for degradation

The mechanisms operating in the late secretory pathway affecting the functional expression of AAPs at the PM have been extensively studied in the yeast S . cerevisiae. These studies have provided important and generally applicable knowledge regarding the multiple trafficking routes that together comprise the late secretory and endosomal-vacuolar pathways.

4.2 Amino acid permeases (AAPs) as model cargo Amino acids are transported across the plasma membrane by high- and low-affinity AAPs (Nelissen et al., 1997; Regenberg et al., 1999). The AAPs constitute a family of about twenty homologous transporter proteins with twelve transmembrane segments (Gilstring and Ljungdahl, 2000). AAPs belong to the secondary transporter

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Targeting and down-regulation of PM proteins

APC (amino acid/polyamine/organocation) super family of proteins, examples of which are found in bacteria, archaea, fungi, plants and animals (Saier Jr., 2000). The individual members of the AAP family exhibit unique substrate affinities, however some permeases transport an overlapping spectrum of amino acids. (Andréasson et al., 2004; Iraqui et al., 1999; Regenberg et al., 1999). Ssy1 is a unique member of the AAP family with a large N-terminal extension. Instead of functioning as an AAP, Ssy1 is the core receptor component of the PM localized SPS-sensor of extra-cellular amino acids (Forsberg and Ljungdahl, 2001). In response to external amino acids, the SPS-sensor initiates signals that induce the expression of several AAP genes (Andréasson and Ljungdahl, 2002; de Boer et al., 2000; Nielsen et al., 2001). Consequently, via the SPS-sensor, amino acids induce their own uptake. Several AAPs are subject to nitrogen regulation (Magasanik and Kaiser, 2002). Nitrogen regulation prevents the utilization of non-preferred nitrogen sources (i.e., proline, allantoin) in the presence of preferred ones (i.e., ammonia, glutamine) and affects the expression of AAP genes, as well as sorting of AAP molecules in the late secretory pathway.

4.3 Sorting of AAPs in the endosomal/MVB pathway Endocytosis, internalization of PM proteins into membrane bound vesicles, plays a key role in physiological responses. Down-regulation of amino acid and other transport systems include their endocytic removal from the plasma membrane and subsequent degradation in the vacuole (Dupre et al., 2004; Haguenauer-Tsapis and André, 2004; Hicke, 1999). The endocytosis of most transporters requires Rsp5 dependent ubiquitylation, and in many cases the ubiquitylation step is preceded by a phosphorylation event (Feng and Davis, 2000; Hicke et al., 1998; Kelm et al., 2004; Marchal et al., 1998). Ubiquitin serves also as a sorting signal for proteins to enter the rather complex MVB-pathway (Katzmann et al., 2004). Studies during the recent years have defined three protein complexes (ESCRT) required for transport in the MVB sorting (Hurley and Emr, 2006). The ESCRT I, -II, and –III complexes recognize ubiquitylated proteins, sort and deubiquitylate them prior to their entry into MVB vesicles. Mutations in genes encoding class E vacuolar protein sorting (Vps) proteins prevent the formation of MVBs, which leads to a malformed late endosomal compartment know as the class E compartment (Katzmann et al., 2002). Cargo proteins that normally target to the vacuole accumulate in the class E compartment. Several studies have shown that mutations in ESCRT complexes leads to the redirection of cargo/transporters from the class E compartment back to the PM (Bugnicourt et al., 2004; Forsberg et al., 2001; Nikko et al., 2003; Rubio-Texeira and Kaiser, 2006). These results suggest that recycling pathways exist that enable cells to reuse PM

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proteins. However, the mechanisms underlying sorting and recycling in the endosomal-MVB pathway are not well characterized. The trafficking of the general amino acid permease (Gap1) has been extensively studied. Gap1 and other yeast PM proteins have been found in compartmentalized stores (Beck et al., 1999; Helliwell et al., 2001). Gap1 appears to cycle between the trans-Golgi and endosomal compartments (Helliwell et al., 2001). The routing of Gap1 is dependent on ubiquitylation (Helliwell et al., 2001). Upon Rsp5 dependent polyubiquitylation, presumably at the trans-Golgi, Gap1 is sorted to the vacuole via the En/MVB pathway without passing through the PM. Gap1 also recycles from the En/MVB pathway to the PM (Rubio-Texeira and Kaiser, 2006). The abundance of amino acids appears to regulate this cycling process. A recent report from Gao and Kaiser identified GSE (Gap1 sorting in endosomes) proteins that selectively bind and sort Gap1 molecules out of late endosomes and restrict their entrance into the MVB pathway for vacuolar degradation (Gao and Kaiser, 2006). Mutations in the GSE genes block Gap1 recycling from the endosome to the PM. Bro1, a class E Vps protein associated with the endosomes (Odorizzi et al., 2003), is required for the sorting of Gap1 into MVB pathway (Nikko et al., 2003). In bro1 null mutant cells, Gap1 is efficiently ubiquitylated and internalized in response to extra-cellular signals, however, it is not sorted into endosomal compartments but instead largely recycled to the PM membrane via the Golgi compartment. In a genetic approach aimed at identifying genes influencing the functional expression of AAPs at the PM, three genes were found (Paper III). These genes encode membrane proteins, Ssh4, Rcr2 and Rcr1, that localize to endosomal-vacuolar compartments. The functional characterization of these proteins suggest that they participate in general sorting events in the endosomal-MVB trafficking pathway and thereby affect the levels of amino acid transporters in the plasma membrane. This work has provided novel insights into the complexity of the poorly defined sorting events in the MVB pathway.

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History of Shr3

5. History of Shr3 SHR3 was found using a genetic approach designed to identify cellular components involved in the regulation of amino acid uptake and compartmentalization (Ljungdahl et al., 1992). Shr3 is an integral membrane protein with four membrane-spanning segments and a hydrophilic cytoplasmically oriented carboxy-terminal region. Shr3 localizes to the ER. In cells lacking Shr3, AAPs accumulate in the ER membrane and fail to become properly targeted to the PM. Accumulating evidence suggests that Shr3 is required for all newly synthesized AAPs to exit the ER, including Ssy1, the primary receptor component of the SPS-sensor of extracellular amino acids (Forsberg and Ljungdahl, 2001; Ljungdahl et al., 1992). The ER export block observed in shr3 null mutant cells is specific for AAPs, other proteins are correctly processed and targeted to their final destination (Fig. 6) (Kuehn et al., 1996; Ljungdahl et al., 1992). Shr3 is a well-conserved protein in fungi, and homologs function similarly in Schizosaccharomyces pombe (Martínez and Ljungdahl, 2000) and Candida albicans (Martínez and Ljungdahl, 2004).

Figure 6: Shr3 is required for the functional expression of AAPs (red and blue balls). Shr3 is an ER membrane protein that is required for AAPs to exit the ER. In cells lacking Shr3, AAPs are properly translocated into the ER membrane but fail to enter the sectretory pathway and thus accumulate in the ER. ER exit of other PM proteins (green balls) is not affected by Shr3.

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In detailed studies using the general amino acid permease (Gap1) as a model protein, it was found that each of the 12 membrane spanning segments were integrated into the ER membrane in the correct topological orientation independently of Shr3 (Gilstring and Ljungdahl, 2000; Gilstring et al., 1999). Also, the AAPs that accumulate in the membrane of the ER of shr3 null mutant cells do not induce the UPR, the ER stress response pathway (Gilstring et al., 1999). Thus, the accumulated AAPs do not expose misfolded sequences in the ER lumen that can be recognized by Kar2 (Bip) (Jarosch et al., 2003). Consistent with its specialized role in promoting the exit of AAPs from the ER, Shr3 was shown to physically associate with Gap1, but not with other polytopic membrane proteins, such as Sec61, Gal2 or Pma1 (Gilstring et al., 1999). The interactions between Gap1 and Shr3 were shown to be transient. At the time this thesis work was initiated, Shr3 was thought to function as an accessory protein, required for events subsequent to translocation and membrane insertion. It was presumed that Shr3 had a major role in facilitating the incorporation of AAPs into ER derived transport vesicles (Nyman et al., 2004). The major aim of my thesis has been to elucidate and understand the mechanistic role of Shr3 in facilitating AAP exit from the ER.

Figure 7: Two-dimensional structure of Shr3 and AAPs. The topology of Shr3 and the amino acid permease Gap1 has been experimentally determined (Paper I; Gilstring and Ljungdahl, 2000).

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Results and discussion

6. Results and discussion This section summarizes and discusses our experiments and results that form the basis of this thesis. For a more thorough description of the work, and the materials and methods used, please refer to the articles (Papers 1 – III) that are reprinted in the final section of this thesis.

6.1 Shr3 – an ER membrane chaperone (Paper I and II) Shr3 consists of two discrete domains, a membrane domain with four hydrophobic segments, each predicted to span the ER membrane, and a hydrophilic C-terminal domain (Fig. 7). To gain a more precise understanding of the significance of these domains we determined the in vivo topology of Shr3 using a modified glycosylation scanning approach (Gilstring and Ljungdahl, 2000). Our results confirmed that each of the hydrophobic segments of Shr3 spans the membrane and that the N- and Ctermini are oriented towards the cytoplasm. Next we constructed the shr3∆CT allele encoding a truncated protein that lacks the entire hydrophilic C-terminus. This allele was introduced into an shr3∆ null mutant and was found to restore amino acid uptake to levels between 30 – 50% of wild-type activity. Consistently, the shr3∆CT allele also restored sensitivity to toxic amino acid analogues. These results indicated that the presence of the hydrophobic region of Shr3 suffices to enable AAPs to exit the ER at appreciable rates and enable them to correctly localize to the plasma membrane. These novel findings indicated that interactions between the membrane domains of Shr3 and AAP are important, and suggested that Shr3 possessed chaperone-like activity. To examine the possibility that Shr3 could function as a chaperone, we used a chemical cross-linking approach to analyze the folding status of the AAP Gap1 in vivo. The results were unequivocal and showed that Shr3 had a clear role in AAP folding. In membranes from shr3∆ mutant cells, Gap1 formed extensive crosslinks as evidenced by the almost complete absence of detectable monomeric forms of Gap1. In wild-type cells no cross-linking of Gap1 was observed. These findings raised the possibility that in the absence of Shr3, AAPs may aggregate, which could explain the ability to efficiently crosslink AAPs. To further test whether Shr3 possesses chaperone-like activity we analyzed the aggregation state of AAPs under nondenaturing conditions using Blue-Native gels (BN-PAGE). Again the results were unambiguous. AAPs in membranes from SHR3 wild-type cells could readily be solubilized as monomers, whereas AAPs in membranes of shr3∆ null mutant cells where not, and migrated exclusively as a high molecular weight smear. Furthermore, we corroborated that the chaperone activity resides within the membrane domain of Shr3. This was accomplished by examining the aggregation state of AAP in cells 37

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expressing C-terminal truncated Shr3∆CT. As in wild-type cells, cells expressing Shr3∆CT, AAP did not aggregate and could be readily solubilized as monomers. We experimentally addressed the possibility that AAP aggregation was an indirect consequence of altered intracellular localization, i.e., AAPs in SHR3 wildtype cells predominantly localize to the PM, whereas AAPs in shr3Δ mutant cells accumulate in the ER membrane. The temperature sensitive sec12-1 mutation was used to block the ER export of AAPs independently of Shr3. At non-permissive temperatures, ER retained Gap1 in sec12-1 SHR3 cells did not form crosslinks and monomers were readily solubilized. Clearly, AAPs do not aggregate merely because they are retained in the ER. We also addressed if Shr3 was affected the folding of other membrane proteins in the ER. Our results show that proteins of similar complexity as AAPs folded properly, and do not aggregate in cells lacking Shr3. Thus, taken together, our data show that Shr3 functions as a membrane chaperone that specifically facilitates folding of AAPs. Next we sought to examine the temporal requirement of Shr3 in AAP folding. Split proteins have been used to study the process of integral membrane protein folding and assembly. In experiments examining the folding of bacteriorhodopsin (Huang et al., 1981), it was shown that truncated N- and C-terminal fragments could incorporate into membranes and properly assemble to attain catalytic activity. Subsequent to this pioneering work, other examples of functional split polytopic membrane proteins have been reported in the literature, including bacterial, yeast and mammalian proteins (Popot and Engelman, 2000). We created a split Gap1 protein consisting of two fragments, an N-terminal fragment comprised of TMS 1-5 and a C-terminal fragment comprised of TMS 6-12. When co-expressed, these non-overlapping Gap1 fragments assembled to form a functional permeases that correctly localized to the PM. Similar to full-length Gap1, the split Gap1 protein required the chaperone activity of Shr3 to functionally assemble. By analyzing the aggregation state of split Gap1 on BN-PAGE gels we could observe that in the absence of Shr3, both the N- and the C-terminal fragments aggregated. Thus, the chaperone function of Shr3 prevents aggregation and facilitates the assembly of the independently co-expressed N- and the C-terminal fragments of Gap1. How does Shr3 chaperone assist the assembly of split Gap1? One could envisage different models explaining this. Shr3 might bind both N- and C-terminal fragments and prevent them from aggregating, a requisite for them to find each other and assemble. Or alternatively, Shr3 may exert its chaperone function after the fragments associate. To address the temporal requirement of Shr3, we expressed the N- and Cterminal fragments individually and analyzed their aggregation state. The data clearly shows that Shr3 prevents the aggregation of the N-terminal fragment of Gap1, whereas Shr3 does not prevent the aggregation of individually expressed C-terminal fragment. Thus, the C-terminal fragment is dependent on the presence of both the N38

Results and discussion

terminal fragment and Shr3 to assemble properly. However, it remains to be determined if Shr3 is required for the C-terminal fragment to properly assemble once it initiates an interaction with the N-terminal fragment. Based on our studies with the split Gap1 protein we propose that the membrane chaperone Shr3 interacts early during the biogenesis of AAP and likely prior to the partitioning of all twelve TMS into the lipid phase of the ER membrane.

6.2 Novel class of membrane chaperones (paper I) Three other ER-localized proteins in yeast that reportedly function similar to Shr3 have been described. Each are integral membrane proteins that specifically affect the ER exit of restricted sets of polytopic PM proteins. The hexose transporters Hxt1 and Gal2 require the assistance of Gsf2 to exit the ER (Sherwood and Carlson, 1999). Similarly, Pho86 and Chs7 are required for the ER exit of the phosphate transporter Pho84 and the catalytic subunit of chitin synthase III (Chs3), respectively (Lau et al., 2000; Trilla et al., 1999). Similar to the highly specific nature of shr3 null mutations, in cells carrying null alleles of one of these accessory proteins only their cognate substrates failed to enter COPII transport vesicles and accumulated within the ER. Although these proteins appear to carry out analogous functions, they do not share identifiable sequence homology with each other or with Shr3 (Fig. 8).

Figure 8: Schematic illustration of the membrane topology of ER membrane chaperones.

Based on our detailed understanding of Shr3, we examined the possibility that Gsf2, Pho86 and Chs7 function as membrane chaperones. Utilizing our cross-linking assay we found that in cells lacking one of these proteins, their cognate substrates specifically formed crosslinked aggregates. These results strongly suggest that these proteins also function as membrane chaperones, which prevent aggregation and assist the folding of their cognate substrates.

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The identification of substrate specific membrane chaperones in yeast established a novel group of proteins involved in the biogenesis of polytopic membrane proteins. The generality of our findings indicates that proteins with multiple membranespanning segments require highly selective membrane chaperones to overcome common structural constraints associated with membrane insertion and folding. Furthermore, since the topogenic signals are interpreted correctly and substrate proteins apparently attain their correct topology (Gilstring and Ljungdahl, 2000), these chaperones likely function to prevent inappropriate contacts between intraprotein TMSs and/or other ER proteins that otherwise would impair folding as they exit the Sec61 translocon.

6.3 Folding and degradation of AAPs (Paper II) As described in Chapter 3, secretory and membrane proteins are subject to ER quality control to ensure that only properly folded proteins exit the ER. Misfolded or incorrectly assembled proteins are recognized and degraded by ERAD. Interestingly, although the observed aggregation of AAPs in shr3Δ mutants is a consequence of misfolding, the aggregated AAPs that accumulate in the ER do not activate the ER unfolded protein response (UPR) (Gilstring et al., 1999). Presumably, Kar2, the lumenal Hsp70 that acts as the primary sensor of misfolded proteins, does not recognize the short hydrophilic and lumenally oriented loops of AAPs. How do then cells recognize and cope with the presence of aggregated AAPs in the ER? In paper II we investigated the fate of aggregated AAPs that accumulate in the ER membrane of cells lacking Shr3. We examined the turnover rate of two AAPs, in wild-type and shr3 null mutant cells, and found that the AAPs in both of these strains were efficiently degraded. Since the aggregated AAPs in shr3Δ mutants cannot exit the ER (Kuehn et al., 1998; Kuehn et al., 1996), we suspected that the aggregated AAPs were degraded by ERAD. Using mutants deficient in vacuolar protease function (pep4) or ERAD (ubc6 and/or ubc7) we found that aggregated AAPs in shr3Δ mutants cells were degraded by ERAD, whereas AAPs in wild-type cells were targeted to and degraded in the vacuole. Next we investigated which of the ERAD pathways, i.e., Doa10 or the Hrd1/Der3 dependent pathways, was responsible for recognizing and degrading AAP aggregates. Mutations that individually inactivated Doa10 or Hrd1/Der3 ERAD pathways did not significantly diminish AAP degradation, however, when both of these pathways were inactivated, the rate of AAP degradation was greatly reduced. These findings indicated that both Doa10 and Hrd1 pathways function in parallel to degrade AAP aggregates. Thus, in the absence of Shr3, the Hrd1 (ERAD-M) pathway could recognize the misassembled TMS of aggregated AAPs, whereas the Doa10 (ERADC) pathway might recognize the misfolded cytosolic regions (Ismail and Ng, 2006). However, it should be noted that AAP aggregates might be comprised of a non40

Results and discussion

homogenous mix of folding intermediates. Alternatively, these ERAD pathways could recognize and degrade discrete AAP folding intermediates. Interestingly, we observed that mutations inactivating both Doa10 and Hrd1/Der3 ERAD pathways partially suppressed the amino acid uptake defects associated with the loss of Shr3 function. Thus, AAPs appeared to be able to attain native structures independent of the chaperone activity of Shr3. We tested this possibility by examining the status of AAP folding using BN-PAGE. Compared to shr3Δ cells with intact ERAD pathways, significantly more AAP monomers were solubilized in shr3Δ cells with impaired ERAD. Thus, in the absence of ERAD, AAPs are afforded additional time to attain functional structures independently of Shr3. As discussed in paper II, it seems likely that AAP aggregates are not comprised of terminally misfolded proteins. AAPs may thus be able to spontaneously rearrange and fold. Alternatively, the proposed chaperone-like activity of the Sec61 translocon or other membrane chaperones may inefficiently substitute for the lack of Shr3 and assist AAP folding. In summary, our findings suggest that in addition to facilitating AAP folding, Shr3 shields partially folded AAPs from being prematurely targeted for degradation. These findings highlight the intimate link between folding and degradation, and clearly demonstrate that membrane-localized chaperones mediate these competing processes.

6.4 Ssh4, Rcr2 and Rcr1- regulating transporters at the PM (Paper III) To further elucidate the requirements for the functional expression of polytopic plasma membrane proteins, we utilized a genetic approach to identify genes that when over-expressed suppress the amino acid uptake deficiency of shr3 null mutant cells. The high-copy suppression analysis identified two genes, SSH4 and RCR2. RCR2 has a close homologue, RCR1 (47% identity), which we found also functions as a high-copy suppressor of the shr3 null mutation. SSH4, RCR2 and RCR1 encode integral membrane proteins, each with one TMS. Based on our recent work on Shr3, we initially addressed whether the suppression was due to the ability of S S H 4 , R C R 2 and RCR1 to function as membrane chaperones. If this was the case, we expected that their overexpression would increase AAP folding. However, overexpression of these suppressors did not influence the aggregation state of Gap1, indicating that they are involved in some other process affecting AAP function. Next we examined the possibility that the overexpression of these suppressors increase AAP gene expression. The SPS-sensor of extracellular amino acids controls the expression of several AAP genes (Andreasson and ljungdahl, 2002; Forsberg and ljungdahl, 2001), and we have previously observed that increased transcription of SPS-sensor dependent genes can partially suppress the amino acid uptake defects of shr3 mutants. However, by 41

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analyzing SPS-sensor regulated gene expression, we could show that the overexpression of SSH4, RCR2 or RCR1 did not affect AAP transcription. Thus, the data collectively indicated that Ssh4, Rcr2 and Rcr1 function at a later step in the secretory pathway to increase AAP activity. To investigate this possibility we first set out to determine the intracellular localization of these proteins, since this would provide clues for further studies. Using immunofluorescence microscopy we successfully determined the intracellular location of Ssh4 and Rcr2. Both these proteins localize to structures corresponding to the vacuole. Additionally, Rcr2 was observed in large vesicular structures resembling endosomes. These results suggested that Ssh4, Rcr2 and presumably Rcr1 function post-ER, and likely affect processes in the downregulation or turnover of AAPs. We examined this by monitoring AAP levels in cells overexpressing Ssh4, Rcr2 and Rcr1. We found that overexpression of these suppressors increased the steady-state levels of two AAPs, Gap1 and Tat2, a finding that accounts for their ability to increase amino acid uptake in shr3 mutant cells. In addition, when we performed a more detailed analysis of Tat2 turnover we observed that Ssh4, Rcr2, and Rcr1 affected the pattern of protein modifications of Tat2 during its downregulation. These modifications most likely act as signals in regulating the trafficking of Tat2 molecules in the late secretory pathway. Finally, the suppressing effects of SSH4, RCR2 and RCR1 were not limited to AAPs, but affect members of other transporter families as well. By analyzing cell growth under various nutritional conditions, we could show that overexpression of SSH4, RCR2 and RCR1 increased the activity of phosphate as well as uracil uptake systems. The pleiotropic effects observed when SSH4, R C R 2 and RCR1 are overexpressed suggest that they encode components of the endosomal-vacuolar pathway that participate in a common sorting process that determines the fate of multiple plasma membrane proteins. In summary, this work, characterizing three proteins in the endosomal-vacuolar trafficking pathway, provides novel insights into sorting processes that affect the functional expression of PM transporters. Additionally, we found that Rcr2 shares a region of homology with the human A-kinase anchoring protein-79 (AKAP79). AKAPs function as dynamic scaffold molecules that bring phosphatases and kinases close to their substrate targets, and thereby participate in controlling various cellular events, including the stability of plasma membrane proteins (Dell'Acqua et al., 2006). The functional significance of the homology region between Rcr2 and AKAP79 needs to be confirmed, but it is tempting to speculate that Rcr2 also functions as a scaffold for enzymes and substrates controlling sorting events affecting the trafficking of nutrient transporters.

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Future prospects

7. Future Prospects The studies described in this thesis identified Shr3 as an ER membrane chaperone and demonstrated the specific and critical role it plays in enabling AAP to fold and attain proper structures. These studies have provided new insights into the general folding and degradation mechanisms of polytopic membrane proteins. Hopefully other researchers will be challenged to address the new lines of questions raised from our analysis. Starting with the results obtained on the split Gap1 construct. We found that both the N-terminal and the C-terminal fragments require Shr3 to fold properly. However, it appears that Shr3 only recognizes the N-terminal fragment. This raises the question: Does Shr3 chaperone function directly facilitate the folding of the Cterminal fragment, or can the C-terminal fold properly once it interacts with the Nterminal fragment? Additional questions arise as to how the N-terminal and Cterminal fragments recognize each other. Can the C-terminal fragment recognize the N-terminal fragment independently of Shr3, or only in the presence of Shr3? Experiments to test the ability of the N- and C-terminal fragments to interact in presence and absence of Shr3 could provide clarification. The discovery that Shr3 recognizes the N-terminal fragment of split Gap1 when expressed without the presence of the C-terminal fragment clearly suggests that Shr3 participates early in the folding process. Could Shr3 interact with Gap1 as soon as the first TMS of Gap1 exits the PCC? Perhaps Shr3 interacts with TMSs containing charged residues, since they may temporarily require assistance prior to folding with appropriate partner TMS? Established in vitro translation/insertion systems may be able to provide answers to these questions. This methodology coupled with sitespecific cross-linkers could provide precise information regarding which membrane segments of Gap1 interact with Shr3, and how early in the folding process this interaction takes place. Several questions remain regarding the nature of unfolded/aggregated AAPs that accumulate in the absence of Shr3. In the second paper, we showed that under conditions where ERAD is impaired, AAPs are able to fold independently of Shr3 leading to increased levels of functional AAPs in the PM. A possible explanation for this observation is that AAP aggregates are not comprised of terminally misfolded proteins. Rather, aggregated AAPs may be in a conformation equivalent to a kinetically trapped folding intermediate, which given sufficient time, could inefficiently fold. If this is the case, the reintroduction of Shr3 may function in a posttranslational manner and “rescue” aggregated AAPs by facilitating their folding. Finally, the high copy suppressor screen described in paper III identified three membrane proteins (Ssh4, Rcr2 and Rcr1) that function within the endosomalvacuolar trafficking pathway. These proteins appear to affect sorting processes that 43

Jhansi Kota

determine whether AAPs and other metabolite transport proteins are targeted for vacuolar degradation or routed to the PM. It remains to be elucidated in what way these proteins affect sorting events. Clues could be obtained by a precise determination of the intracellular distribution of AAPs; are AAPs mostly localized to the PM, or to internal compartments in the late secretory pathway? Also, how does the overexpression of Ssh4, Rcr2 and Rcr1 affect the known post-translational modifications of AAPs? It would also be interesting to determine the importance of the homology region within Rcr2 and the neuronal scaffolding protein AKAP79. Does Rcr2 also function as a scaffolding protein? Interaction studies could provide information about their respective function.

44

Acknowledgements

8. Acknowledgements Being a graduate student is an absorbing commitment lasting for several years. Most of the time it is a stimulating and positive experience, but as every major challenge, it has its tough days too. However, no day felt completely unrewarding thanks to all you superb people who encouraged and helped me. A special thanks to the following people: My supervisor Per Ljungdahl, who has been there to enthusiastically discuss results and always allowing me a high degree of independence and professional freedom. Thanks for taking me on as a PhD student and thank you for the excellent team of people that you always gathered to your lab. Speaking of the people in the lab: When I first came to our group, I didn't know what to expect, but you quickly became like a second family to me. Thank you for all the encouragement and support these years. Whenever and whatever, you have been an email or a lab bench away. Thank you for sharing many ups and downs with me! Claes – I have always admired your talent and passion for science, which is truly awesome. Being lab-neighbor with you (and your fish-tank) meant a lot of fun and interesting discussions about everything. Thanks for all your ideas and suggestions and helping me out whenever needed. Paula – I really missed you when you left for Barcelona. We had so much fun together both in the lab, and out of the lab. You are such a wonderful combination of fire and earth, flamenco and pop, hot-blooded enthusiasm and calm, rational comfort. Hasta luego! Until we meet again in Spain. Arezou – You are such a wonderfully kind and caring person. You always looked on things from the bright side and you filled the whole lab with good mood. We had such a good time together when you were at Ludwig. I am still waiting for kick-off of the Persian-tapas concept…… Stijn – I am happy that it was someone like you who came to claim Arezous lab bench after she left us. It was very nice to have you to discuss everything from life to projects with. Mirta - my dear friend, I am so grateful for all your help, support, providing ideas, proofreading….. everything! Thanks for always being there. I have gradually learned that you are a person with many hidden talents. For instance, it was only last year we all learned that you are an A-grade better on Football Championships. Now you are the last Mohican of the lab… Kör hårt! Tomas – It has been great having you in the lab. With your great sense of humour and inventive lab techniques, you lifted the spirits of the lab. It’s been empty here without you (I even miss your teasing humour). Fredrik – Thanks for teaching me all the ins and outs of Shr3 and all the help you gave me when I came as a little "freshman" to the lab. Jessica – Unfortunately you were not in the lab for very long, but I really enjoyed the time while you were here. Hanna – I cannot thank you enough for all you done this last year and especially the last days. What a great time we had. I still cannot believe that we managed to put together a paper in 6 months. After Paula left, I thought that my partying days were over. Thanks to you I was wrong. I could not have had a better company to be lost in Prague with… even at five in the morning. Thanks for being such a great friend! 45

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Ralf Pettersson - my co-supervisor and Branch Director for the Ludwig Institute during my time. We have you to thank for many things, including employing all our excellent administrative staff, and basically making the Ludwig Institute in Stockholm what it is today. Not the least, thank you for welcoming me. I also want to thank Ulf Eriksson - for always taking interest in my work and helping me out whenever I needed it. Thomas Perlmann - for keeping the Ludwig Institute an excellent Institute. Sincere thanks to the administrative personnel. You where the ones who kept the wheels turning: Charlotta, Eva, Birgitta, Mats, Erika, Angelica – Thanks! Dear Inger - you always took especially good care of us PhD-students, organizing parties for us and playing golf with me, but what is most remarkable is that you always managed to sort things out in no-time even when I was in trouble. Thank You! Finally, to all the other “Ludwigers”, past and present: Thanks to all of you, the Ludwig Institute is a very special place. I can only hope that my next work place will have an atmosphere remotely as fun and positive. Especially, Ph.D students and other party animals for all the fun! Etienne - for taking an interest in my work and for the great times in Nice. Banafsheh, Stina, Maria, Carolina, Anna, Elisabeth and Theresa for your friendship. Theresa – we have been rather synchronized with our PhDs, which was great because we could always help and support each other. Thanks to the colleagues during my time at College Station in Texas and Professor Art Johnson for inviting me to your lab. It was a great experience and I learned a lot. Now I know how a real steak should be like - Texan! Matti Nikkola - Your help with all the practicalities concerning my doctoral thesis, especially coming close to the end have meant more to me than you can possibly imagine. Thank you! To all my friends on the “outside”: Thanks for reminding me that there actually exists a reality outside of the lab. I promise to visit this alternative reality and all of you much more frequently than I have been able to do this last year. My dear friend Nancy - congratulations to your Ph.D. Thanks for being such a super good friend all the time since when we first met in Heidelberg. You are a mountain of fun and one of the few people not being taller than I am. Also, thank you for introducing me two such fun people as Katarina and Stefania. Susanne - thanks for being the best of friends for more than 20 years. The Elvnert family - Thank you for all your support and for welcoming me into your family. Thank you for introducing me to golf – Fore! Sorry for my bad temper some rounds, but I am really working on improvement… Thanks for being my 112 number here in Stockholm, thanks for everything. Eva - we have so many interests in common. Now when I finally feel free, you have moved to Dubai. But I promise that won’t stop us. Let’s have some fun! Hannes - I wish you super success in your new job! We will visit you after having cheered for our home sports team here in Råsunda only once more. 46

Acknowledgements

To Mom and Dad - You have always supported me to 100 percent whatever crazy things I decided to do. Thank you for always believing in me. I can not express how proud I am of both of you and how much you mean to me. I love you so much. I am the person I am today thanks to you. Dilip - my dear brother, I am so proud of you and I take such comfort from knowing that I can always rely on you no matter what happens. Johan - Thank you for sharing this with me every step on the way. Thank you for your constant support, love, encouragement and advice. Thank you for making sacrifices without a blink of an eye. I love you.

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