Mechanism of protein quality control in the cytosol in budding yeast

2.4 Microscopy techniques 50 2.5 Genetic screening method used in this study 51 65 3.5 San1p-dependent pathway is a general mechanism of CytoQC substrates 67 3.5.1 Nuclear E3 ligase San

MECHANISM OF PROTEIN QUALITY CONTROL IN

THE CYTOSOL IN BUDDING YEAST

RUPALI PRASAD

(M.Sc, IIT Bombay)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE

2011

ACKNOWLEDGEMENTS

This research work is by far one of the most significant scientific accomplishments in

my life and it would have been impossible without the following people, who supported me and had belief in me

First and foremost, I want to express my wholehearted gratitude to my mentor and research advisor Associate Professor Davis Ng, for his expert guidance and motivation throughout my research work I am grateful to him for his invaluable support and also for introducing me to the wonderful and interesting field of protein quality control

I would also like to express my sincere thanks to Dr Shinichi Kawaguchi and Ms Alisha for assisting me and being a part in the projects I am also thankful to Dr Kazue Kanehara, Dr Guillaume Thibault, and Songyu Wang for fruitful discussions and suggestions I owe very special thanks to all current and previous members of Cell Stress and Homeostasis Group, especially Dr Nurzian Ismail, Dr Chia Ling Hsu and Dr Xie Wei and to all my friends at TLL I want to thank them for all their help, support, interest and valuable hints

I gratefully acknowledge the financial support rendered by the National University of Singapore in the form of Research Scholarship I am also grateful to the academic and technical staffs at the Temasek Life Sciences Laboratory who have helped me in one way or the other in my research work

Above all, I want to thank my family, which continuously supported me at all times I thank my parents for teaching me the value of education at a young age and instilled

in me a desire for higher education I wish to thank my sisters for their love and support Words cannot express the love, encouragement and unequivocal support I received from my beloved husband Anil without whose constant help and support, my PhD research work would have remained a daydream The loving family environment and support I enjoyed from all my family members was greatly instrumental in providing me the tranquility and enthusiasm to pursue my research with a piece of mind

1.3.1 Recognition of damaged proteins and repair mechanism 10

1.5.2 Substrate unfolding, translocation and proteolysis 21

1.6 Structure and function of selected cytosolic chaperones and

1.6.4 Hsp90 29

2.1 S cerevisiae strains, growth media and genetic techniques 33

2.1.3 Mating, sporulation and tetrad dissection 36

2.1.4.1 Low efficiency plasmid transformation via simple and

2.1.4.2 High efficiency transformation using lithium acetate 37

2.2.2 List of oligonucleotide primers used in this study 39

2.3.2 TCA precipitation of yeast whole Cell Lysate 44

2.3.10 Cell labeling and Immunoprecipitation analysis 48

2.4 Microscopy techniques 50

2.5 Genetic screening method used in this study 51

65

3.5 San1p-dependent pathway is a general mechanism of CytoQC substrates 67 3.5.1 Nuclear E3 ligase San1p is required for degradation 67 3.5.2 CytoQC substrates polyubiquitination is dependent on E3 ligase San1p 69

3.6 San1p can interact with CytoQC substrate in vivo 70 3.7 San1p pathway is a constitutive mechanism of CytoQC 74 3.8 CytoQC substrates are polyubiquitinated and degraded inside the nucleus 76 3.8.1 Substrate degradation is independent of nuclear export 76 3.8.2 Nucleus is the site for CytoQC substrates degradation 77 3.9 E3 ligase Doa10p is not required for degradation of DssPrA and D2GFP 78 3.10 Ubr1p augments, but is not required for, DssPrA and D2GFP degradation 79

CHAPTER 4: Roles of molecular chaperones in the cytosolic quality control 85

4.2 The Hsp70 chaperone machinery is essential for the degradation of

4.3 The Hsp70 chaperone can interact directly with cytosolic misfolded

4.4 The Hsp70 chaperone system is required for efficient nuclear transport of

4.5 Effect of temperature on CytoQC substrate localization 93 4.6 The Hsp70 co-chaperone Ydj1p is directly involved in nuclear import of

4.7 Nucleotide exchange factor Sse1p is essential for CytoQC pathway 97 4.8 Lack of Hsp90 inhibit substrate degradation and has paltry effect on nuclear

5.2 Folding state of proteins in the cytosol or membrane tethered forms, are

5.3 Ste6C requires all the factors of cytosolic quality control 112 5.4 Genetic screen to identify new components required for cytosolic quality

5.5 The qcc mutants are defective in cytosolic quality control pathway 121

5.7 Identification of new candidates in CytoQC pathway 123

SUMMARY

Intracellular quality control systems monitor protein conformational states Irreversibly misfolded proteins are cleared through specialized degradation pathways Their importance is underscored by numerous pathologies caused by aberrant proteins In the cytosol, where most proteins are synthesized, quality control remains poorly understood Stress-inducible chaperones and the 26S proteasome are known mediators but how their activities are linked is unclear In this thesis, I have used

Saccharomyces cerevisiae as a model organism to study the quality control of

cytosolic misfolded proteins To better understand quality control of cytosolic proteins in chapter 3 and 4 of this thesis, a panel of model misfolded substrates was analyzed in detail Surprisingly, their degradation occurs not in the cytosol but in the

nucleus (Prasad et al., 2010) Degradation is dependent on the E3 ubiquitin ligase

San1p, known previously to direct the turnover of damaged nuclear proteins (Gardner

et al., 2005) San1p, however, is not required for nuclear import of substrates Two reasons can account for nuclear trafficking of misfolded cytosolic proteins First, in S cerevisie, nucleus accounts for over 80 % of proteasomes at steady state throughout

the cell cycle, suggesting the requirement of nuclear import of misfolded cytosolic proteins Second, by trafficking misfolded proteins in the nucleus, cells provide enough time for newly synthesized proteins to fold in proper conformation One view asserts that a key strategy of protein quality control is the integration of timing devices to permit folding (Helenius and Aebi, 2004) As such, proteins failing to fold within a set window are targeted for degradation Experimental precedence comes from ERAD studies where a sophisticated timing mechanism utilizes a series of

glycosidases to set a time limit for folding (Clerc et al., 2009) Proteins still unfolded

after the final trimming step by Htm1p are detected by the Yos9p ERAD factor (OS-9

in mammals), which binds the resulting glycan signal (Quan et al., 2008) In CytoQC,

nuclear import of substrate can provide an analogous function The detailed analyses

of cytosolic substrates have provided a clue that the Hsp70 family proteins Ssa1p and Ssa2p and its co-chaperone Ydj1p are needed for efficient import and degradation In chapter 5 of this thesis, I have described a genome wide genetic screen to identify the genes involved and to decipher the mechanism for quality control of cytosolic protein Among the genes identified, there are genes that encodes for proteasomal subunits

(RPN7 and RPN11) and UMP1, a chaperone required for assembly of 26S proteasome

Together all our data reveal a new function of the nucleus as a compartment central to the quality control of cytosolic proteins

LIST OF TABLES

Table 2.1 List of yeast strains used in this study 33

Table 2.3 List of oligonucleotide primers used in this study 39

LIST OF FIGURES

Figure 1.1 ERAD recognition of misfolded protein by N-glycan signal 6

Figure 1.3 Role of molecular chaperones in the balance of folding,

Figure 1.5 The ubiquitin-proteasome system for intracellular protein

Figure 1.6 The 26S complex ofSaccharomyces cerevisiae 22

Figure 3.1 A fraction of the misfolded protein ΔssCPY* translocates

into the ER without a signal sequence and ΔssCPY* degrades

Figure 3.2 DssPrA and D2GFP are substrates of CytoQC 62

Figure 3.3 Turnover of DssPrA and D2GFP requires cytosolic quality

Figure 3.4 DssPrA and D2GFP are localized in the cytosol and nucleus 66

Figure 3.5 Intracellular localization and degradation of ΔssPrA-3A 66

Figure 3.6 San1p is required for DssPrA and D2GFP degradation 68

Figure 3.7 San1p is required for DssPrA and D2GFP ubiquitination 70

Figure 3.8 San1p overexpression enhances substrate degradation 72

Figure 3.9 Effect of San1p-V5 overexpression on its localization and

Figure 3.10 Effect of substrate expression level on degradation 75

Figure 3.11 Substrate degradation in nuclear export defective

Figure 3.12 Visualization of intracellular substrate decay 78

Figure 3.13 Doa10p is not required for ΔssPrA and Δ2GFP degradation 79

Figure 4.1 Analysis of SSA1-4 single and double mutants in

Figure 4.2

Figure 4.3 Ssa1p and Ssa2p are required for the nuclear localization

Figure 4.4 Temperature effect on substrate localization 94

Figure 4.5 Substrate loses its ability to be transported into the nucleus

Figure 4.6 Sse1p is required for substrate degradation 98

Figure 4.7 Hsp90 is required for substrate degradation and partly for nuclear

Figure 4.8 Model of CytoQC-degradation pathway for the transport of

misfolded cytosolic proteins into nucleus 104

Figure 5.1 Ste6C degradation does not depend on the Doa10p E3

Figure 5.2 Cdc48 ATPase complex is required for membrane anchored

Figure 5.3 Ste6C is present both in cytosolic and membrane fraction 113

Figure 5.4 Ste6C is a cytosolic quality control substrate 115

Figure 5.5 A schematic diagram representing the strategy taken to obtain

genes involved in cytosolic quality control 118

Figure 5.6 Stabilization of Ste6C-Ura3p fusion protein in qcc mutant strains 119

Figure 5.7 The qcc mutant strains can also stabilize other cytosolic

Figure 5.8 Introduction of complementary gene restores the cytosolic

Figure 5.9 Introduction of complementary gene suppresses the

thermosensitive growth effect of temperature sensitive

ERAD ER-associated protein degradation

ERQC ER quality control

Endo H Endoglycosidase H

HRD HMG-CoA reductase degradation

Hsp Heat shock protein

Htm1 Homologous to mannosidase I

IPODs Insoluble protein deposits

JUNCs Juxtanuclear quality controls

NEF Nucleotide exchange factor

NES Nuclear export signal

NLS Nuclear import signal

OST

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PDI Protein disulfide isomerase

PMSF Phenylmethylsulphonylfluoride

QCC Quality control in the cytosol

RING Really Interesting New Gene

RPM Round per minute

SC Synthetic complete

SBD Substrate binding domain

SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

SEM standard error of the mean

TAP Tandem affinity purification

TCA Trichloroacetic acid

TPR Tetratricopeptide repeats

UPS Ubiquitin-proteasome system

VHL von Hippel-Lindau

Yos9 Yeast osteosarcoma 9

YPD Yeast peptone dextrose

LIST OF PUBLICATIONS RELATED TO THIS STUDY

Prasad, R., Kawaguchi, S and Ng, D.T (2010) A nucleus-based quality control

mechanism for cytosolic proteins Molecular Biology of the Cell 21, 2117-2127

Prasad, R., Kawaguchi, S and Ng, D.T Nuclear import of misfolded cytosolic

protein is mediated by Hsp70-Hsp40 chaperone system Manuscript in preparation

CHAPTER 1

Introduction

The central dogma of molecular biology—DNA to RNA to protein—concisely describes the information flow of protein synthesis Errors arising at any step can disrupt protein folding and lead to potentially toxic products Although DNA replication is highly accurate, transcriptional and translational error rates can be as high as 10-4 and 10-3, respectively (Zaher and Green, 2009) Even with a correct sequence, the need for chaperones and, in some cases, modifying enzymes for folding makes an already complex process even more precarious The consequences of accumulating aberrant proteins are so serious that numerous sophisticated protein quality control mechanisms (PQC) have evolved to protect cells

Efficient protein folding is very important for proper function and viability and to do

so cells contain elaborate enzymatic machinery called molecular chaperones Some bind to non-native polypeptide and promote their folding in an ATP-dependent manner (Hartl and Hartl, 2002) Protein misfolding can occur by genetic mutation, biosynthetic errors and cellular stress such as chemical and temperature perturbation

If a cell fails to eliminate these misfolded non-native proteins, it can lead to formation

of toxic aggregates, non-functional proteins and ultimately cell death A number of diseases associated with aberrant protein conformation highlight the importance of protein quality control for cell survival (Dobson, 2004)

To repair or remove damaged or unfolded proteins, both prokaryotes and eukaryotes use protein quality control system that includes numerous molecular chaperones and proteases In general intracellular misfolded proteins have three fates: they either get

rescued by molecular chaperones or destroyed via proteases or form aggregates Chaperones are a diverse group of proteins defined by size, cellular compartment and function They work together to prevent protein aggregation and facilitate the correct folding of nascent polypeptide into native conformation Misfolded proteins are degraded by a variety of proteases; however, the main intracellular degradation site is the proteasome

1.1 Protein quality control (PQC)

As there is a high degree of compartmentalization in eukaryotic cells, proteins are more prone to damage in specific organelles For example, in the endoplasmic reticulum (ER), proteins are vulnerable to errors in glycosylation, disulfide bond

formation and membrane insertion (Alexander et al., 2010) In contrast, proteins in

the cytosol are more sensitive to stress, while ER proteins are more stable because of the effect of disulfide bonds and glycosylation The protein quality system is adapted

to handle any situation that results in a change in protein conformation from its native state Protein quality control degradation systems have been identified in several organelles such as cytosol, ER, mitochondria and nucleus In the cytosol and ER, degradation is mainly brought about by polyubiquitination of misfolded proteins by protein-ubiquitin-complexes that mark the substrate for proteasomal degradation (Hampton, 2002; McDonough and Patterson, 2003; Trombetta and Parodi, 2003) In these two compartments, PQC can also occur via transport to the lysosome/vacuole (Trombetta and Parodi, 2003) In the mitochondria, localized proteases function in PQC and degradation (Arnold and Langer, 2002) Although, PQC is found everywhere proteins are made or mature, the best understood PQC mechanisms are in the endoplasmic reticulum (ER)

1.2 ER quality control

The ER is a protein factory, where secretory and membrane proteins enter and are folded, which account for one-third of total cellular proteins ER has a quality control system that monitors the correctly folded proteins and sends them to their final destination Proteins which misfold or unfold, are recognized by the quality control

system in the ER which retain and refold them (Ellgaard and Helenius, 2003)

Accordingly, ER quality control mechanisms have the added responsibility to control trafficking, to prevent the premature exit of folding intermediates (Vembar and Brodsky, 2008) For proteins that fail to fold, the integration of ER associated degradation (ERAD) pathways removes and destroys aberrant products ERAD includes the involvement of general folding factors like Kar2p and protein disulfide isomerase as well as specialized factors that recognize and target misfolded proteins

to ERAD processing sites These sites, made up of factors organized by E3 ubiquitin ligases, function to translocate and ubiquitinate substrates before they are degraded by

the 26S proteasome (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006)

1.2.1 Role of ER-lumenal chaperones

Misfolded glycosylated proteins in the ER lumen are first recognized by Hsp70 homologue, Kar2p Kar2p is also required for posttranslational protein translocation into the ER and protein folding in the ER lumen (Kabani et al., 2003, Vashist and Ng,

2004) Kar2p binds to hydrophobic patches of misfolded or unfolded proteins to prevent them from forming aggregates (Kabani et al., 2003; Flynn et al., 1991) Two

mutants of Kar2p have been identified; kar2-1 and kar2-133 and both of them showed

reduced affinity for its substrate and ERAD defect In these mutant strains, ERAD

substrates get aggregated in the ER lumen and this defect can be rescued by coexpression of wild type Kar2p

Another abundantly present ER chaperone, protein disulfide isomerase (PDI) is a thiol oxidoreductase This chaperone is involved in disulfide bond formation, protein

folding and recognition and targeting of misfolded protein for ERAD (Nishikawa et al., 2005; Tu and Weissman, 2004) Yeast PDI contains a characteristic CXXC motif that is located in the active site of the enzyme (Tian et al., 2006) PDI binds to

hydrophobic patches of proteins in the folding process in its groove, breaks the wrong disulfide-bonds and catalyzes the formation of correct ones

1.2.2 Recognition of ERAD substrate

The best understood mechanism for recognition of ERAD substrate is the glycan-dependent pathway Majority of polypeptides, which are synthesized in the ER are modified by N-linked glycans The oligosaccharyltransferase covalently add Glc3Man9GlcNAc2 glycan to asparagines in Asn-X-Ser/Thr motif of polypeptides Additions of glycans to polypeptides make them more hydrophilic and direct them for folding Moreover, it provides information about the folding status of glycoproteins The outermost glucose residues are further removed by Gls1p and Gls2p to label polypeptide as in the process of folding (Figure 1.1) The resulting glycoproteins which bear, Glc1Man9GlcNAc2 glycan, can associate with glycan-dependent chaperones, which promote their maturation Once liberated from these chaperones, Gls2p trims the final glucose residue from polypeptide to attain their native structure

If a polypeptide fails to mature within its folding window, further trimming of glycans trigger its destruction ER resident Mns1p (α1,2-mannosidase 1) trims the outermost

glycan residue from the B branch of N-glycan of a polypeptide, resulting in Man8GlcNAc2 glycan However, Mns1p can process glycoproteins regardless of their folding status, thus Man8 itself is not sufficient to be a degradation signal (Jakob et al.,

1998; Jelinek-Kelly and Herscovics, 1988) Therefore additional signature glycan is needed to distinguish defective polypeptides from the mature protein Htm1p (mannosidase 1) a lectin, further removes the C branch from the glycan, resulting in Man7GlcNAc2 structure which flags potential ERAD substrates for degradation (Clerc

et al., 2009) Man7GlcNAc2 structure, in turn is recognized by glycan binding protein

Yos9p (yeast osteosarcoma 9) (Quan et al., 2008) Recent studies have shown that

Yos9p binds misfolded proteins but is unable to interact with their folded counterparts Yos9p is part of the Hrd1 complex, as it directly interacts with the large luminal domain of Hrd3p, indicating that it can links the recognition of misfolded glycoproteins to the ubiquitin-proteasome system

Figure 1.1 ERAD recognition of misfolded protein by N-glycan signal . In yeast, glucosidases 1 and 2 trim the two outermost glucose residues to generate Glc1Man9GlcNAc2 Final sugar is removed by glucosidases 2, generating Man9GlcNAc2and thus protect the glycoprotein from degradation Next, Mns1 cleaves the middle α1,2-linked mannose to generate the Man8GlcNAc2 glycan Htm1 then processes the terminal mannose rsidue from the C-branch yielding the terminal α1,6 mannose

residue as the Yos9p ligand

ERAD-C pathways degrade them respectively (Carvalho et al., 2006; Vashist and Ng,

2004) Thus, the site of the lesion is an important determinants for the pathway used for degradation of misfolded proteins

E3 ubiquitin ligase Hrd1p forms a complex with Hrd3p, Yos9p and Kar2p (Figure 1.2) This complex is involved in the degradation of luminal misfolded proteins (ERAD-L

pathway) Yos9p, a lectin, interact with Hrd3p and Kar2p and recruits misfolded

proteins to this complex for ubiquitination (Bhamidipati et al., 2005; Kim et al., 2005; Quan et al., 2008; Szathmary et al., 2005) In addition, there are extra components

found in the Hrd1p complex that function to recognize and deliver misfolded proteins

to the ERAD machinery (Carvalho et al., 2006; Denic et al., 2006) which include

Ubc7p/Cue1p (E2 complex), Der1p, Ubx2p and Usa1p Misfolded proteins that have lesions present within transmembrane domain are also polyubiquitinated via Hrd1p complex (ERAD-M pathway) Proteins with misfolded cytosolic regions are degraded via ERAD-C pathway, which requires the Doa10 ubiquitin-ligase All the three pathways share common factors such as the E2 ubiquitin conjugating enzyme, Ubc7p

and Cdc48 complex (Carvalho et al., 2006; Denic et al., 2006; Vashist and Ng, 2004)

Figure 1.2 Distinct ERAD complexes in yeast (A) Dao10 complex recognizes ERAD-C

substrates which contain a misfolded cytosolic domain (B) The Hrd1p complex recognizes proteins with a misfolded luminal or TM domain and defines ERAD-L and ERAD-M pathway Figure taken from Ismail and Ng, 2006

1.2.4 Retrotranslocation and the Cdc48 complex

Once selected and polyubiquitinated, misfolded proteins need be to retrotranslocated from the ER to the cytosol for proteasomal degradation Membrane proteins must translocate either prior to or during degradation from the lipid bilayer To do so, ERAD substrates require a retrotranslocon channel The proposed translocon

complexes are Sec61 (Plemper et al., 1997) and the Derlins (Knop et al., 1997 and Ye

et al., 2004) and these protein complexes are bound to factors linked to substrate

selection, ubiquitination and translocation E3 ubiquitin ligases such as Hrd1

(Hampton et al., 1996 and Plemper et al., 1999) and Doa10 (Swanson et al., 2001)

have also been proposed to be protein-conducting channels, as they are made up of multispanning transmembrane domains, which can from oligomers to form channels Whether retrotranslocation occurs through the Sec61 translocon or require Der1p or ubiquitin ligases is still a matter of debate

In nearly all cases, ERAD substrates are ubiquitinated and released from the ER by a

ubiquitin-specific chaperone complex consisting of Cdc48p, Ufd1p and Npl4p (Ye et al., 2001; Braun et al., 2002; Jarosch et al., 2002) This complex binds to

polyubiquitinated substrates and couples ATP hydrolysis with retrotranslocation ATP hydrolysis couples force on the substrate protein to extracts it from the translocation channel Cdc48p is an AAA-ATPase, which binds to the proteasome and has a high

affinity for ubiquitin chain (Dreveny et al., 2004) Ubx2p is an integral ER membrane

protein, which recruits Cdc48p-Ufd1p-Npl4p complex to E3 ubiquitin ligase complex

and to ERAD substrates (Neuber et al., 2005; Schuberth et al., 2005) Once the

substrates are polyubiquitinated and extracted from the membrane, they are further targeted to the proteasome via the action of Rad23p and Dsk2p Both proteins have a C-terminal UBA (Ubiquitin associated) motif that recognize the polyubiquitinated substrates and target them to proteasome with the help of subunits Rpn10p and Rpn13p Before proteasomal degradation, substrates are deubiquitinated by deubiuinating enzymes to remove the polyubiquitin chain from the substrates

(Hirsch et al., 2009)

1.3 Cytosolic quality control

Protein misfolding in cytosol is toxic to cells and the accumulated toxic proteins can lead to protein misfolding diseases (For examples, Parkinson’s and Alzheimer’s diseases) Misfolding of proteins can expose hydrophobic surfaces that result in unnecessary binding to normal proteins which disrupt the essential interactions between cellular proteins To avoid this situation, quality control systems present in the cytosol monitor protein folding and remove misfolded proteins in the cytosol Hydrophobic patches of misfolded proteins are recognized by molecular chaperones that mask them and transfer the misfolded species to the ubiquitin-proteasome system and chaperone-mediated autophagy to eliminate them The entire quality control systems in cytosol are regulated by stress-inducible transcription factors, molecular

chaperones and other factors for the effective elimination of toxic proteins

1.3.1 Recognition of damaged proteins and repair mechanism

Recognition of non-native cytosolic proteins is the first step towards their elimination, which rely mainly on the interaction between chaperones and non-native folding intermediates Once recognized, cell can respond to their presence in three ways (Figure 1.3) First, cytosolic factors may attempt to rescue the non-native folding intermediates by folding them to a functional native state Second, in order to prevent toxic interactions, cells can sequester misfolded proteins Finally, proteins that cannot

be folded into native conformation must be eliminated by ubiquitin-proteasome pathway

There are different chaperones systems present in the cytosol, which possess specific mode of substrate binding that determine substrate specificity and range The highly

conserved Hsp70 chaperone system interacts with a variety of substrate conformers like unfolded proteins, folded native structure and aggregates (Mayer and Bukau, 2005) All these interactions rely on the transient interactions of Hsp70 with exposed

hydrophobic patches (Zhu et al., 1996; Rüdiger et al., 1997) Binding of substrates

with Hsp70, not only prevent protein aggregation but it also assist in the folding of proteins through one or several ATPase cycle of binding and release and also promote the disaggregation of proteins with the help of another chaperone family Hsp100

(Diamant et al., 2000; Goloubinoff and De Los Rios, 2007) Similar to Hsp70, Hsp90

chaperone family is also thought to bind and stabilize partially folded substrates in cooperation with other chaperones The determinants for substrate recognition for Hsp90 are still not known in detail; however, it may in part reside within specific co-chaperones In comparison to Hsp70 and Hsp90, Hsp60 (Chaperonin) family member recognizes a smaller range of substrates (Bukau et al., 1998) Chaperonins are large double-ring complexes (~800 kDa) encloses a central cavity There are two groups of chaperonin, group I is found in bacteria, mitochondria and chloroplast while group II chaperonins exist mainly in eukaryotic cytosol (TRiC/CCT) It encapsulates the substrates in the central cavity and thus provides a protecting environment for folding As in the case of Hsp70s, substrate binding by chaperonin is also, ATP regulated, but, unlike the Hsp70s, it promotes the protein folding through cycles of global encapsulation In addition to these well established chaperone systems, the cytosol also possesses small heat shock proteins families (Hsps), which bind to misfolded proteins to prevent their aggregation, disaggregation and to facilitate refolding of proteins

Figure 1.3 Roles of molecular chaperones in the balance of folding, degradation and aggregation Molecular chaperones interact with newly synthesized polypeptides to fold and

direct native conformation of proteins They can also bind with non-native intermediates to either refold or eliminate by UPS If fails to refold or degrade, misfolded proteins may form soluble or detergent resistant aggregates Figure taken from McClellan et al., 2005

The exact role of molecular chaperones in elimination of non-native proteins is still not clear According to older model, chaperones would be primarily involved in the stabilizing and refolding of non-native polypeptides This means that, the primary role

of chaperones in the quality control is just to maintain the solubility of misfolded intermediates and facilitate the sampling by the ubiquitination machinery However, recent analysis of the quality control mechanisms of mutant von Hippel-Lindau (VHL) tumor suppressor protein suggests that chaperones have an active role in selecting

proteins for degradation (McClellan et al., 2005) Additionally, observation that some

chaperones specifically interact with E3s, suggest that at least in some cases, chaperones could recognize misfolded proteins and subsequently directly recruit an

E3 ligase (McClellan et al., 2005; Esser et al., 2004)

1.3.2 Transcriptional regulation

Several stress condition like heat stress, oxidative stress and proteasome inhibition can increase the rate of misfolding (Figure 1.4) Many genes, including genes encoding chaperones are induced under stress to protect cells against the potential toxicity caused due to misfolding of proteins (Morimoto, 2008) Induction is nearly instantaneous as heat shock genes (Hs genes) are induced and their intensity is proportional to the duration and type of stress Primary form of regulation is at the level of transcription but it can also be regulated post-transcriptionally by stress-induced mRNA stability and by translation control

Heat shock response is mediated by a family of heat-shock transcription factor (HSFs) which generally are expressed and maintained in the cytosol as monomers in a

non-DNA-binding state under normal condition Saccharomyces cerevisiae expresses

only HSF1 (Heat shock factor 1), which under stress conditions get activated and translocate into the nucleus as trimers This induced HSF1 binds to a specific

cis-acting element in the promoter region of the heat shock response element (HSE)

of stress-responsive genes (Shi et al., 1998) The binding induces several factors; one

of them being ubiquitin expression indicating its role in the regulation of the degradation system

Under normal conditions, in the cytosol, HSF1 monomer activation is inhibited by the

association of Hsp90 (Zou et al., 1998) In stress conditions competitive binding of

denatured proteins to Hsp90 releases HSF1 HSF1 is transiently activated in response

to stress condition and is rapidly attenuated, as the presence of excess chaperones is toxic for the cells Interaction of HSF1 with Hsp70 attenuates the further induction of

chaperones and thus indicates a negative feedback mechanism for the stress response

(Shi et al., 1998)

Figure 1.4 Cell stress response The expression of HS genes including chaperones and

components of the degradation machinery is induced in response to several classes of stresses, including environmental stress, pathophysiological stress, and protein conformational disease, and cell growth and development (Adapted from Morimoto, 2008)

1.3.3 Autophagy-lysosome system

Autophagy is a cytoplasmic event wherein molecules or organelles are sequestered in vesicles that fuse to lysosome and get degraded by lysosomal proteases The autophagy-lysosome system plays an important role in the elimination of cytosolic

misfolded proteins (Mizushima et al., 2008; Nakatogawa et al., 2009) For example,

in addition to proteasomal degradation, polyglutamine-expansion proteins also require autophagy system for their elimination It has been shown that mice deficient in

autophagy develop neurodegenerative diseases (Komatsu et al., 2006; Hara et al.,

2006) Two protein p62 and the neighbor of BRCA1 gene 1 (NBR1) are shown to link polyubiquitination and autophagy by making a bridge between degradation tag,

et al., 2009) The ubiquitin-proteasome system degrades soluble polyubiquitinated

misfolded proteins but is unable to degrade irreversibly oligomerized species These species are thus degraded via autophagy-lysosome pathway through the recognition of polyubiquitin by p62 and NBR1

There is another pathway called chaperone-mediated autophagy (CMA) that can direct cytosolic proteins to degrade in the lysosome CMA does not require sequestration of target proteins into autophagosomes (Dice, 2007); instead a specific motif (KFERQ) in the target proteins is recognized by Hsc70 which form a complex with Hsp40 and co-chaperones Around 30% of cytosolic proteins possess KFERQ-like sequences, suggesting the importance of CMA-mediated degradation Once recognized by the chaperone complex, it transfers the target proteins to the lysosomal lumen through the lysosome-associated membrane protein type 2A (LAMP-2A) CMA is maximally activated during ageing, prolonged starvation, mild

oxidation and other conditions that cause proteins damage (Kiffin et al., 2004) In

neuronal cells, it helps in the degradation of α-synuclein, a main component of

amyloid aggregates formed in Parkinson’s disease (Cuervo et al., 2004) Parkinson’s

disease-associated mutation in α-synuclein blocks the degradation pathways CMA activity decreases with age, which caused mainly due to an–age dependent decline in the levels of the CMA receptor LAMP-2A

1.3.4 Sequestration into large aggregates

Many cytosolic misfolded proteins that are prone to form toxic aggregates can form a large perinuclear aggregate, called aggresome Recent studies have suggested that formation of aggresome or aggresome-like inclusions generally have cytsoprotective

effect (Kawaguchi et al., 2003 and Kaganovich et al., 2008)) Aggregation can be

divided into two types: aggregate formation by misfolded proteins to small soluble aggregates which is cytotoxic, and another is formation of aggresome or inclusion bodies caused due to forced aggregation of small soluble aggregates which are cytoprotective In yeast two major inclusion-like compartments, both distinct from each other in their ubiquitination and aggregation states have recently been identified:

a juxtaneculear quality control compartment (JUNQ) and a perivaculor insoluble

protein deposits (IPODS) (Kaganovich et al., 2008) Misfolded or polyubiquitylated

proteins that cannot be refolded or degraded, are temporarily stored in JUNQ The stored proteins of the JUNQ can exchange with cytosol and can be refolded or degraded IPODs in contrast, consist of terminally sequestered aggregated, immobile and mostly nonubiquitinated proteins from cytosol to protect cells from toxicity Studies have indicated that IPODs are cleared by autophagy, as it co-localizes with

autophagosome marker Atg8 (Alexander et al., 2010) Formation of aggresomes,

JUNQ and IPODs is dependent on the active transport of misfolded protein via

microtubules and dynein motors (Kopito, 2000; Kaganovich et al., 2008) This

microtubule dependent sequestering of misfolded proteins is a protein quality control system for cell survival

1.3.5 Degradation of misfolded proteins by UPS

Once a misfolded protein is recognized by cellular protein quality control system, the next step is to either repair or eliminate it by degradation If fails to repair, damaged proteins need to be redirected from futile cycles of chaperone interactions to rapid degradation Irreparably damaged proteins are either directly recognized through specific features of substrates or by timer mechanism based on the dwell time of

irreparable proteins on chaperones The major pathway present in the cytosol to degrade misfolded proteins is ubiquitin-proteasome pathway (Ciechanover and Brundin, 2003)

Multiple degradation pathways have been identified in eukaryotic cytosol The decision to either repair or target for degradation critically depends on E3 ubiquitin ligases, their direct interaction with Hsp70 and Hsp90 and on dedicated co-chaperones E3 ubiquitin ligase CHIP in higher eukaryotes is an important factor responsible for polyubiquitinated substrates in a chaperone dependent manner

(Murata et al., 2001; Arndt et al., 2007) CHIP possesses a ubiquitin ligase and

interacts with both Hsp70 and Hsp90 via TPR domain However, mode of action of CHIP activity is very different from Hsp70 and Hsp90 systems Both modes of activity seem to interfere with chaperone cycles It inhibits the formation of Hsp70-susbtrate complex by inhibiting Hsp40 induced stimulation of ATP hydrolysis

of Hsp70 chaperone (Ballinger et al., 1999; Stankiewicz et al., 2010) In Hsp90, CHIP

interferes in the late maturation step of Hsp90 substrate by inhibiting binding of the

co-chaperone p23 to Hsp90 (Connell et al., 2001 )

While in higher eukaryotes CHIP appears to be central to triage decision of the Hsp70 and Hsp90 chaperone system, in yeast that lacks CHIP homologs, proteasomal degradation of cytosolic misfolded proteins depends on which ubiquitin ligases is yet

to be identified E3 ubiquitin ligase Doa10 has a well-established role in the ERAD to remove misfolded protein with lesion present in the cytosol (Vashist and Ng, 2004;

Carvalho et al., 2006) Recently, Doa10 is also been shown to be involved in the

degradation of some misfolded cytosolic proteins (Metzger, 2008 & Ravid et al.,

2006)

1.4 Ubiquitin-proteasome system

Cellular proteins are targeted for degradation in an ATP-dependent manner by 26S proteasome The process of ubiquitin mediated substrate delivery to 26S proteasome occurs by the successive action of a cascade of three enzymes (Figure 1.5) The E1 ubiquitin-activating enzymes covalently activate ubiquitin by using ATP and transfer ubiquitin to a ubiquitin-conjugating enzyme E2 (UBC); E2 enzymes then transfer ubiquitin to substrates with the help of an ubiquitin ligase E3 (Hershko and Ciechanover, 1998) This repetitive process tags the substrates with a polyubiquitin chain, which is finally recognized by proteasome, leading to degradation of the ubiquitinated substrates In yeast, there is one E1, 10 E2s and nearly one hundred E3 ubiquitin ligases operating to ubiquitinate the substrates Although 26S proteasome recognize polyubiquitin as a degradative determinant, E3 ligases recognize substrates having different degradation signals and contribute to specificity (Weissman 2001; Pickart, 2001)

Figure 1.5 The ubiquitin-proteasome system for intracellular protein degradation

Ubiquitin activating enzyme E1, activates ubiquitin by using ATP Ubiquitin conjugating enzymes E2 are charged by transfer of ubiquitin from E1 Ubiquitin ligase E3, transfers the ubiquitin to target substrates, producing ubiquitinated proteins This process is repeated again and again to produce polyubiquitinated substrates Figure adapted from Hampton and Garza,

2009

up of six essential ATPase and three non-ATPase subunits (AAA-ATPases RPT1-6 and Rpn subunits 1-2) Multiubiquitin binding protein Rpn10 links the cap to base The proteolytic core 20S is composed of four heptameric rings (α7β7β7α7) (Rechsteiner and Hill, 2005) The outer ring is composed of α-subunits and the inner 2 rings are made up of β-subunits α-subunits are the sites for the binding of various regulatory factors, entry and exit of substrates, while β-subunits harbour the catalytic site, which can cleave after acidic, basic and hydrophobic residues

1.5.1 Substrate recognition by 26S proteasome

Polyubiqutin chain is the main targeting signal recognized by 26S proteasome Its substrate specificity is diverse and is determined by a variety of E3-substrate-signal interactions Some studies have shown that Rpn10p either can bind polyubiquitin chain tightly or has a direct role in the chain recognition (Pickart and Cohen, 2004;

Deveraux et al., 1994; Hartmann et al., 2003) However, recent studies have shown

that Rpt5p, one of the six ATPase subunits of the base can specifically contact a

proteasome bound polyubiquitin chain (Figure 1 6B) (Pickart and Cohen, 2004; Lam

et al., 2002)

1.5.2 Substrate unfolding, translocation and proteolysis

26S proteasome accomplishes unfolding of polyubiquitinated substrates in two steps:

an initial reversible interaction of the substrates with the 19S complex through the

ployubiquitin chain (Pickart and Cohen, 2004; Lee et al., 2001; Verma and Deshaies,

2000) followed by a more stable engagement of loosely folded polypeptide region of the substrate with proteasome This stable engagement of substrate finally initiates the mechanical unfolding Once the substrates reach the catalytic chamber of 20S, they get destroyed by nonspecific peptide hydrolysis Proteasomes cleave their substrates

to produce 3-20-residue peptides, which are either degraded into single amino acids

(Tamura et al., 1998) or escape complete hydrolysis and are presented by MHC class

I molecules (Rock et al., 1994)

1.5.3 Cellular localization of 26S proteasome

Temporal and spatial control of proteolysis is not only achieved by the selective degradation of ubiquitinated proteins, but also by changing the cellular localization of proteasome Studies have indicated that it is present in the cytosol as well as in the nucleus (Reits et al., 1997) By using specific localization signal and according to the requirement, 26S proteasome can be deployed to different locations in the cytosol or nucleus Nuclear localization of proteasome depends on the cell type and growth, and the relative proportion of nucleus versus cytosolic varies between 20 to 80% (Russell

et al., 1999; Laporte et al., 2008) Studies performed with a fusion of proteasomal

β-subunit with GFP in human cell lines revealed the distribution of proteasome in

eukaryotes was found mainly in the cytosol and in nucleus The proteasomes either reach the nucleus by active transport via nuclear pore or are trapped in the nucleus after cell division In lower eukaryotes for example in yeast, the majority of proteasome is present in the nucleus, where the nuclear membrane persists during the mitosis These studies suggest that in yeast, nucleus is the major sites of protein degradation

Figure 1.6 The 26S complex of Saccharomyces cerevisiae (A) Composition of 26S

complex (B) Steps in substrate proteolysis by 26S proteasome: a Polyubiquitin chain is recognized by Rpt5p b Engagement site is found by Base complex c Substrate translocation d Substrate hydrolyzed in short prptide e Peptides exit the catalytic chamber

f Rpn11p (subunit of lid complex hydrolyses the isopeptide bond between polyubiquitin and substrate) Figures taken from Pickart and Cohen, 2004

1.6 Structure and function of selected cytosolic chaperones and co-chaperones

Chaperones are diverse protein families that have the ability to bind and stabilize non-native conformations of other proteins Molecular chaperones aid in most aspects

of protein maturation, including: folding, assembly into multi-protein complexes, protein activation, membrane translocation and degradation (Hartl, 2002; Bukau, 1998) They are ubiquitously expressed and are present in every compartment of eukaryotic cells, which reflects their importance in living organisms Many molecular chaperones for example, heat shock proteins (Hsps) were initially characterized as

proteins upregulated in response to heat shock and other cellular stresses (Bukau et al.,

2006)

1.6.1 Hsp70/Hsc70

Hsp70s are perhaps best-studied and highly conserved class of chaperones (Mayer and Bukau, 2005) which are present in all organisms Function of Hsp70 is tightly regulated by co-chaperones, Hsp40 and nucleotide exchange factors (NEFs)

(Dragovic et al., 2006; Kabani et al., 2002) Members of the Hsp70/Hsc70 class of

molecular chaperones are essential for a vast majority of the chaperone dependent processes like translation, translocation across membranes, presentation of substrates for degradation, assembly and disassembly of macromolecular complexes or

aggregates (Deshaies et al., 1988; Glover and Lindquist 1998; Szabo et al., 1994; Ungewickell et al., 1995)

Cytosolic Hsp70 are well conserved and have been divided in two categories, stress inducible Hsp70 and constitutively expressed Hsc70 which is an isoforms of Hsp70

S cerevisiae has six cytosolic Hsp70s which are subdivided into two classes, Ssa

identity and possess some common functions but functionally they are not identical (Craig, 1995) Functional difference among Ssa proteins have still not been resolved though Ssa1 and Ssa2 share 98 % primary sequence identity, whereas identity between Ssa3 and Ssa4 is around 88 % (Sharma and Masison, 2009)

All Hsp70s consist of three structural domains, a 44 kDa amino-terminal ATPase domain (NBD), an 18 kDa substrate binding domain (SBD) and a 10 kDa C-terminal

lid domain (CTD) (Flaherty et al., 1990; Wang et al., 1993; Zhu et al., 1996) The

C-terminus contains an EEVD motif that interacts with co-chaperones that contain several degenerate 34 amino acid repeats, called tetratricopeptide repeats (TPRs) Hsp70s function by binding substrates and using the energy from ATP hydrolysis to

influence substrate binding (McCarty et al., 1995)

Hsp70 function requires coordinated action of all three domains Substrate binding occurs in a hydrophobic pocket in the SBD of Hsp70 with an affinity and kinetics dependent on the nucleotide bound state of the NBD When Hsp70 is present in the ATP bound state, the lid is open and substrates have a low affinity for the peptide-binding domain, resulting in a high on/off rate Substrate binding is stimulated by Hsp70 ATP hydrolysis that causes the lid to clamp down onto the

substrate (Flynn et al., 1989; Jordan and McMacken, 1995) ADP bound state of

Hsp70 can interact with substrate with high affinity, resulting in a low on/off rate

(McCarty et al., 1995; Russell McCarty et al., 1999)

The major difference between high and low affinity bound states is the location of CTD It is positioned over SBD when ADP is bound and thus reduces the release of

bound substrates (Han, 2003) After ATP hydrolysis, structural changes in NBD induce conformational changes in the SBD and CTD that leads to substrate trapping Binding of substrate to SBD alters SBD structure in such a way that transmits a signal

to the NBD that stimulates ATP hydrolysis and thus increases its affinity of binding to Hsp70 Nucleotide exchange factors (NEF) subsequently releases ADP and facilitates rebinding of ATP and thus restores Hsp70 to the low affinity state, allowing substrate release Released substrates then can either obtain native state or else rebind to Hsp70,

to other chaperones or form aggregates

The ability of Hsp70 to hydrolyze ATP and release ADP is very low and thus it represents the rate-limiting step (McCarty, 1995, Theyssen, 1996) The Hsp70 ATPase cycle is stimulated by various co-factors such as Hsp40s and NEFs (Figure 1.7) Simultaneous interaction of Hsp70s and substrates with Hsp40 synergistically

stimulates ATPase activity of Hsp70s (Lu et al., 1998; Wegele et al., 2003) whereas NEFs helps in the release of ADP (Kabani et al., 2002; Dragovic et al., 2006)