University of Groningen

Membrane protein targeting to the outskirts of the endoplasmic reticulumKralt, Annemarie

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1.1.An introduction on the insertion, quality control and targeting of endoplasmic reticulum-inserted integral membrane proteins

ChapterChapter

In 1999 the Nobel Prize in Physiology or Medicine was awarded to Günter Blobel for the discovery that “proteins have intrinsic signals that govern their transport and localization in the cell” (Nobel prizes and laureates, 1999). Now, fi fteen years later, one can still be intrigued by the diverse and complex mechanisms that involve protein targeting to their destined location. Especially for membrane proteins, much is to be gained on the identifi cation of sorting signals and mechanisms. In this thesis we describe the targeting of integral membrane proteins to two separate subdomains of the endoplasmic reticulum, the inner nuclear membrane and endoplasmic reticulum-plasma membrane junctions, respectively.

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1.1 Insertion and assembly of integral membrane proteins into the ER membrane

Integral membrane proteins are present in the plasma membrane (PM) and membranes of organellar compartments. They fulfi l functions in numerous cellular processes, including signalling and transport and biogenesis of biomembrane components. Most membrane proteins are initially inserted into the lipid bilayer of the membrane of the endoplasmic reticulum (ER), from where they have to be assigned to the destined subcellular membrane or ER subdomain. The insertion of transmembrane segments is mediated by the Sec61 translocon or the Guided-Entry of Tail-Anchored protein insertion (GET) pathway. The latter is dedicated to the posttranslational insertion of C-terminal (tail)-anchored proteins (Denic et al., 2013). The Sec61 complex is an ER membrane-embedded translocon that both translocates soluble proteins from the cytoplasm to the ER lumen, and forms the core of the complex for the membrane insertion of integral membrane proteins (Rapoport, 2007). A (signal) sequence, consisting of hydrophobic residues (Lee and Bernstein, 2001), in the nascent chain of secretory or membrane proteins is recognized and captured by the signal recognition particle (SRP) (Walter and Johnson, 1994;Hainzl et al., 2011) positioned at the exit tunnel of the ribosome (Krieg et al., 1986;Halic et al., 2004;Halic and Beckmann, 2005). The SRP slows down translation and shields the transmembrane (TM) domains from the bulk cytosol, preventing aggregation and inappropriate interactions. The SRP interacts with an integral ER membrane protein, the SRP receptor (Halic et al., 2006), after which the translating ribosome is transferred to the Sec61 translocon (Rapiejko and Gilmore, 1997;Jiang et al., 2008;Akopian et al., 2013), which assists in protein translocation to the ER lumen or insertion and assembly of TM segments into the ER membrane (Alder and Johnson, 2004;Rapoport, 2007).

The assembly of polytopic proteins (membrane proteins that contain multiple membrane spanning domains) is a complex and strictly coordinated process. Sec61 incorporates the TM segments of a membrane protein co-translationally, but the conducting, lateral channel of Sec61 is too small to hold multiple transmembrane segments simultaneously, which would be required for a co-translational assembly mechanism (Shao and Hegde, 2011). The TM segments of immature polytopic proteins thus have to await the insertion of the other TMs and this may cause unwanted inter- and intramolecular interactions with folding intermediates or the lipid phase of the membrane. In principle after the stage of insertion as just described, a second stage involves proper folding. This stage is regulated by integral ER accessory proteins. Several of these chaperone proteins are identifi ed and have distinct substrates; Shr3 for amino acid permeases, Gsf2 for hexose transporters, Pho86 for phosphate transporters and Chs7 for chitin synthase III (Kota et al., 2005). Once inserted into the ER membrane, integral membrane proteins are subjected to quality control and posttranslational modifi cations.

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Figure 1.1: Th e endoplasmic reticulum in yeast. Th e plasma membrane, endoplasmic reticulum, nuclear envelope with embedded nuclear pore complexes and inner and outer nuclear membrane (INM and ONM, respectively) are indicated. Fluorescence image shows a yeast cell expressing a GFP-fused ER marker protein (GFP-HDEL), N: Nucleus.

plasmamembrane

nuclearenvelope

corticalendoplasmic

reticulum

nuclearpore

complex

GFP-HDEL

N

INM

ONM

1.2 Quality control at and exit from the endoplasmic reticulumThe ER has a robust quality control system called ER-associated protein degradation

(ERAD). The function of ERAD is twofold; it eliminates misfolded proteins and it plays a role in protein homeostasis by specifi cally degrading folded proteins, so as to control their levels in response to certain stimuli (Ruggiano et al., 2014). Substrate proteins are selected for degradation by ubiquitylation involving the covalent attachment of ubiquitin molecules. Ubiquitylation is mediated by ubiquitin-activating (E1) enzymes, ubiquitin-conjugating (E2) enzymes, and ubiquitin-ligating (E3) enzymes facilitating the transfer of ubiquitin from an E2 enzyme to the substrate.

In yeast two complexes act in recognizing ERAD substrates in the ER, named after their core transmembrane E3 ligase proteins; the Doa10 complex and the Hrd1 complex. The Doa10 complex ubiquitinates membrane proteins that are misfolded in their extralumenal domain and extralumenal soluble proteins (Swanson et al., 2001;Ravid et al., 2006). Proteins with misfolded regions in their transmembrane or lumenal domain are prone to ubiquitination by the Hrd1 complex (Bordallo et al., 1998). Degradation of ubiquitinated ER (transmembrane) substrates occurs by the cytoplasmic proteasome, implying that ubiquitinated proteins have to be removed from the ER, which is done by a more recently discovered process called retrotranslocation (Hampton and Sommer, 2012). Well described targets for controlled protein turnover are proteins involved in ergosterol biosynthesis (Foresti et al., 2013). Very recently a third branch in the ERAD pathway was identifi ed, involving the proteins Asi1, Asi2 and Asi3 (Foresti et al., 2014;Khmelinskii et al., 2014). The Asi transmembrane proteins that localize at the inner nuclear membrane are described to be involved in the repression of expression of amino acid permeases (AAPs) in the absence of extracellular amino acids (Forsberg et al., 2001a;Boban and Ljungdahl, 2007). This complex seems to specifi cally ensure protein homeostasis at the INM and works synergistically with the Doa10 complex that is localized in both the ER and the interconnected INM membrane. The Hrd1 complex does not play a role in this process as it is excluded from the INM (Deng and Hochstrasser, 2006).

ER stress caused by accumulation of misfolded proteins escaping the ERAD due to saturation of the system is monitored by the unfolded protein response (UPR). Activation of

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Box 1.1: Membrane protein selection for packaging in COPII coated vesicles

Several motifs in the cytoplasmic C-terminus of membrane proteins are described that mediate selectivity towards the packaging of the cargo proteins in COPII vesicles, including the di-acidic (D/E)x(D/E) motif, the di-hydrophobic (FF, YY, LL or FY) motif, YNNSNP and triple Arg motif. Those signals interact with Sec24 or Sar1, which are components of the COPII machinery (Sato and Nakano, 2007;Venditti et al., 2014).

Enrichment of the general amino acid permease Gap1 relies on the presence of the di-acidic signal at its C-terminus (Malkus et al., 2002) and this motif is highly conserved among proteins belonging to the family of AAPs, including the amino acid sensor Ssy1. Exit of AAPs from the ER is also facilitated by the accessory protein Shr3 (Kota and Ljungdahl, 2005;Kota et al., 2007). The partitioning of insertion and assembly in two stages (as described in paragraph 1.1) implicates that in the absence of Shr3 the 12 transmembrane domains of AAPs are correctly inserted into the ER membrane by the Sec61 translocon, where they are prone to aggregation into large, ER exit-incompetent complexes due to the lack in assembly assistance.

this intracellular signal transduction pathway results in reduction of proteins entering the ER and an increase of the ER protein folding capacity. Prolonged UPR, occurring when protein folding homeostasis cannot be restored, ultimately leads to apoptosis or cell death (Ron and Walter, 2007;Walter and Ron, 2011). UPR increases the size of the ER and detection of overabundance of the ER leads to organelle-selective autophagy or ER-phagy (Bernales et al., 2007;Schuck et al., 2014).

In higher eukaryotes a third quality control mechanism for ER proteins is described, named the rapid ER stress-induced Export (RESET) (Satpute-Krishnan et al., 2014). Where the UPR-response involves the transcriptional upregulation of target genes requiring a temporal lag phase, RESET allows a subset of misfolded ER proteins to escape to the Golgi, rapidly reducing the burden of misfolded proteins in the ER to avoid aggregation. RESET may also function to remove unfolded proteins that form poor substrates for ERAD.

The vast majority of integral membrane proteins that are initially inserted into the ER membrane, but are destined for other organellar membranes, exit the ER via vesicular transport of the secretory pathway. Recruitment of components of the coat protein II (COPII) complex to specialized sites of the ER leads to the formation of COPII-coated vesicles (Lord et al., 2013), by which proteins and lipids are traffi cked from the ER to the Golgi network (Lee et al., 2004;Venditti et al., 2014). The Golgi provides another set of modifi cations (glycosylation, sulfation, proteolytic cleavage) before proteins are sorted into vesicles and targeted to their destination e.g. the vacuolar or PM (Hong and Tang, 1993;Keller and Simons, 1997). How membrane proteins, specifi cally AAPs, are selected for packaging in COPII-coated vesicles to exit the ER is described in box 1.1.

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1.3 Localization of membrane proteins to ER subdomains

1.3.1 Lateral mobility of proteins in a membrane Incorporation in or anchoring to a lipid bilayer restricts proteins to move in a

2-dimensional manner in a crowded environment, and as such limits diffusion of a protein with at least an order of magnitude (Meinema et al., 2013). The ER membrane in Saccharomyces cerevisiae forms a network of cisternal and tubular domains with a single lumen (West et al., 2011) that extends from the interconnected nuclear envelope (NE) to the peripheral or cortical ER aligning the PM (Fig 1.1). Integral membrane proteins are generally free to move laterally throughout this network. Localization to specifi c subdomains is dependent on molecular interactions with stable residents of specifi c membrane domains locally slowing down diffusion. The NE-embedded nuclear pore complex (NPC) provides a barrier for diffusion to the nucleus and inner nuclear membrane (INM). We will describe two ER subdomains where concentration of the specifi c membrane proteins discussed in this thesis occurs, namely the INM and membrane contact sites.

1.3.2 Localization of membrane proteins to the inner nuclear membraneThe NE separates the nucleoplasm from the cytoplasm, protecting the genetic

material and strictly separating the processes of transcription from translation. The NE is a double lipid bilayer that consists of the INM, the highly curved pore membrane (POM) that aligns the NPC and the outer nuclear membrane (ONM), which is continuous with the ER membrane (Fig 1.1). Despite being a continuous membrane system, the membrane protein composition of the INM is distinct from the ONM and ER (Schirmer et al., 2003). How this dissimilarity is achieved is discussed in paragraphs 1.3.2.1 and 1.3.2.2

1.3.2.1 Accumulation of membrane proteins in the INM by diffusion and retentionNE embedded NPCs form the gate through which bidirectional movement of

molecules and proteins between the compartments can occur. NPCs are high molecular weight protein complexes with an 8-fold rotational symmetry, build of approximately 31 individual nucleoporins (Nups) that assemble in subcomplexes, which are present in several copies (Rout et al., 2000;Cronshaw et al., 2002;Alber et al., 2007). Especially with electron microscopy studies much data has been gained about the architecture of the NPC, where the complex appears as a donut-shaped ring structure composed of 8 identical spokes. Two channels are described in the NPC; the central channel, fi lled with fi laments of phenylalanine-glycine (FG) repeat containing Nups, and the lateral channel between the scaffold of the NPC and the pore membrane (Hinshaw et al., 1992;Akey and Radermacher, 1993;Yang et al., 1998;Fahrenkrog et al., 1998;Walther et al., 2002;Lutzmann et al., 2002;Stoffl er et al., 2003;Beck et al., 2004;Kiseleva et al., 2004;Alber et al., 2007;Beck et al., 2007;Gaik et al., 2015).

Integral membrane proteins are able to freely diffuse through the pore membrane, while the extralumenal domain slides through the lateral channel of the NPC (Fig 1.2C). This

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RanGTP

βα α

βα

βα

β

α

?

βα

RanGTP

β

α

β

α

A

C

B D

nucleoplasm

cytoplasm

ONM

INM

Figure 1.2: Simplifi ed scheme for import through the nuclear pore complex (NPC, depicted in dark grey). (A) Small proteins readily diffuse through the with FG-nup fi laments-fi lled central channel of the NPC (A). (B) The accumulation of (larger) proteins or protein complexes requires energy supplied by RanGTP. In the cytoplasm, an NLS (indicated in red) is recognized and bound by transport factors (represented by importin α and importin β, α and β, respectively); in the nucleoplasm release of the NLS-cargo is stimulated by RanGTP. (C) Membrane proteins with smaller extralumenal domains enter the nucleus by diffusing through the pore membrane, which connects the inner and outer nuclear membrane (INM and ONM), while their globular domain traffi cs through the lateral channel between the NPC and the POM. (D) Transport receptor mediated import of Src1/Heh1 and Heh2 starts with the interaction between the NLS (red) and the receptor in the cytoplasm, after which translocation through the NPC occurs.

event can only occur as long as the extralumenal domain is not exceeding the size limit of the lateral channel (~9-10 nm) (Hinshaw et al., 1992;Ohba et al., 2004;Beck et al., 2007;Bui et al., 2013). In order to accumulate at the INM, proteins have to be retained by interacting with nuclear components. Indeed, often interactions between INM proteins and chromatin or nuclear proteins like lamins are described, preventing diffusion of those proteins out of the nucleus (Soullam and Worman, 1993;Furukawa et al., 1998;Lin et al., 2005). This mode of INM accumulation is known as diffusion and retention (Powell and Burke, 1990;Ellenberg et al., 1997).

1.3.2.2 Active transport through the nuclear pore complex Integral membrane proteins with a larger extralumenal domain are more hindered

in their passage through the lateral channels. In human systems it was concluded that an

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extralumenal domain exceeding dimensions of 10 nm, a size that correlates with a globular domain of approximately 60 kDa, is too large to accumulate at the INM through diffusion and retention (Hinshaw et al., 1992;Soullam et al., 1993;Wu et al., 2002;Ohba et al., 2004) and passage through the lateral channel will also be obstructed when obligate complexes are formed with lumenal domains of membrane proteins. Recently for yeast it was described that the size of an extralumenal domain of 90 kDa was still small enough to pass the NPC, albeit signifi cantly hindered, while 136 kDa was too big (Popken et al., 2015). For soluble proteins traffi cking through the central channel of the NPC, the same observation has been made; diffusion is relatively unhindered for smaller proteins and protein complexes (Fig 1.2A), but more restricted for larger proteins and protein complexes. To overcome the diffusion barrier of the NPC, that is established by the fi laments of FG-nups, soluble proteins contain localization signals and are transported using an energy-consuming mechanism (Fig 1.2B) (Stewart, 2007). Exposed nuclear localization signals (NLSs) or nuclear export signals (NESs) on soluble proteins are recognized and bound by transport factors/receptors in the cytoplasm. Those transport factors mediate transport through the pore by specifi c interactions with FG-repeats of the fi laments in the central channel of the NPC (Fiserova et al., 2010;Peleg and Lim, 2010;Yang, 2013). Most transport through the NPC is driven by a RanGTP gradient between the nucleoplasm and the cytoplasm, dictating transport receptor-cargo binding and release (Kalab et al., 2002;Fried and Kutay, 2003;Madrid and Weis, 2006;Cook et al., 2007).

The transport factors importin α and importin β co-ordinately escort the import of a wide range of soluble proteins to the INM. Importin α forms the NLS binding moiety of the complex and contains a C-terminal domain of 50 kDa with two NLS binding sites, the major and the minor binding site, respectively (Conti et al., 1998;Conti and Kuriyan, 2000;Fontes et al., 2003;Giesecke and Stewart, 2010;Marfori et al., 2012;Roman et al., 2013). One binding site will be occupied by a monopartite NLS consisting of a stretch of basic residues, while bipartite NLSs consist of two clusters of basic residues spaced by a region of 10-12 residues (Robbins et al., 1991), with each cluster interacting with one of the binding sites. The 10 kDa N-terminal domain of importin α, called the importin β-binding (IBB) domain (Kobe, 1999) interacts with the NLS binding sites in the absence of importin β, and this autoinhibition prevents binding of classical NLSs. Upon binding of the IBB domain by importin β, the NLS binding sites are exposed and available to interact with an NLS (Gorlich et al., 1996;Moroianu et al., 1996;Lott et al., 2010). The interaction between importin α and the NLS sequences is described in more detail in the introduction of chapter 3.

For a long time the question was whether a mechanism for active transport could also explain the accumulation of integral membrane proteins at the INM. Ohba et al. (2004) indeed described energy-requirement for the translocation of some membrane proteins through the NPC. More recently, it was shown that two integral membrane proteins in Saccharomyces cerevisiae, Heh2 and Src1/Heh1, are actively transported to the INM by the yeast homologues of importin α and importin β, Karyopherins 60 and 95, respectively (King et al., 2006) (Fig 1.2D). In addition to Heh2 and Src1/Heh1, in a number of INM proteins an NLS-like

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Box 1.2: Intrinsically disordered proteins and protein regionsID proteins or protein regions lack a persistent ordered secondary structure and

are characterized by low hydrophobicity and high net charge (Romero et al., 2001;Vucetic et al., 2003). Due to increasing amounts of sequence availability and development of techniques to characterize structural propensities of proteins, the functions and advantages of ID proteins and protein regions became subjected to extensive investigations in the last decade. It has been found that ID proteins and protein regions are surprisingly widespread throughout the proteome (Uversky, 2013) and are involved in various cellular processes including signalling, transcriptional regulation and cell cycle progression. ID regions are highly fl exible and dynamic; in some cases coupled folding and binding of ID regions occurs upon interaction with a biological partner, but in other cases the region simply function as a linker between two domains that allows some orientational freedom, and at the same time increases the local concentration of one domain in proximity of the other domain. A detailed description of the role of intrinsically disordered proteins and protein regions is beyond the scope of this introduction chapter, and is reviewed in Dyson and Wright (2005) and Liu and Huang (2014).

sequence is predicted (Lusk et al., 2007). The NLS-regions of Sun2 (Turgay et al., 2010), LBR (Ma et al., 2007) and Pom121 (Yavuz et al., 2010;Doucet et al., 2010;Funakoshi et al., 2011) were shown to interact with importin α and/or β and contribute to their INM localization.

However, translocation of Heh2 and Src1/Heh1 to the INM did not only require binding of the bipartite NLS to Karyopherin (Kap) 60/95, but also the presence of an intrinsically disordered (ID) region between the NLS and the transmembrane domain (Meinema et al., 2011) (Fig 1.2D). General characteristics of ID regions can be found in box 1.2. A reporter protein composed of the minimal requirements for INM import (the bipartite NLS (h2NLS), intrinsically disordered region (L) and fi rst transmembrane segment (TM) of Heh2) was strongly accumulated but mobile in the INM (Meinema et al., 2011;Meinema et al., 2013), arguing against nuclear retention. When the linker between the h2NLS and the TM has insuffi cient length, accumulation at the INM is completely abolished, demonstrating its vital role in INM import. It is suggested that the ID linker stretches from the membrane to the central channel of the NPC (where the transport factors interact with FG-repeats) through ‘lateral gates’ between two spokes, and as such the linkers slides through the NPC structure. The mechanisms underlying the translocation of Heh2 and its derived reporter proteins (e.g. how is the NPC traversed, which Nups interact with the transport vectors) are yet unknown (Meinema et al., 2012) and subject of present studies.

1.3.3 Localization of membrane proteins at contact sites between heterologous membranesMembrane contact sites (MCSs) are formed when membranes of two organelles are in

close proximity without being fused (typically within 30 nm). MCSs were fi rstly described in the 1950s upon visualization by electron microscopy (Bernard and Rouiller, 1956;Copeland and

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Dalton, 1959). It turns out that numerous MCSs are formed in a living cell, between especially the ER and virtually all other organellar membranes. In yeast for example, extensive contact sites are formed between the ER membrane and the PM, where 20-45% of the PM surface is underlined with cortical ER (West et al., 2011). The two membranes can form very close associations, the endoplasmic reticulum-plasma membrane (ER-PM) junctions. Nowadays, a number of molecular players in the formation of contact sites are identifi ed (examples can be found in Pan et al., 2000;Kornmann et al., 2009;Manford et al., 2012) and emerging evidence arose for the importance of MCSs in various cellular processes. Contact sites seem to play a major role in the nonvesicular traffi cking of lipids and lipid homeostatis (Gaigg et al., 1995;Pichler et al., 2001;Kornmann and Walter, 2010;Elbaz-Alon et al., 2014;Honscher et al., 2014;Lahiri et al., 2015), but are also involved in signalling, autophagy and organellar division and inheritance (Friedman et al., 2011;Elbaz and Schuldiner, 2011;Helle et al., 2013;Klecker et al., 2014). Integral membrane proteins that act in the formation of membrane contacts or interact with those proteins are subsequently limited in lateral diffusion in the membrane and therefore are selectively concentrated and retained at those areas.

1.4 Aim and outline of the thesisWith the characterization of the minimal domain requirements of Heh2 and Src1/

Heh1 to be targeted to the INM in an energy-dependent manner by Kap60/95 (King et al., 2006;Meinema et al., 2011), several questions remained to be elucidated and those questions formed the starting point of this thesis.

The fi rst question we addressed was whether other protein besides Heh1 and Heh2 contain an ‘NLS-L-TM’ signature that mediates INM import. In chapter 2 we report about a genome-wide screen in Saccharomyces cerevisiae to identify candidate membrane proteins with a putative NLS and ID linker region. The role of these ‘NLS-L’ regions in subcellular localization of a membrane protein was determined using fl uorescence microscopy. In this study no new membrane proteins were found that accumulate at the INM in a Kap95-dependent manner. Unanticipated, we found few reporter proteins localized to the cell periphery, and the sorting of one of them is studied in chapter 4.

The second question that remained was whether the INM import mechanism described for Heh2 and Src1/Heh1 was conserved in higher eukaryotes. In chapter 3 we extended the screen for ‘NLS-L’ containing membrane proteins to the mammalian proteome, focussing on known INM localized membrane proteins with a predicted ID linker and NLS. One of our candidates, Pom121, has the reversed topology (‘TM-L-NLS’). We fi rst provide evidence that the reversed topology also facilitates INM import and present data that the Pom121NLS interacts with importin α adopting a similar fold as the NLSs of Src1/Heh1 and Heh2, although it is not able to compete with the IBB-domain in the absence of importin β, like the NLSs of Src1/Heh1 and Heh2 The Pom121NLS can functionally replace the NLS of Heh2, restoring INM localization of Heh2Δh2NLS and rescuing synthetic sickness of Heh2Δh2NLS in a strain lacking Nup84, confi rming the similarities between the NLS of Pom121 and Heh2 in

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vivo. Furthermore, we show NLS-dependent accumulation of h2NLS-L-TM at the INM in HEK293T cells. These data suggest that the active transport routes for membrane proteins to the INM that was described in yeast, is conserved in higher eukaryotes.

Our fi ndings of peripheral localized membrane proteins with ‘NLS-L-TM’-like sorting signals prompted us to investigate the role of ID linkers in this sorting route. We asked if ID linker regions could be suffi cient to span the distance between two heterologous membranes at membrane contact sites. In chapter 4 we show that the basic residues in the N-terminus of the amino acid sensor, Ssy1, function as a plasma membrane-binding (PMB) domain that, together with the ID linker region, forms a membrane-tethering module. A similar domain composition was identifi ed in Ist2, an ER-embedded integral membrane protein involved in the formation of ER-PM contact sites. We provide data that both Ssy1 and Ist2 are cortical ER residing membrane proteins of which the PMB domain interacts with components of the PM, while the ID linker, that separates the PMB from the TM, provides length and fl exibility to span the distance between the two membranes.

In chapter 5 we study where and how Ssy1 may sense extracellular amino acids, while residing in these membrane junctions and not the PM as previously thought. We show that Ssy1 is not able to sense a sudden increase of intracellular amino acids and found that the SPS sensing pathway (initiated by Ssy1), is still mildly activated in the absence of the chaperone Shr3 that is required for proper folding and targeting of AAPs to the PM. We discuss possible models for sensing from membrane junctions.

In chapter 6 we summarize the data presented in this thesis and give an outlook to future studies that will give a better insight in the targeting of ER-inserted integral membrane proteins, the role of ID linkers in the sorting process and the biological implications of effi cient targeting of membrane proteins to their destined location.

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