MISFOLDED WSC1P UNDERGO POST ER QUALITY CONTROL

61 Chapter 3 Golgi quality control captures misfolded Wsc1 proteins that evade ERQC .... 112 Chapter 4 The multi-vesicular body pathway is essential in the complete degradation of misfol

THE SECRETORY PATHWAY USES MULTIPLE

MECHANISMS FOR PROTEIN QUALITY CONTROL

WANG SONGYU

(B.Sc (Hons.), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE

2010

ACKNOWLEDGEMENTS

I am deeply grateful for my supervisor, Dr Davis Ng, for his valuable guidance and support throughout the course of this work His wide knowledge and interesting ideas have never failed to impress me His constant encouragement has always given me full confidence and is one of the major driving forces for me to complete my PhD

I would like to thank my thesis committee, Dr Wanjin Hong, Dr Snezhana Oliferenko and

Dr Cynthia He for their valuable comments and suggestions Special thanks are also given to Dr Graham Wright and Cristiana Barzaghi for their great help on confocal microscopy and for their patience to answer my numerous questions

Many thanks to all the past and current members of Davis’ lab, especially Dr Guillaume Thibault, Dr Kazue Kanehara, Dr Nurzian Ismail and Dr Chia Ling Hsu All of them have taught me how to be a good scientist and they are always there when I need help I would like to express my appreciation to Rupali, Chengchao, Alisha, Dr Shinichi Kawaguchi, Liu Ying, Sylvia, Gerard, Yu Jun, Sandy and Jeremy for stimulating scientific discussions and friendship Thanks also go to Hong Xin, Jing Jing, Xue Jing, Lu Song, Anbu and Sook Keat for our happy friendships

Last but not least, I dedicated this thesis to my beloved husband Seng Kah and parents for their love, support, help and encouragement throughout these years Without them, I could not have completed my PhD smoothly

TABLE OF CONTENTS

SUMMARY v

LIST OF TABLES vii

LIST OF FIGURES viii

LIST OF VIDEOS viii

LIST OF SYMBOLS AND ABBREVIATIONS xi

LIST OF PUBLICATIONS xiii

Chapter 1 Introduction 1

1.1 Quality control in the ER 1

1.1.1 ER retention of misfolded and unassembled proteins 2

1.1.1.1 By BiP and other molecular chaperones 2

1.1.1.2 Thiol-mediated retention 4

1.1.1.3 By chaperone-like molecule Rer1p 5

1.1.2 Substrate recognition during ERAD 6

1.1.3 Classification of ERAD pathways 8

1.1.4 Retrotranslocation and degradation by the ubiquitin-proteasome system11 1.1.5 Degradation of endogenous proteins 12

1.2 Balance among folding, ER export and quality control 13

1.2.1 ER export 13

1.2.1.1 COPII coat formation 14

1.2.1.2 Signals in transmembrane cargoes 16

1.2.1.3 Export of soluble cargoes from the ER and transmembrane sorting receptors 17

1.2.1.4 Packaging chaperones that modulate ER exit 19

1.2.1.5 Oligomeric assembly 20

1.2.2 Competition between ER export and ER retention for misfolded proteins 21

1.3 Post-ER quality control 23

1.3.1 Substrate recognition in the Golgi apparatus 24

1.3.1.1 Receptor-mediated mechanism 24

1.3.1.2 A Golgi environment-specific recognition 27

1.3.1.3 Golgi modifications mark mutant proteins abnormal 28

1.3.1.4 Aggregation in the Golgi lumen 29

1.3.2 Plasma membrane quality control 29

1.4 The ESCRT machinery and the multi-vesicular bodies 31

1.4.1 Function of MVBs in the biosynthetic and endocytic pathway 31

1.4.2 The multivesicular body biogenesis requires ESCRT complexes 34

1.5 Ubiquitin signals in the biosynthetic and endocytic pathways 39

1.5.1 Ubiquitin-dependent endocytosis 40

1.5.2 Ubiquitin as a signal for MVB sorting 42

1.5.3 Ubiquitin-dependent sorting at the trans-Golgi network 42

1.5.4 Deubiquitinating enzymes 43

1.6 Wsc1p as a model substrate 43

1.7 Thesis objectives 47

Chapter 2 Materials and methods 50

2.1 S cerevisiae strains and growth media 50

2.1.1 List of strains 50

2.1.2 Growth media 50

2.2 Genetic and molecular methods 50

2.2.1 Yeast transformation 50

2.2.1.1 Plasmid transformation via a simple and rapid way 50

2.2.1.2 High efficiency DNA fragment transformation 51

2.2.2 Strain construction via mating, sporulation and tetrad dissection 51

2.2.3 Yeast genomic DNA extraction 52

2.3 Plasmid construction 52

2.3.1 Site-directed mutagenesis 55

2.3.2 Oligonucleotide primers used in this study 55

2.4 Protein biochemistry and cell biology 55

2.4.1 Antibodies 55

2.4.2 SDS-PAGE and immunoblot analysis 56

2.4.3 Preparation of yeast extracts 56

2.4.4 Co-immunoprecipitation 57

2.4.5 Cell labeling and Immunoprecipitation analysis 58

2.4.5.1 Metabolic pulse-chase analysis and denaturing immunoprecipitation 58

2.4.5.2 PEGylation-based protein-folding assay 59

2.5 Microscopy 60

2.5.1 Indirect immunofluorescence 60

2.5.2 Live cell imaging 61

Chapter 3 Golgi quality control captures misfolded Wsc1 proteins that evade ERQC 69

3.1 Introduction 69

3.2 Wsc1p variants are misfolded 71

3.2.1 Wsc1p variants are transported from the ER to the Golgi via COPII vesicles 71

3.2.2 All Wsc1p variants are grossly misfolded 74

3.3 Misfolded Wsc1p is an obligate substrate of Golgi quality control 79

3.3.1 The variants are subject to protein quality control 79

3.3.2 Wsc1p variants are degraded independent of ERAD 80

3.3.3 Misfolded Wsc1p traffics to the vacuole for degradation 84

3.3.4 The degradation is autophagy independent 87

3.3.5 Golgi quality control recognizes misfolded Wsc1p 88

3.4 Misfolded Wsc1p evades ER surveillance 91

3.4.1 ERQC does not recognize Wsc1p variants when they are retained in the ER 91

3.4.2 Wsc1p lacks an ERAD determinant in its luminal domain 95

3.4.3 ER chaperone Kar2p does not recognize misfolded Wsc1p 100

3.5 Discussion 105

3.5.1 Reported substrates involved in Golgi QC 105

3.5.2 The machinery of Golgi QC 107

3.5.3 ER retention of soluble misfolded Wsc1p 110

3.5.4 ER export of misfolded proteins 111

3.5.5 Poor recognition of misfolded Wsc1p by Kar2p 112

Chapter 4 The multi-vesicular body pathway is essential in the complete degradation of misfolded membrane proteins in Golgi quality control 113

4.1 Introduction 113

4.2 Misfolded Wsc1 proteins are degraded in the vacuolar lumen 116

4.3 ESCRT mutants alter the vacuolar localization pattern of misfolded Wsc1p 122

4.4 The MVB pathway is essential for complete degradation of misfolded Wsc1p128 4.5 Re-routing of misfolded Wsc1p to the plasma membrane in ESCRT mutants 131 4.6 Entry of misfolded Wsc1p into the MVB pathway is ubiquitination dependent 133

4.7 Discussion 141

4.7.1 The dual functions of the cytoplasmic domain of Wsc1p 143

4.7.2 The cell surface re-routing pathway 143

4.7.3 The ubiquitination of misfolded Wsc1p 144

4.7.4 The importance and physiological relevance of the MVB pathway in protein quality control 145

Chapter 5 Conclusions and future perspectives 148

REFERENCES 151

SUMMARY

Quality control (QC) mechanisms monitor the folding and assembly of newly synthesized proteins The most well characterized QC pathway occurs in the ER and is termed ER quality control (ERQC) which targets misfolded proteins to be degraded via ER-associated degradation (ERAD) Post-ER QC pathways, albeit poorly understood, function to capture proteins that exit the ER prematurely In our study, we reported a yeast plasma membrane protein Wsc1p to be a substrate that demonstrates the fundamental role of the Golgi in protein QC A panel of Wsc1p variants misfolded in the extracellular/luminal domain was generated The variants are degraded in an ERAD-independent pathway Instead, they traffic to the Golgi from where they are delivered to the vacuole for degradation Two reasons can account for the ERQC evasion of Wsc1p First, a strong export signal in the cytoplasmic domain renders its efficient ER exit whether it is folded or not and whether it contains an ERAD determinant Second, the luminal domain of Wsc1p lacks functional ERAD signals and a chaperone binding site The identification and characterization of Wsc1p as an endogenous and obligate substrate reinforces the importance of the Golgi QC as a primary surveillance mechanism in the secretory pathway and provides a physiological basis for its existence

Golgi QC generally recognizes misfolded proteins in the Golgi apparatus and targets them to the vacuole/lysosome for degradation For misfolded membrane proteins, there are two fates They can be localized to either the limiting vacuolar/lysosomal membrane

or the lumen To understand how Golgi QC delivers its misfolded membrane proteins to

the vacuole, we examined Wsc1p variants with a misfolded luminal domain that are bona fide substrate of Golgi QC We found that the mutants are transported from the Golgi to the vacuolar lumen via the multi-vesicular body (MVB) pathway MVB sorting requires ubiquitination at the lysine residue(s) in the cytoplasmic domain of misfolded Wsc1p and the endosomal sorting complex required for transport (ESCRT) machinery Most importantly, mislocalization of the variants at the limiting vacuolar membrane results in a series of degradation fragments suggesting incomplete elimination This provides a physiological basis for the vacuolar lumen targeting of misfolded membrane substrates in Golgi QC It ensures efficient degradation of the entire molecules and prevents the accumulation of potentially toxic fragments

LIST OF TABLES

Table 1.1 ER export signals in transmembrane and soluble cargoes 17

Table 1.2 Transmembrane cargoes of the MVB pathway 33

Table 2.1 Strains used in chapter 3 62

Table 2.2 Strains used in chapter 4 64

Table 2.3 Plasmids modified by site-directed mutagenesis in chapter 3 66

Table 2.4 Plasmids used in chapter 4 67

Table 2.5 Oligonucleotide primers used in chapter 3 68

Table 2.6 Oligonucleotide primers used in chapter 4 68

Table 4.1 Effect of ESCRT mutants on the localization of Wsc1-L63R-GFP 127

LIST OF FIGURES

Figure 1.1 ERAD recognition of misfolded N-glycosylated proteins 8

Figure 1.2 ERAD pathways in yeast 10

Figure 1.3 Bidirectional transport between the ER and Golgi apparatus 14

Figure 1.4 COPII vesicle formation 15

Figure 1.5 Model for post-ER quality control 23

Figure 1.6 The ubiquitination pathway 27

Figure 1.7 The ESCRT machinery 38

Figure 1.8 Molecular mechanism of MVB biogenesis 38

Figure 1.9 A cartoon depicting Wsc1p 46

Figure 1.10 O-mannosylation in yeast and mammalian cells 47

Figure 3.1 Generation of Wsc1p variants 72

Figure 3.2 Wsc1p and its variants show similar mobility 73

Figure 3.3 The principle of the PEGylation-based protein folding assay 76

Figure 3.4 CPY* is grossly misfolded 77

Figure 3.5 Wsc1p variants are misfolded 78

Figure 3.6 Wsc1-L63R is degraded rapidly 79

Figure 3.7 Wsc1-L63R is degraded independent of ERAD 81

Figure 3.8 Degradation of Wsc1p variants does not require ERAD 82

Figure 3.9 Wsc1-L63R is degraded independent of the proteasome 83

Figure 3.10 Wsc1-L63R is transported to the vacuole for degradation 84

Figure 3.11 Misfolded Wsc1p degrades in the vacuole 85

Figure 3.12 The stabilization results in strong vacuolar staining of misfolded Wsc1p in ∆pep4 cells 86

Figure 3.13 Visualization of the vacuolar ATPase by indirect immunofluorescence 86

Figure 3.14 Wsc1-L63R is not degraded via the autophagy pathway 87

Figure 3.15 Wsc1-L63R is transported to the vacuole via the Golgi 89

Figure 3.16 Misfolded Wsc1p is degraded by Golgi QC 90

Figure 3.17 Wsc1p mutants are stabilized when the transport from the ER to the Golgi is blocked 92

Figure 3.18 Generation of the soluble version of misfolded Wsc1p 93

Figure 3.19 The soluble forms of Wsc1p variants are retained in the ER 93

Figure 3.20 Wsc1-L63RLuminal and Wsc1-68-80Luminal are stable in the ER 94

Figure 3.21 The slight mobility shift of Wsc1-L63RLuminal and Wsc1-∆68-80Luminal is due

to ER modifications 94

Figure 3.22 An ERAD determinant is appended to Wsc1p variants 97

Figure 3.23 ED-Wsc1-L63R and ED-Wsc1-∆68-80 are ERAD substrates 98

Figure 3.24 ED-Wsc1-L63RLuminal and ED-Wsc1-∆68-80Luminal are completely dependent on ERAD for degradation 99

Figure 3.25 Misfolded Wsc1p is not recognized by the major ER chaperone BiP/Kar2p 103

Figure 3.26 Misfolded Wsc1p fused with an ERAD determinant binds Kar2p efficiently 104

Figure 3.27 Degradation of misfolded Wsc1p is partially Vps10p dependent 109

Figure 4.1 Wsc1-L63R is localized to the vacuolar lumen in the ∆pep4 strain 118

Figure 4.2 (PGAS1)Wsc1-L63R behaves similarly to (PPRC1)Wsc1-L63R 120

Figure 4.3 Wsc1-∆68-80 is localized to the vacuolar lumen in ∆pep4 cells 121

Figure 4.4 ESCRT proteins are essential in transporting Wsc1-L63R-GFP to the vacuolar lumen 124

Figure 4.5 Wsc1-L63R-GFP is degraded in a Pep4p-dependent manner 124

Figure 4.6 ESCRT mutants alter the localization of Wsc1-L63R 125

Figure 4.7 The ESCRT mutants affect the localization of Wsc1-∆68-80 126

Figure 4.8 Misfolded Wsc1p is degraded into multiple fragments in ESCRT mutants 129 Figure 4.9 ESCRT mutants affect the degradation of (PGAS1)Wsc1-∆68-80 130

Figure 4.10 Misfolded Wsc1p is re-routed to the plasma membrane in ESCRT mutants 132

Figure 4.11 Wsc1-L63R is ubiquitinated by Rsp5p before entry into the MVB pathway 136

Figure 4.12 The entry of Wsc1-∆68-80 into the MVB pathway requires Rsp5p 137

Figure 4.13 Ubiquitination at the lysine residue(s) of Wsc1-L63R provides the MVB sorting signal 139

Figure 4.14 Wsc1-∆68-80-3R is not sorted to the vacuolar lumen via the MVB pathway 140

Figure 4.15 Model of the MVB-dependent pathway for the transport of misfolded Wsc1p 142

Figure 5.1 The basis of the genetic screen using invertase-misfolded Wsc1p fusion protein 150

LIST OF VIDEOS (Please see attached CD)

Video 4.1 Wsc1-L63R is localized to the vacuolar lumen in ∆pep4 cells

Video 4.2 Wsc1-∆68-80 is localized to the vacuolar lumen in ∆pep4 cells

Video 4.3-4.5 Wsc1-L63R is localized to the class E compartment (exaggerated

prevacuolar compartments) and the limiting vacuolar membrane in ∆pep4∆vps27 (Video 4.3), ∆pep4∆vps36 (Video 4.4) and ∆pep4∆vps37 (Video 4.5) cells

Video 4.6-4.7 Wsc1-L63R is localized to the limiting vacuolar membrane in rsp5-1

(Video 4.6) and ∆pep4 rsp5-1 (Video 4.7) cells

Video 4.8-4.9 Wsc1-L63R-3R is localized to the limiting vacuolar membrane in both

wild-type (Video 4.8) and ∆pep4 (Video 4.9) cells

LIST OF SYMBOLS AND ABBREVIATIONS

COPII Coat protein complex II

ERAD Endoplasmic reticulum associated degradation

ERAD-C Endoplasmic reticulum associated degradation-Cytosol

ERAD-L Endoplasmic reticulum associated degradation-Lumen

ERAD-M Endoplasmic reticulum associated degradation-Membrane

ERGIC ER-Golgi intermediate compartment

ERQC Endoplasmic reticulum quality control

ESCRT Endosomal sorting complex required for transport

FM4-64 N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl)

pyridinium dibromide Golgi QC Golgi quality control

Mal-PEG Methoxypolyethylene glycol 5000 maleimide MVB Multi-vesicular body

NEF Nucleotide exchange factor

PI(3)P Endosomal lipid phosphatidylinositol 3-phosphate PrP Prion protein

PVC Prevacuolar compartment

QC Quality control

RTK Receptor tyrosine kinase

S cerevisiae Saccharomyces cerevisiae

SC Synthetic complete

SD Standard deviation

TGN Trans-Golgi network

UPS Ubiquitin-proteasome system

VPS Vacuolar protein sorting

LIST OF PUBLICATIONS

Wang, S., and Ng, D.T (2010) Evasion of endoplasmic reticulum surveillance makes

Wsc1p an obligate substrate of Golgi quality control Mol Biol Cell 21, 1153-1165

Wang, S., Thibault, G., and Ng, D.T The multi-vesicular body pathway is essential in the complete degradation of misfolded membrane proteins in Golgi quality control

Manuscript in preparation

Wang, S., and Ng, D.T (2008) Lectins sweet-talk proteins into ERAD Nat Cell Biol

10, 251-253

Chapter 1 Introduction

Proteins carry out the vast majority of cellular functions To work properly, they must fold into correct and specific conformations during synthesis The complexity of folding

pathways causes up to a third of all proteins to fold improperly (Schubert et al., 2000)

Since misfolded proteins can be toxic, cells have evolved various mechanisms such as molecular chaperones and protease systems to monitor folding These quality control (QC) systems play a vital role in the health of an organism by segregating and degrading aberrant proteins Their importance is underscored by the numerous protein

conformational diseases (e.g., Alzheimer’s, Huntington’s, and cystic fibrosis) that afflict

tens of millions annually

1.1 Quality control in the ER

In eukaryotic cells, secretory and transmembrane (TM) proteins enter the endoplasmic reticulum (ER) for maturation To ensure that only properly folded and assembled proteins are exported, mechanisms collectively termed ER quality control (ERQC) allow only properly folded proteins to be transported to their sites of functions Misfolded or unassembled proteins that fail to pass ERQC are retained in the ER, retrotranslocated from the lumen and rapidly degraded by the cytoplasmic ubiquitin-proteasome system (UPS) This terminal step has been termed ER-associated degradation (ERAD) (for

reviews, see Hegde and Ploegh, 2010; Hirsch et al., 2009; Vembar and Brodsky, 2008)

The stringent ERQC process ensures the fidelity of proteins that are destined to the

extracellular matrix, plasma membrane and the compartments that are involved in secretion and endocytosis

1.1.1 ER retention of misfolded and unassembled proteins

One important aspect of ERQC is to retain misfolded and unassembled proteins in the ER Retention can be passive or active Proteins that lack ER exit signals are passively retained but the exact mechanism is unclear Active retention requires cooperative or sequential actions of molecular chaperones or chaperone-like proteins and will be discussed in detail

1.1.1.1 By BiP and other molecular chaperones

The 70 kDa heat-shock protein (Hsp70) chaperone family interacts with substrates that expose hydrophobic patches The ER luminal Hsp70-family member BiP (immunoglobulin heavy chain binding protein) associates with unfolded proteins that are

in the process of folding and misfolded ERAD substrates In fact, Bip was originally identified as an immunoglobulin (Ig) heavy chain (HC) binding protein HCs interact with BiP transiently in the presence of light chain (LC) They subsequently assemble with LCs and are capable of ER export In the absence of LC expression, HCs synthesized in a

pre-B cell line associate with BiP and are retained in the ER (Bole et al., 1986; Haas and

Wabl, 1983) Similarly, unassembled LCs associate with BiP as partially oxidized species

whereas secreted LCs bind to BiP briefly and are fully oxidized after release (Knittler et

al., 1995) Its yeast homologue Kar2p similarly retains misfolded proteins in the ER For

example, CPY* which is a mutant form of CPY (carboxypeptidase) and is a

well-established ERAD substrate contains several binding sites for Kar2p (Finger et al., 1993; Xie et al., 2009) BiP/Kar2p contains a KDEL consensus sequence at its C-terminus

which is an ER retention motif (Munro and Pelham, 1987) This in turn retains its associated proteins in the ER Once the protein is folded, it is released from BiP/Kar2p and transported to its destination Prolonged interaction with BiP/Kar2p leads to ERAD

After discovery of the interaction between BiP and unassembled HCs, it was subsequently found that other chaperones interact with misfolded or unassembled proteins after BiP interaction One example is GRP94, an Hsp90 chaperone BiP first associates with unassembled Ig chains in an early disulphide intermediate form and dissociates afterwards Next, GRP94 binds fully oxidized molecules and dissociates much

more slowly (Melnick et al., 1994) Two other examples are VSV-G (a vesicular

stomatitis virus glycoprotein protein) and thyroglobulin VSV-G is first bound by BiP and is subsequently associated with calnexin, a lectin-like ER protein (Hammond and Helenius, 1994) On the contrary, thyroglobulin interacts with calnexin first, followed by BiP (Kim and Arvan, 1995) The difference in the interaction order could be due to specific structural features of substrates

In addition to the sequential action between BiP and other chaperones, BiP is able to act synergistically with chaperones like PDI (protein disulfide isomerase) in retaining misfolded ERAD substrates in the ER BACE457 is a pancreatic isoform of human beta-secretase It folds inefficiently in the ER and is degraded by ERAD The folding is sequentially assisted by calnexin and BiP/PDI before being dislocated into the cytosol for

degradation After being released from calnexin, it forms intermolecular disulfide-bonded complexes PDI acts as an oxidoreductase and a redox-driven chaperone to retain and

disassemble complexes for dislocation (Molinari et al., 2002) Another example is

unassembled procollagen chains which are retained by PDI within the ER They are

bound to PDI and the retention depends on the ER retrieval signal of PDI (Bottomley et

al., 2001) In addition, the PDI family is also critical in thiol-mediated retention

mechanism which will be discussed in the next section

1.1.1.2 Thiol-mediated retention

Individual subunits of an IgM polymer are linked by disulfide bonds involving Cys575 which is located in the C-terminal tailpiece of µ chains Cys575 is a key residue

regulating assembly, retention and degradation of unpolymerized IgM (Fra et al., 1993)

In B lymphocytes, polymerization is slow This leads to ER retention and degradation of

nearly all unassembled µ chains (Sitia et al., 1990) Unpolymerized IgM subunits are secreted by B cells if Cys575 is mutated (Sitia et al., 1990) Thiol-mediated retention is

mediated by at least in part ERp44, a member of the PDI family It is an ER resident protein with an RDEL motif and recycles between ER and post-ER compartments (the

ER-Golgi intermediate compartment [ERGIC] and cis-Golgi) (Anelli et al., 2007; Gilchrist et al., 2006; Wang et al., 2007) In the cis-Golgi, ERp44 captures

unpolymerized IgM subunits which are capable of ER exit and retrieves them back to the

ER in an RDEL-dependent manner (Anelli et al., 2002; Anelli et al., 2003; Anelli et al.,

2007)

Another substrate that is subject to thiol-dependent mechanism is adiponectin, a plasma

membrane protein secreted by adipocytes (Scherer et al., 1995) Plasma adiponectin can form trimers, hexamers and oligomers (Bobbert et al., 2005; Lara-Castro et al., 2006; Tonelli et al., 2004) ERp44 retains folded adiponectin trimers by forming mixed

disulfides with Cys39 in one of the subunits Ero1-Lα, an oxidoreductase in the ER lumen releases adiponectin from ERp44 and facilitates secretion of adiponectin oligomers

(Qiang et al., 2007; Wang et al., 2007) Together, these studies demonstrate that ERp44

plays an essential role in thiol-dependent retention mechanism during protein oligomerization

1.1.1.3 By chaperone-like molecule Rer1p

Rer1p is a highly conserved TM protein It is localized primarily to the Golgi apparatus

and recycles between Golgi compartments and the ER via COPI vesicles (Sato et al.,

2001) Initially identified in yeast genetic screens for mutants that failed to retain an ER resident protein Sec12p, Rer1p was subsequently found to be required to retain other ER

localized proteins like Sec63p, Sec71p and Mns1p (Boehm et al., 1994; Massaad et al., 1999; Nishikawa and Nakano, 1993; Sato et al., 1996) Interestingly, it serves as a QC

receptor functioning to retrieve unassembled iron transporter subunit Fet3p Rer1p interacts with Fet3p in the early Golgi and brings it back to the ER so that it can assemble

with its partner Ftr1p (Sato et al., 2004) Human Rer1 is also suggested to play a QC role

in regulation of secretase activity It can bind directly to unassembled subunits of the

γ-secretase complex and retrieve them to the ER (Kaether et al., 2007; Spasic et al., 2007)

The exact mechanism of Rer1p-dependent retrieval is unclear but it seems that it depends

on direct binding to sorting motifs in the TM domain of cargoes (Sato et al., 2001; Sato et

al., 2003; Sato et al., 2004)

1.1.2 Substrate recognition during ERAD

The major challenge of ERAD is to distinguish terminally misfolded proteins from newly synthesized proteins that are in the process of folding Recent studies have mostly focused on glycoproteins which are co-translationally modified on asparagines in a consensus Asn-X-Ser/Thr motif (X is any amino acid except proline) with an N-linked oligosaccharide to yield a glucose3-mannose9-N-acetylglucosamine2 (GlcNAc2-Man9-Glc3) glycan (Figure 1.1) N-glycan modification is to increase the solubility of proteins and to facilitate their folding processes More remarkably, it provides information about the folding status of glycoproteins First, removal of the two terminal glucose residues by Gls1p and Gls2p (glucosidases 1 and 2) marks the polypeptide that is in its folding process Further trimming of the final glucose residue by Gls2p results in a folded glycoprotein bearing a GlcNAc2-Man9 glycan They can be demannosylated by Mns1p (an α1,2-mannosidase) to yield a GlcNAc2-Man8 structure before ER exit (for review, see Xie and Ng, 2010)

Misfolded N-glycosylated molecules are distinguished from its folding counterparts based on the glycan structure If a glycoprotein bearing a GlcNAc2-Man9 glycan is unable

to fold within its folding window, Mns1p will trim Man9 to Man8at the B branch (Figure

1.1) (Camirand et al., 1991) This indicates that the protein requires prolonged ER

retention and folding Because Mns1p also trims folded proteins to yield similar glycans,

Man8 itself is not sufficient to be a degradation signal (Jakob et al., 1998; Jelinek-Kelly

and Herscovics, 1988) Work from the Aebi group has demonstrated that Htm1p (homologue to mannosidase 1) further processes the C branch, yielding a GlcNAc2-Man7

structure with an exposed α1,6-mannose (Clerc et al., 2009) This is in turn recognized

by Yos9p (yeast osteosarcoma 9) which contains an MRH (mannose-6-phosphate

receptor homology) domain (Quan et al., 2008) A luminal complex that consists of Yos9p, Hrd3p and Kar2p has been shown to be a receptor for ERAD substrates (Denic et

al., 2006) The subsequent delivery of misfolded glycoproteins by the Yos9p-Hrd3-Kar2p

complex to the Hrd1p E3 ubiquitin ligase leads to ERAD (Figure 1.2)

If a glycoprotein is modified with N-glycans at multiple sites, will Htm1p trim all of them

to a Man7 structure? This seems unlikely as analysis of two typical ERAD substrates CPY* and PrA* shows that only a specific glycan can trigger ERAD (Spear and Ng, 2005) Examination of the folded structures of CPY and PrA (proteinase A) reveals a unique feature of the two signal glycans Both are positioned at a local segment that seems to be unstructured If a protein is properly folded, the signal glycan will fold into the overall structure; otherwise, it will be processed by Htm1p to yield a Man7 sugar The resulting bipartite signal which is a Man7GlcNAc2 glycan attached to an unfolded/disordered structure signals ERAD Thus, this ERAD determinant functions as

an intrinsic sensor for the overall folding of the polypeptide (Xie et al., 2009)

Figure 1.1 ERAD recognition of misfolded N-glycosylated proteins

In yeast, Gls1p and Gls2p first remove the two outermost glucose residues to generate a GlcNAc2-Man9-Glc glycan Once the third sugar is removed by Gls2p, the protein is protected from degradation Mns1p trims a mannose from branch B, yielding a Man8

structure which can be recognized by Htm1p It generates a Man7 glycan by trimming a mannose at the C branch The exposed α1,6-mannose is in turn recognized by Yos9p

1.1.3 Classification of ERAD pathways

Studies based on both genetic and biochemical approaches carried out in the budding yeast have indicated that the misfolded lesion site of a substrate is a key factor to determine which ERAD pathway the substrate will be target to (Figure 1.2) Soluble and membrane proteins with misfolded luminal domains use ERAD-L (-Lumen) and ERAD-

M (-Membrane) pathways, respectively Membrane proteins with lesions in cytoplasmic

domains are destined to the ERAD-C (-Cytosol) pathway (Carvalho et al., 2006; Vashist

and Ng, 2004) The divergence is at the two distinct E3 ligase complexes assembled at the ER membrane The Hrd1p complex defines the ERAD-L/M pathway and the Doa10p complex refers to the ERAD-C pathway Both complexes function to capture and polyubiquitinate ERAD substrates by exposing them to the E3 ubiquitin ligase active site which is located at the cytosolic face of the ER membrane They share some common components such as the E2 ubiquitin conjugating enzyme Ubc7p (anchored to the ER

membrane via Cue1p) and Cdc48p together with its cofactors Ufd1p and Npl4p

(Carvalho et al., 2006; Denic et al., 2006; Vashist and Ng, 2004) In addition, extra

players are found in the Hrd1p complex and function to recognize and deliver misfolded

proteins to the ERAD machinery (Carvalho et al., 2006; Denic et al., 2006) These include Hrd3p, a TM protein that forms a complex with Hrd1p (Gardner et al., 2000) and Yos9p which is bound to Hrd3p (Denic et al., 2006; Gauss et al., 2006) and serves as a lectin (Bhamidipati et al., 2005; Kim et al., 2005; Quan et al., 2008; Szathmary et al.,

2005) It is important to note that ERAD-L/M and -C pathways are only defined in yeast

In higher eukaryotes, the ERAD machinery is more complicated and the boundary among the pathways is probably more blurred as there is an expansion of molecular components

Figure 1.2 ERAD pathways in yeast

(A) The Hrd1p complex recognizes proteins with a misfolded luminal or TM domain and defines ERAD-L/-M Soluble substrates are retrotranslocated via a translocon whose identity remains controversial (B) ERAD-C substrates contain a misfolded cytoplasmic domain and are recognized by the Doa10p complex Both pathways converge at the Cdc48/Ufd1/Npl4 extraction step After substrates are dislocated into the cytosol, they are subject to ubiquitin-proteasomal degradation

1.1.4 Retrotranslocation and degradation by the ubiquitin-proteasome system

The catalytic sites of both E3 ubiquitin ligases are located at the cytosolic side of the ER membrane Membrane substrates may access the ubiquitination site by lateral diffusion in the plane of the membrane and their cytosolic domains may be placed close to the catalytic sites Thus, a retrotranslocon is probably not necessary in ERAD-M and ERAD-

C On the contrary, luminal substrates must cross the ER membrane in order to be accessible to E3 The identity of the protein conduit is still an enigma Nevertheless, several candidates have been proposed and they may exhibit substrate specificity These include Sec61p which is a translocon for forward transport of the nascent polypeptides

into the ER, Derlins, Hrd1p and Doa10p (for review, see Hebert et al., 2010)

Alternatively, misfolded proteins may exit the ER through lipid droplets independent of a

proteinaceous channel (Ploegh, 2007) Perhaps, an in vitro retrotranslocation assay is the

best way to resolve the issues

Once in the cytosol, substrates are polyubiquitinated by E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases The polyubiquitin chain is in turn recognized by the Cdc48p (p97 in mammals) complex, a hexameric AAA+ ATPase with two associated cofactors Ufd1p and Npl4p (Ufd1 and Npl4 in mammals) (for review, see Hegde and

Ploegh, 2010; Hirsch et al., 2009 and references therein) The recruitment of the cytosolic

complex to the ER membrane is probably mediated by UBX2 (a UBX-domain-containing membrane protein) in yeast or VIMP (valosin-containing protein-interacting membrane

protein) in mammals (Ye et al., 2004) How the Cdc48/p97 complex detects ERAD

substrates is currently unknown

The energy to extract substrates from the membrane into the cytosol is provided by the ATPase activity of the Cdc48/p97 complex The exact molecular mechanism of the process remains explored After extraction, substrates are delivered to the proteasome by Rad23p and Dsk2p which have domains to recognize polyubiquitin chains The proteasome subunit Rpn10p and Rpn13p recognize polyubiquitinated substrates Before proteasomal degradation, substrates are deubiquitinated so that ubiquitin moieties are

recycled (for review, see Hirsch et al., 2009 and references therein)

1.1.5 Degradation of endogenous proteins

Many endogenous proteins mature inefficiently It has been reported that 30–75% of the proteins that are synthesized are degraded within 20 min, and this includes proteins that

are being degraded as growing nascent chains (Schubert et al., 2000) It is proposed that

degraded newly synthesized and nascent proteins are in fact a source of peptides to be loaded onto MHC class I molecules and this is crtical in immune defense One potential reason for the inefficient folding of many native proteins is that they are often optimized for function but not folding and assembly during evolution For instance, more than 40%

of newly synthesized CFTR (cystic fibrosis TM conductance regulator) and the δ opioid

receptor are degraded (Kopito, 1999, Petaja-Repo et al., 2001) On the other hand, many

disease-associated mutant proteins may retain some biological activity but they are rapidly recognized by QC and degraded One typical example is CFTR-∆508 which

contains a point mutation in the cytoplasmic domain and is degraded by ERAD (Cheng et

al., 1990) However, at low temperature or in the presence of chemical chaperones, a

fraction of CFTR-∆508 is rescued from ER retention and traffics to the plasma membrane

(Denning et al., 1992, Gelman and Kopito, 2002)

Similarly, orphan subunits of an oligomeric complex and partially assembled oligomers are often retained in the ER and degraded This may provide a protective function for cells For example, the light chains of immunoglobulin complex are often synthesized in excess of the heavy chains which are aggregation prone and may form toxic aggregates in the absence of light chains Therefore, this ensures all heavy chains are assembled into the complex (Sitia, 1995)

In spite of the positive sides, many proteins form aggregates when they fail to fold and those aggregates often lead to various diseases Aggregates like amyloid plaques are frequently not recognized by the QC system and their accumulation is toxic to cells (Dobson, 2002) A lack of recognition can be attributed to a failure in QC Althernative,

it is possible that their conformation is somehow not “seen” as misfolded by the QC system In either case, this results in poor degradation and the onset of neurodegenerative diseases

1.2 Balance among folding, ER export and quality control

1.2.1 ER export

Properly folded secretory proteins are ready for ER export in order to be delivered to sites

of function From the ER, the coat protein complex II (COPII) forms transport vesicles and collects cargoes with appropriate sorting signals into these vesicles Forward

transport via COPII vesicles to the Golgi is balanced by a retrograde transport using COPI coats back to the ER (Figure 1.3) (for reviews, see Dancourt and Barlowe, 2010;

Lee et al., 2004; Watanabe and Riezman, 2004) Although ERQC ensures most

transported proteins are fully folded and assembled, some misfolded or unassembled proteins are exported out of the ER and delivered to post-ER compartments (for reviews,

see Anelli and Sitia, 2008; Arvan et al., 2002)

Figure 1.3 Bidirectional transport between the ER and Golgi apparatus

The anterograde/forward transport mainly refers to cargoes exiting the ER via COPII vesicles The traffic is balanced by the retrograde transport which involves budding of COPI vesicles from the Golgi compartments The recycling process ensures a balance of vesicle components and the retrieval of ER resident proteins that have escaped the ER Overall, there is a net anterograde transport of secretory proteins from the ER and resident proteins remain localized in their respective organelles (Adapted from Dancourt and Barlowe, 2010)

1.2.1.1 COPII coat formation

The COPII machinery is assembled at specialized regions of ER membrane known as ER

the nucleotide exchange factor (NEF) Sec12p It results in the insertion of the N-terminal helix of Sar1p-GTP into the bilayer to initiate membrane curvature This stabilizes Sar1p

on the ER membrane and subsequently attracts the Sec23p-Sec24p complex via the interaction between Sar1p-GTP and Sec23p Soluble cargoes through TM cargo receptors

or TM cargoes are recruited by interaction with Sec24p The outer layer Sec13p-Sec31p assembles around Sar1p-Sec23p-Sec24p-cargo complexes and the entire complexes are concentrated due to the self-assembly ability of Sec13p-Sec31p This forms a cuboctahedral caged structure that deforms the ER membrane and buds COPII vesicles Finally, GTP hydrolysis by Sar1p allows cargoes to be released from sorting receptors and COPII coat proteins to be disassembled from budded vesicles for recycling (Figure

1.4) (for reviews, see Dancourt and Barlowe, 2010; Gurkan et al., 2006)

Figure 1.4 COPII vesicle formation

Sar1p is first activated by Sec12p and becomes membrane bound Activated Sar1p (Sar1p-GTP) recruits the inner coat layer Sec23p-Sec24p complex Sec24p recognizes sorting signals in TM cargoes and TM receptors Finally, the outer layer Sec13p-Sec31p complex is recruited Assembly of the entire cargo complex leads to coat polymerization and budding from the ER membrane (Adapted from Dancourt and Barlowe, 2010)

1.2.1.2 Signals in transmembrane cargoes

In both mammalian and yeast cells, some TM cargo proteins possess an ER exit signal (For review, see Barlowe, 2003; Watanabe and Riezman, 2004) VSV-G is a type I TM protein that traffics to the cell surface It contains a diacidic (DXE) motif in its cytoplasmic domain and similar motifs are found in other secreted type I TM proteins (Table 1.1) It is shown that those diacidic residues can directly bind to Sec23p–Sec24p (Votsmeier and Gallwitz, 2001) and they are necessary to form pre-budding complexes

with Sar1p and Sec23p–Sec24p (Malkus et al., 2002) In addition to diacidic motifs, other signals including dihydrophobic or diaromatic and dibasic motifs in the cytoplasmic tails

of TM cargoes have been reported All these signals are able to bind to COPII subunits

(Giraudo and Maccioni, 2003; Kappeler et al., 1997; Nufer et al., 2002).

Table 1.1 ER export signals in transmembrane and soluble cargoes

Proteins Signals Recognition Mechanism Reference

Transmembrane

cargoes

Direct recognition by COPII components Diacidic (DXE)a

1997; Sevier et al., 2000

2001

Dihyrophobic (FF, LV)

Axl2p, Hxt1p,

Gap1p, Mid2p Unknown Erv14p Castillon et al., 2009; Powers and Barlowe,

1998 ALP, Ktr3p Unknown Erv26p Bue et al., 2006;

Inadome et al., 2005

a Underlined residue is critical in ER export of the cargo

1.2.1.3 Export of soluble cargoes from the ER and transmembrane sorting receptors

Export of soluble cargoes from the ER is proposed to be mediated by the bulk flow or

receptor-mediated models The bulk flow process is passive and non-selective (Wieland

et al., 1987) After folding, cargo proteins enter transport vesicles at their prevailing

concentrations and no signal is required One example is amylase and chymotrypsinogen

from the ER of pancreatic exocrine cells (Martinez-Menarguez et al., 1999) They are

concentrated into tubular structures instead of COPII-coated buds The enrichment is

mostly like due to exclusion from retrograded COPI vesicles However, in vitro assay

using three bulk flow markers show that only ~0.6-2% of cargoes are packaged into

COPII vesicles, indicating the inefficiency of the bulk flow mode of transport (Malkus et

al., 2002) Thus, it is possible that this mode is used by certain secreted proteins

Receptor-mediated transport mode operates by enrichment of soluble cargo proteins in transport vesicles at concentrations ~3- to 50-fold higher than the bulk flow markers This active process requires luminal cargoes to be recognized by their respective TM cargo receptors which interact with the COPII coat Examples for such membrane receptors include ERGIC53, the p24 proteins and Erv29p that cycle between the ER and Golgi

compartments (Appenzeller et al., 1999; Belden and Barlowe, 2001; Muniz et al., 2000; Nichols et al., 1998) ERGIC53 acts in ER export of fully folded glycoproteins (Itin et al.,

1996) Yeast cells lacking the p24 protein Emp24p or Erv25p display selective trafficking defects including the delayed transport of GPI-anchored protein Gas1p (Belden and

Barlowe, 1996; Schimmoller et al., 1995) Another receptor is Erv29p which is required

for transport of soluble cargo proteins pro-α-factor, CPY and PrA (Belden and Barlowe,

2001; Malkus et al., 2002) In addition, Erv14p which is a highly conserved protein is

required for ER export of many TM proteins including Axl2p, Hxt1p, Gap1p and Mid2p

(Castillon et al., 2009; Powers and Barlowe, 1998) Erv26p is required for efficient ER

exit of ALP (alkaline phosphatase) and the Ktr3p mannosyltransferase (Bue and Barlowe,

2009; Bue et al., 2006) Although cargoes are not necessarily to be soluble, all of them

share a common feature The ER exit signals in the cytoplasmic domain, if present, do not play a role in interacting with TM receptors Instead, it seems that the interaction is via a yet to be identified exit motif in the luminal domain It is likely that the motif is a

structural basis rather than being a consensus primary amino acid sequence The motif is only formed after the cargo is properly folded

1.2.1.4 Packaging chaperones that modulate ER exit

Some membrane proteins have dual functions They act as a molecular chaperone and a sorting receptor to facilitate folding and ER exit of the cargo For example, a multispanning ER membrane protein Shr3p is specifically required for ER export of amino acid permeases like Gap1p The interaction between Shr3p and the COPII coat

delivers permeases into transport vesicles and Shr3p itself is not packaged into (Gilstring

et al., 1999) In ∆shr3 cells, amino acid permeases aggregate, accumulate in the ER and

are degraded by ERAD (Kota and Ljungdahl, 2005; Kota et al., 2007) Another ER

membrane protein Gsf2p is required for export of hexose transporters from the ER (Sherwood and Carlson, 1999) Chs7p facilitates ER exit of a plasma membrane chitin

synthase III (Trilla et al., 1999); Pho86p is required for ER transport of the phosphate transporter Pho84p (Lau et al., 2000) Yeast cells lacking Gsf2p, Chs7p and Pho86p lead

to aggregation of their respective substrates (Kota and Ljungdahl, 2005) In addition, Vma22p is also required for the proper ER exit of Vph1p, a 100 kDa membrane subunit

of the yeast V-ATPase In the absence of Vma22p, Vph1p is rapidly degraded by ERAD (Hill and Cooper, 2000) Similar examples are observed in higher eukaryotes A

Drosophila protein, NinaA, seems to function as an isomerase and regulates ER export of

rhodopsin (Baker et al., 1994; Colley et al., 1991) The ER membrane proteins BAP31

and BAP29 promote ER export of secretory proteins such as MHCI (major

histocompatibility complex class I) and cellubrevin (Annaert et al., 1997; Paquet et al.,

2004) A recently identified protein TANGO1 is required for transport of collagen out of

the ER (Saito et al., 2009) In all these cases, these receptors play a role in folding and

delivery of the cargo to ER exit sites However, how they assist in folding and ER export remains to be characterized

1.2.1.5 Oligomeric assembly

Some membrane proteins use assembly instead of accessory proteins to facilitate ER export Assembly can mask retention signals present in the TM domain One example is unassembled T cell receptor α (TCRα) with two basic residues in the TM domain that is

retained in the ER and subject to ERAD (Bonifacino et al., 1990a; Bonifacino et al.,

1990b) Once TCRα assembles with its CD3 partners of which TM domain contains

acidic residues, the resulting complex is capable of ER exit (Wileman et al., 1990) The

retention signal is the two basic residues and assembly helps neutralize the charges and therefore shield the signal The other well-characterized example is the unassembled µ

HC of IgM (Stevens et al., 1994) It contains two hydrophilic residues in the TM domain

which is an ER retention signal in the absence of LC expression

Alternatively, it seems that assembly promotes ER export via other mechanisms One possibility is a conformation dependent ER exit signal is only achieved after oligomerization It is also possible that one ER export signal is not sufficient until the combination of multiple signals after the assembly This may increase the affinity of the COPII coat for the cargo protein Homo-oligomerization and disulfide-linked stabilization of ERGIC53is necessary for its efficient ER export even though it has an

export signal in the cytoplasmic domain (Nufer et al., 2002) The yeast homologues of

ERGIC53, Emp46 and Emp47 both contain ER exit signals in the cytoplasmic domain and interact with COPII components However, assembly is required for efficient export (Sato and Nakano, 2002; Sato and Nakano, 2003) Another similar example is the complex of Erv41p and Erv46p ensures ER export despite the presence of ER exit motifs

in both proteins (Otte and Barlowe, 2002) Together, these studies suggest that assembly acts together with the ER retention machinery to exclude unassembled proteins from COPII vesicles This in turn explains why proper assembly is an important element in ERQC

1.2.2 Competition between ER export and ER retention for misfolded proteins

The fundamental principle of ERQC is to ensure only properly folded and assembled proteins are allowed to traffic to their final destinations However, some misfolded proteins are capable of ER exit with a functional ER export signal despite active ER retention mechanisms imposed on them The classical ERAD substrate CPY* contains a

Kar2p binding site where active ER retention mechanism applies (Xie et al., 2009)

Interestingly, it possesses a functional ER export signal in its prodomain even though the

entire protein is grossly misfolded (Kawaguchi et al., in press) This ER export signal

may be conformation dependent and CPY* misfolding does not destroy its structure and function When CPY* is expressed under endogenous level, it is retained in the ER due to Kar2p binding and subject to ERAD The functional ER export signal does drive the ER-to-Golgi transport of some CPY* molecules in complex with Kar2p with a C-terminal HDEL signal At the Golgi, CPY* is brought back to the ER via the HDEL-dependent

retrieval mechanism This in turn explains why blocking ER-to-Golgi transport inhibits

CPY* degradation (Caldwell et al., 2001; Vashist et al., 2001) The export signal is essential in survival when cells are under stress (Kawaguchi et al., in press)

Overexpressed CPY* is degraded by ERAD and vacuolar proteases (Spear and Ng, 2003) Under this condition, ER retention mechanism and ERAD is probably saturated Thus, elimination of excess CPY* requires its export signal which in turn transports molecules

to the vacuole for degradation

In another example, a misfolded form of Yor1p (Yor1p-∆F) which is a homolog of CFTR

is ER retained despite the presence of ER export signals (Pagant et al., 2007) However,

it is not known whether these export signals are conformation dependent If this is the case, signals are likely destroyed in misfolded Yor1p-∆F Another instance is a misfolded cargo ALP* which is unable to be packaged into COPII vesicles via its cargo receptor Erv26p (Dancourt and Barlowe, 2009) The export signal of ALP is likely to be conformation or assembly dependent and hence, it is no longer functional in ALP* Consequently, the binding between ALP* and Erv26p is diminished These two studies imply that lack of a functional export signal is one of the reasons for ER retention of some misfolded proteins Once they have a functional ER export signal, they are capable

of ER exit Thus, ER export can override ER retention in some cases Although intriguing, the exact relationship among protein folding, ER retention and ER export requires further studies Since misfolded proteins can evade ERQC, post-ER QC mechanisms are essential in capturing those escaped molecules

1.3 Post-ER quality control

As mentioned in section 1.2.2, the escape of aberrant proteins from the ER highlights the importance of post-ER QC which mainly involves two post-ER compartments the Golgi apparatus and the cell surface The endosomal system is generally responsible for the degradation of these non-ERAD substrates (Figure 1.5) The interplay among QC systems in various organelles in turn maintains cellular homeostasis Any disruption in the fidelity of secretory pathway QC may result in disease such as neurodegneration and

viral/bacterial infection (Lilley and Ploegh, 2004, Muchowski, 2002, Tsai et al., 2001, Ye

et al., 2004)

Figure 1.5 Model for post-ER quality control

Non-native proteins that escape ERQC are recognized in the Golgi apparatus and they are targeted to the vacuolar/lysosomal degradation (pathway 1) Some membrane proteins that are not captured by Golgi QC are delivered to the plasma membrane Plasma membrane QC recognizes and delivers them into the vacuole/lysosome for degradation (pathway 2) MVB, multi-vesicular body; PVC, prevacuolar compartment

1.3.1 Substrate recognition in the Golgi apparatus

Golgi QC recognizes aberrant proteins in the Golgi apparatus and subsequently delivers them to vacuoles in yeast or lysosomes in animal cells for degradation Although there is

an increasing number of reported mutant proteins that are targeted directly from the Golgi

to the vacuole/lysosome, the exact mechanism of substrate recognition in the Golgi is relatively poorly understood Based on results from numerous groups, several potential mechanisms have been proposed and will be discussed in detail

1.3.1.1 Receptor-mediated mechanism

When mutant proteins are sorted at the Golgi, they are likely to be recognized by a protein receptor Such a receptor may be itself packaged into the endosomal deriving vesicles or promote the sorting of mutant cargoes to be incorporated into vesicles The first candidate is Vps10p which is a type I TM protein receptor localized in the late Golgi

It is required for sorting of multiple soluble vacuolar hydrolases like CPY and PrA

(Cooper and Stevens, 1996; Marcusson et al., 1994) Vps10p binds proCPY in the late

Golgi and the complex is delivered to prevacuolar compartments (PVCs) which are

functionally equivalent to late endosomes in mammals (Mulholland et al., 1999) At

PVCs, Vps10p dissociates and is recycled back to the Golgi where it is ready to bind new substrates (Cooper and Stevens, 1996) Its role in recognizing misfolded proteins was suggested by two studies The Kaiser group has found that a fusion protein between invertase and mutant forms of bacterial lambda repressor (invertase-repressor fusion) is rapidly degraded in the vacuole in a Vps10p-dependent sorting pathway Absence of

Vps10p leads to secretion of the molecules (Hong et al., 1996) In a separate study, the

luminal domain of Vps10p was dissected The authors found that it contains two different binding sites One is for the recognition of biosynthetic cargoes like CPY and the other

one binds to unfolded structures displayed by aberrant proteins (Jorgensen et al., 1999)

The second proposed receptor is Tul1p which is a TM ubiquitin ligase in the Golgi and functions to recognize polar residues in the TM domain of non-native proteins The model substrate is a mutant form of Pep12p, an endosomal SNARE protein Wild-type Pep12p is normally localized to the limiting vacuolar membrane When acidic amino acids are introduced into the TM domain of Pep12p, the protein is ubiquitinated by Tul1p and sorted into the multi-vesicular body (MVB) pathway, albeit still functional (Reggiori

and Pelham, 2002; Reggiori et al., 2000) Thus, it is proposed that Tul1p can recognize

mutant proteins with polar residues in the TM domain However, the fact that Pep12p with an aberrant TM domain is still functional suggests that it is probably folded and not degraded Thus, whether it is a true substrate of Golgi QC remains unclear

Another candidate is Rsp5p which is a HECT (homologous to E6-AP carboxy terminus)

domain E3 ubiquitin ligase (for review, see Rotin et al., 2000) The N terminus of Rsp5p

is a C2 domain which can bind proteins and lipids (Hofmann and Bucher, 1995), followed by three WW domains which mediate protein-protein interaction by binding to

proline-rich sequences and a HECT domain (Chen et al., 1997; Wang et al., 1999) The

HECT domain forms a thiolester bond with ubiquitin when an ubiquitin conjugating E2 enzyme is transferring ubiquitin to a substrate (Figure 1.6) On the contrary, a RING (really interesting new gene) domain E3 ligase binds both the substrate and an E2 during

an ubiquitination reaction (Figure 1.6) (for review, see Hershko and Ciechanover, 1998; Weissman, 2001) Rsp5p is a cytosolic protein that can be recruited to multiple sites of cells including the cell surface, ER, Golgi, endosomes and mitochondria (Fisk and Yaffe,

1999; Gajewska et al., 2001; Helliwell et al., 2001; Hoppe et al., 2000; Wang et al.,

2001) Since Rsp5p ubiquitinates the cytoplasmic domain of membrane proteins, it is likely that it detects structural defects in proteins with an aberrant cytoplasmic domain This may be the case for Pma1-7p which is a mutant form of Pma1p, a plasma membrane

H+-ATPase It contains two mutations with one near the conserved catalytic phosphorylation domain, and the one predicted to be in a cytoplasmic loop between TM segments 8 and 9 (Chang and Fink, 1995) The Chang group has demonstrated that Pma1-7p is polyubiquitinated by Rsp5p at the Golgi and is subsequently targeted for the vacuolar degradation by GGA proteins which are Golgi-localized ubiquitin binding proteins (Pizzirusso and Chang, 2004) However, exactly how Rsp5p recognizes its cargoes and which domain is required for binding remains unclear