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REGULATION OF CELLULAR HSP70

Jayasankar Mohanakrishnan Kaimal

Regulation of cellular Hsp70

Proteostasis and aggregate management

Jayasankar Mohanakrishnan Kaimal ©Jayasankar Mohanakrishnan Kaimal, Stockholm University 2017

ISBN print 978-91-7649-998-6 ISBN PDF 978-91-7649-999-3

Printed in Sweden by Universitetsservice US-AB, Stockholm 2017 Distributor: Department of Molecular Biosciences, The Wenner-Gren Institute To my family,

Late Mr.Mohanakrishnan G. Mrs. Jayasree Kaimal Mrs. Jisha Raj Mr. Jayakrishnan M. Kaimal

Research summary

Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis.

Vetenskaplig sammanfattning

Proteiner måste veckas till sina nativa strukturer för att uttryckas funktionellt. Felveckade proteiner är giftiga och påverkar cellen negativt. För att hålla proteomet funktionellt så kvalitetkontrolleras proteiner kontinuerligt av molekylära chaperoner. I den här avhandlingen används modellorganismen Saccharomyces cerevisiae för att utreda de mekanismer som celler använder för att upprätthålla proteinhomeostas (proteostas). I delarbete I utreds hur den molekylära chaperonen Hsp110 är involverad i extraktion och återaktivering av aggregerade proteiner. Vi fann att Hsp110 krävs för cellulär proteindisaggregering tillsammans med Hsp40, Hsp70 och Hsp104 samt karaktäriserade dess nukleotidutbytesfaktorfunktion i regleringen av Hsp70 ATPasaktivitet. I delarbete II beskrivs hur Hsp110 uttrycks genom att translationen skiftar läsram vilket resulterar i att proteinet skickas till cellkärnan. Kärnlokaliserad Hsp110 interagerar med Hsp70 och programmerar om cellens proteostassystem så att det bättre kan hantera stress och öka cellers livslängd. Delarbete III beskriver hur nukleotidutbytesfaktorn Fes1 reglerar Hsp70-funktion i kvalitetskontroll av proteiner. Vi upptäckte att Fes1 uttrycks via en ovanlig form av alternativ splitsning som reglerar dess lokalisering i cytoplasma och cellkärna. Den cytoplasmatiska formen av Fes1 har en nyckelroll i kvalitetskontroll av proteiner. I delarbete IV beskriver vi mekanismen som Fes1 använder för att reglera kvalitetskontroll av proteiner. Vi visar att Fes1 bär på en specialiserad domain som säkerställer att proteinsubstrat släpps loss från Hsp70. Resultaten förklarar hur Fes1 håller Hsp70-systemet fritt från skadliga felveckade proteiner. I delarbete V beskriver vi hur en ny biolumeniscent rapportör (Nanoluc) kan användas i jäst för att mäta genexpression och proteinnivåer. Sammanfattningsvis så bidrar denna avhandling till förståelse av de molekylära mekanismer som utgör chaperonberoende kvalitetskontroll av proteiner, både vad gäller de individuella komponenterna och hur de samspelar för att upprätthålla proteostas.

List of publications

1) Kaimal JM, Kandasamy G, Gasser F, Andréasson C. (2017). Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae. Molecular and Cellular Biology 37

2) Kaimal JM, Habernig L, Büttner S and Andréasson C. Nuclear targeting of Hsp110 modifies the proteostasis system by mobilizing latent Hsp70 chaperones. Manuscript

3) Gowda NKC, Kaimal JM, Masser AE, Kang W, Friedländer MR, Andréasson C. (2016). Cytosolic splice isoform of Hsp70 nucleotide exchange factor Fes1 is required for the degradation of misfolded proteins in yeast. Molecular Biology of the Cell 27: 1210-1219

4) Gowda NKC*, Kaimal JM*, Kityk R, Daniel C, Liebau J, Öhman M, Mayer MP, and Andréasson C. (2017). Substrate-mimicking domain of nucleotide-exchange factor Fes1/HspBP1 ensures efficient release of persistent substrates from Hsp70. Nature Structural and Molecular Biology (in revision)

* Contributed equally to the study  5) Masser AE, Kandasamy G, Kaimal JM, Andréasson C. (2016). Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae. Yeast 33(5):191-200

Studies not included in this thesis

1) Suhm T, Kaimal JM, Hanzén S, Dawitz H, Ambrozic M, Smialowska A, Björck ML, Brzezinski P, Nyström T, Andréasson C, and Ott M. Mitochondrial translation efficiency controls cytoplasmic protein homeostasis. Cell Metabolism (in revision)

2) Suhm T, Habernig L, Rzepka M, Kaimal JM, Andréasson C, Büttner S and Ott M. A system to follow mitochondrial translation in yeast with optical methods. Manuscript

Abbreviations

AAA+ ATPases Associated with diverse cellular activities APOD Age-dependent protein deposits CLS Chronological life span CMA Chaperone mediated autophagy CTD C-terminal domain Fes1 Factor exchange for Ssa1 HSE Heat shock element HSF Heat shock factor HSP Heat shock protein HSR Heat shock response INQ Intranuclear quality control compartment IPOD Insoluble protein deposit JUNQ Juxtanuclear quality control compartment MD Middle domain NBD Nucleotide binding domain NEF Nucleotide exchange factor NTD N-terminal domain PN Proteostasis network PQC Protein quality control RD Release domain SBD Substrate binding domain Snl1 Suppressor of Nup116-C lethal Ssa Stress-seventy A Ssb Stress-seventy B Ssc Stress-seventy C Sse1 Stress-seventy E1 UPS Ubiquitin proteasome system

Contents Research summary ...... i Vetenskaplig sammanfattning ...... ii List of publications ...... iii Studies not included in this thesis ...... iv Abbreviations ...... v Preface ...... ix 1 Introduction ...... 11 Protein structure ...... 11 Protein folding ...... 11 Protein aggregation ...... 12 Proteotoxicity ...... 15 2 Proteostasis maintenance ...... 17 Proteostasis ...... 17 Protein quality control ...... 17 Spatial protein quality control ...... 19 Protein degradation and clearance ...... 20 3 Molecular chaperones and their regulation ...... 22 Molecular chaperones ...... 22 A glimpse into the eukaryotic chaperone network ...... 25 The heat-shock response ...... 26 4 Saccharomyces cerevisiae as a model organism ...... 28 5 The Hsp70 system ...... 29 Hsp70 chaperones ...... 29 Allosteric chaperone mechanism of Hsp70 ...... 30 Hsp70 in yeast ...... 32 Hsp40 in yeast ...... 33 Nucleotide exchange factors ...... 35 The BAG family-Snl1 ...... 36 The Armadillo family- Fes1 ...... 36 The Hsp110 family- Sse1 and Sse2 ...... 37 6 Protein disaggregation ...... 40 The Hsp104 disaggregase ...... 40 Bi-chaperone disaggregase machinery ...... 42 Aims ...... 45 Summary of individual studies ...... 46 Study I ...... 46 Study II ...... 48

Study III ...... 50 Study IV ...... 52 Study V ...... 54 Conclusion and Outlook ...... 55 Acknowledgement ...... 57 References ...... 59

Preface

Proteins are ubiquitous in the cell with wide range of activities including sensing internal and external cues, DNA replication, metabolic activities, transport of molecules etc. To perform their functions proteins have to be folded to their native structures. Failure of the protein folding results in loss of function causing pleiotropic physiological disturbances that downgrades the cellular fitness. Therefore, understanding maintenance of proteostasis is crucial for human health and longevity. In this thesis using Saccharomyces cerevisiae as the model organism, I have focused on understanding how cells employ protein quality control (PQC) pathways to maintain proteostasis. Specifically, PQC mediated by the molecular chaperone Hsp70 and its cofactors have been investigated in this thesis. The work offers insight into the cellular disaggregation machinery. Moreover, the data provides mechanistic insights into how nucleotide exchange factors (NEFs) regulate Hsp70 in PQC. The results support the notion that Hsp70 function is regulated in the cell to participate in different pathways of PQC by a conserved pair of NEFs.

1 Introduction

Protein structure

Proteins are macromolecular structures comprised of polypeptide chains that have structural, enzymatic and regulatory roles in cells. For example globular proteins are water-soluble proteins 1 that achieve their conformations by the tertiary and quaternary structures where the hydrophobic amino acids are hidden in the interior and the hydrophilic amino acids are exposed on the surface. Globular proteins are involved in many functions, e.g. as enzymes, structural components, regulators messengers and also as transporters. The secondary structure is formed by hydrogen bonds between the peptides leading to a locally folded conformation of alpha helices and beta sheets. The alpha and beta structures are arranged in a tertiary structure that finally confers the functional conformation of the protein. The final level of protein structure is the quaternary structure that involves association of polypeptide chains. Taken together protein structure and function are highly interdependent.

Protein folding

Protein folding is a process by which polypeptides fold into a structured and biologically active protein. In vitro experiments by Anfinsen and colleagues showed that the primary amino acid sequence contains the information sufficient for folding the protein into its native conformation 2. The folding of proteins into their native structures based on the information intrinsic to the primary amino acid sequence is termed Anfinsen’s dogma or the thermodynamic hypothesis. Yet protein folding should not be viewed as a spontaneous process independent on the environmental context. Instead in the crowded cellular milieu folding is highly dependent on environmental conditions (pH, solvent, ionic strength, temperature etc.) 3. Specifically, protein folding relies on the assistance of many molecular factors that are described later in detail. 11 The natively folded conformation of a protein generally represents its most thermodynamically stable state. Proteins achieve their stable low energy states by a trial and error process 4. It has been found that in protein folding residues interact with each other forming a folding nucleus on which other residues build on to finally form the low energy state of the natively folded structure 5. For a fully folded functional protein all possible native interactions have to be attained resulting in the hydrophobic residues buried deep inside the structure 4.

Together with size and stability, the topology of the polypeptide also dictates the protein folding rate 6. It was shown that the average sequence distance between all pairs of contacting residues (contact order) correlates with folding rate 6. α-helical proteins are found to fold faster compared to their counterparts having mixed α helices and β sheets or β sheets alone, due to the fact that α-helical proteins have extensive short range contacts 7. In vitro experiments have demonstrated that small single domain proteins fold faster than larger proteins 8.

In vivo, protein folding happens both co-translationally close to ribosomes and also post-translationally in the cytosol or sometimes in specific compartments including mitochondria and endoplasmic reticulum 4. Proteins have evolved to fold efficiently in the context of their native cellular milieu. For this purpose cells employ a set of factors called molecular chaperones that assist in proper protein folding, in protein export and import to different compartments. Molecular chaperones through a series of action mediates protein folding and reduce the competition of other processes, including aggregation and off-pathway folding, thereby offering a net positive effect in folding 9-11. Chapter 3 and the publications included in the thesis cover in depth molecular chaperones and their regulation.

Protein aggregation

Protein aggregation is the process by which proteins interact inter- or intra- molecularly resulting in accumulation of the aggregated states. Protein aggregation can be both deleterious and advantageous for cellular function. This section focuses in detail on the features and mechanisms of protein aggregation.

12 The aggregation propensity of a protein is mainly governed by its amino acid sequence, the conformational stability of the native state and its concentration in the cells. The presence of hydrophobic amino acids on the surface of a polypeptide increases the likelihood of aggregation. A systematic protein sequence analysis has shown that proteins have evolved to avoid hydrophobic residue clusters 12. It has been shown that the vulnerability of proteins to aggregation is also concentration dependent, for example haemoglobin S, a protein in the red blood cell responsible for delivering oxygen is highly susceptible to aggregation depending on its concentration in the cell 13. Stability of the protein also leads to aggregation as mutating the buried polar residues leads to aggregation of proteins during stressful conditions 14,15. Aggregates are normally formed when the otherwise buried hydrophobic residues are exposed to the environment giving rise to an increase in their number of interacting protein partners and also to β-sheet stacking 16. Loss of the native structure drives proteins to adopt non-functional structures including partially folded, misfolded, aggregated and amyloid states. Amyloids are protein structures with fibrous morphology that are formed predominantly by the association of β-sheets, and are highly resistant to degradation. Native protein structures are also susceptible to aggregation resulting in the formation of amyloids or fibrils 16- 18. An example of natively folded aggregation is the yeast prion Ure2, where fibrils are formed by the association and polymerization of native-like subunits 19. Protein aggregation has also been offered a regulatory function in the cell. In dormancy stage (a state with reversible cell cycle arrest and reduced metabolic activity) or in stressful conditions like nutrient starvation several microscopic structures are seen in yeast cells that harbours protein and RNA molecules 20-24. Aggregated filamentous structures of enzymes have been observed in this condition that are readily activated when favourable condition returns 25. Recent studies by Alberti and colleagues have shown that during starvation or dormancy state several proteins get clustered and accumulate into microscopic structures. Here, they found that the soluble cytosolic compartment gets converted into a solid-like compartment resulting in lower mobility of molecules as a result of protein aggregation 20. This adaptable cellular strategy is highly important for survival under stressful conditions and also evolutionarily favourable. Figure 1 illustrates the various structural conformations a protein undergoes from its synthesis until degradation.

13

Figure 1: Schematic representation of protein conformations. Adapted from 17.

Proteins are prone to aggregation starting from their synthesis to degradation. Transcriptional and translational accuracy is crucial to protein structure and function and the fidelity has been reported to deteriorate with aging 26. Transcriptional errors are reported to occur once in every 10-5 nucleotide 27. In addition the mis-incorporation frequency during translation ranges from 10-3 to 10-4 28,29. Finally, any faulty translation drives the misfolding of proteins. Recent evidence from independent studies suggests that aggregation-prone proteins exhibit high translation rates and are at extensive surveillance of chaperones and other cofactors during translation 30,31. Also mutations may code for destabilized proteins that do not fold well. The ribosome structure also impacts on protein folding. The exit tunnel of the large ribosome subunit is almost 100Å long and 20Å wide 32 with the result that folding beyond α-helical structures are hindered 33,34 moreover the length of the tunnel prevents the last 40-60 amino acid residues to form interactions 35. Consequently, efficient folding occurs after complete protein

14 synthesis of at least one domain (≈50-300 amino acid) has been completed and emerged from the translating ribosome 10,36,37. Non-native interactions during translation can lead to aggregation. From translation and onwards aggregation is induced by stressful conditions for example non-optimal temperature or pH and disruptions in the refolding and degradation pathways. Misfolded proteins that accumulate under those conditions act as seeds for further aggregation by non-specifically interacting with other partly folded and misfolded proteins 38,39.

One must not assume that all protein aggregation events are spontaneous and deleterious off-pathway folding events. Recent evidence suggests that cells employ specialized aggregation factors for the targeting of unfolded or misfolded proteins into specific aggregate structures in the cell potentially reducing their toxicity (discussed below in detail). Protein aggregation is also used in physiological regulation. Cells take advantage of controlled aggregation to confer protease resistance, regulating the cell cycle and biofilm formation 40. For example, in mammalian cells, it has been shown that amyloids act as a storage reservoir for peptide hormones 41. Another example of physiological regulation relates to the cell cycle. Cdc19, an important regulator of metabolism and cell growth have been shown to form reversible aggregates, that ensures efficient cell cycle restart after stressful condition by protecting the otherwise degradation prone protein from proteolysis 42. The amyloid aggregates help in bacterial adhesion, biofilm formation and host invasion 43,44. Prions constitute another classical example of aggregates of physiological relevance. The proteinaceous infectious particle in amyloid form is capable of self-propagation and transmitting genetic information between cells 45,46. These findings highlight evolutionary advantages of harboring regulated protein aggregation mechanisms.

Proteotoxicity

Proteotoxicity is the toxicity caused by proteins that occupy non-native conformations. In yeast, it has been shown that misfolded proteins are proteotoxic and affect thermo sensitivity and cellular life span 47,48. Defect in mitochondrial machinery due to the accumulation of α-synuclein is an example of proteotoxicity. 49-51. Cells counteract the toxicity caused by the misfolded proteins by sequestration and thereby reduce toxicity and improve cell fitness 52,53. Recent studies suggest that the stably misfolded

15 conformations are usually not the toxic species but instead the intermediate structures enroute misfolding are the culprit for toxicity 54. It was shown that the cytotoxicity stems from the organization of the aggregates. Rapidly formed non-fibrillar aggregates possessed higher cytotoxicity compared to the organized fibrillar structures. Recent studies have shown that the oligomeric intermediate non-native protein structures are more toxic than the stable mature fibrils 54-56. The reason for these observations can be attributed to the structural organization of the conformers. Oligomeric species expose more hydrophobic residues as compared to the fibres. They are smaller in size compared to its mature fibres, thereby increasing the mobility and interacting partners. In summary, accumulation of non-native protein structures lead to toxicity and perturbs cellular function.

Proteotoxicity is considered to be the outcome of both loss and gain of function of the misfolded proteins. Proteotoxicity from loss of function arises from non-native conformations that lack the destined function of a protein. For example, mutations in Parkin an E3 ubiquitin ligase results in loss of ubiquitin ligase activity, which in turn leads to the accumulation of toxic parkin 57,58. Other examples of disease conditions caused by loss of function include cystic fibrosis and lysosomal storage disorders 59,60. In contrast, gain of function toxicity is the result of when protein misfolding dominates over clearance pathways 59,61. Huntington’s disease is caused by gain of function proteotoxicity, in which the accumulation of polyglutamine proteins leads to aggregation in the cytosol and causes neurotoxicity 62. Other examples include cancers, neurodegenerative diseases like Alzheimer’s and amyotrophic lateral sclerosis 63. All these conditions arise from accumulation of non-natively folded and aggregation prone proteins that interfere with cellular protein homeostasis 59,61,64,65. Although, it is not clear whether the proteotoxicity is solely caused by the direct effect of the aggregates or if it is an indirect effect where toxicity have arisen during the aggregation process. Studies have shown both aggregates and intermediates contribute to toxicity and impair cellular processes including mitochondrial function 66,67 and the proteasomal system 49,68.

16 2 Proteostasis maintenance

Proteostasis

Proteostasis or protein homeostasis is a phenomenon by which cells control the concentration, conformation, binding partners and localization of proteins depending on the physiological milieu by controlling the transcription and translation 64. From protein synthesis till its degradation proteins are under the surveillance of a network of systems comprising the synthesis, folding, degradation and translocation machinery, that is defined as the proteostasis network (PN) 69. Proteostasis is essential for enabling cells to adapt and readapt according to the environment. Cells employ several strategies to ensure proteostasis. These strategies include spatial sequestration of protein aggregates and increased activity of folding and degradation machineries. An imbalance in proteostasis causes widespread and pleiotropic proteotoxic effects. The failure of the proteostasis system to manage specific proteins may also results in proteotoxicity. For example, Alzheimer’s, Parkinson’s and Huntington’s disease all stem from altered protein homeostasis in the cell 64. Impairment in the activity of PN have been reported during cell development, aging and also with environmental stress 70. Several studies have identified a reduced fidelity of PN during the early stages of aging affecting protein folding and increased propensity of protein aggregation 71-73. The following section deals in detail with the factors controlling PN.

Protein quality control

Proteins are under thorough surveillance by the protein quality control (PQC) system from synthesis till degradation. Cells maintain protein homeostasis by monitoring the folding of proteins to their native conformations. When misfolded proteins are detected they are routed to refolding and degradation pathways to ensure their clearance from the cell (Figure 2). Chaperones support folding and ensure the targeting of the 17 misfolded proteins for degradation via the ubiquitin proteasome system (UPS) 10,74,75.

Figure 2: Representation of PQC.

PQC occurs both co- and post-translationally. Co-translational PQC monitors proper folding to avoid the formation and accumulation of misfolded, non-native proteins during protein synthesis. In parallel, defective nascent polypeptides from stalled ribosomes are routed for degradation via the proteasome, thereby reducing the proteotoxicity caused by the defective translation product 5,76-78.

The second layer of PQC occurs after the successful polypeptide synthesis in the cytoplasm, termed post-translational quality control 79. Here, the PQC system ensures correct folding followed by challenges from the physiological milieu, translational errors and aging. PQC occurs in a compartment specific manner but are linked via the integrated organellar organization of the cell. For example nuclear and cytosolic PQC are highly interdependent with protein synthesis localized to the cytosol and degradation system highly active in the nucleus. From a mechanistic point of view, PQC systems are based on that the hydrophobicity exposed by the substrates is recognized by chaperones that mediate refolding, disaggregation or degradation by the UPS 80-83. The chaperone ensures the selective degradation of substrates by the assistance of ubiquitin ligases that tag the substrate for proteasomal degradation (discussed later in detail) 80,84,85. The degradation of misfolded proteins is mainly performed by the proteasomal and autophagy systems. Proteasomes are specialized proteolytic

18 complexes that localize to the cytoplasm and nucleus. Autophagy on the other hand clears proteins by isolating them from the cytosol inside a double walled vesicle that is targeted to the vacuole/lysosome compartment 86,87. Yeast cells harbour proteasomes predominantly in the nucleus, where the misfolded proteins from the cytosol are sent for degradation 88-90. In conclusion, the misfolded proteins are selectively targeted for degradation by the sequential action of chaperones and ubiquitin ligases. First, the chaperones bind to the exposed hydrophobicity in the misfolded proteins. Second, a degradation signal is attached on the substrate by the action of ubiquitin ligases that are degraded by the proteasomes.

Spatial protein quality control

In parallel to refolding and degradation PQC pathways, cells employ spatial sequestration of misfolded proteins to distinct compartments. Using yeast as a model organism several studies have highlighted the presence of these compartments. Upon temperature stress proteins misfold and aggregate in the cytosol to form peripheral aggregates, also known as Q bodies, CytoQ or stress foci, 52,53,91-94. In parallel, recent studies have shown the existence of two different compartment specific aggregate structures called JUNQ/INQ and IPOD. JUNQ/INQ was initially found as JUxtaNuclear Quality control compartment (JUNQ). Later Bukau and colleagues have identified it within the nucleus and proposed the name IntraNuclear Quality control compartment (INQ) 91,92. IPOD (Insoluble Protein Deposit) on the contrary harbors misfolded amyloidogenic protein that might be destined for degradation or for dissolution 91-93.

For the formation of these specific compartments several chaperones and aggregation promoting factors have been shown to be important, namely Hsp104, Ssa1/Ssa2, Sis1, Mca1, Btn2, Hsp26, Tsa1, Cur1 in the JUNQ/INQ and Hsp42 and Ltn1 in the IPOD 92. Q-body formation does not depend on cellular ATP however the clearance and dynamics of the inclusions are ATP-dependent processes 52. Ubiquitination have been thought to be the signal for sorting misfolded proteins to the nucleus as blocking ubiquitination results in IPOD formation and enhanced ubiquitination moves the protein population form IPOD to JUNQ/INQ 91,95. However recent studies have shown that ubiquitination is not required for JUNQ/INQ formation 92. Other important sorting factors are the stress inducible proteins

19 Btn2 and Cur1. Complex formation between Btn2-Sis1-substrates and Cur1- Sis1 targets misfolded client proteins and Sis1 to JUNQ/INQ. On the contrary, Btn2-Hsp42 targets proteins to the peripheral deposition site 96. Recent studies have discovered another site for protein sorting during aging termed Age-dependent PrOtein Deposits (APOD) that are formed during early aging phase. APOD formation enhances cytosolic protein quality control. The chaperones Hsp42, Ssa1 and Hsp104 colocalizes with APOD. Hsp42 enhances the formation and Hsp104 increases the dissolution of APOD 97. Interestingly, it has been shown that during cell division asymmetric inheritance of aggregates is favoured and the aggregates in the QC compartments (JUNQ/INQ, IPOD, APOD) sites gets retained in the mother cell and are not passed on to the daughter cells 53,95,97. All these findings highlight the importance of these specific structures in the PQC and suggest important roles in enhancing stress tolerance, fitness and cellular longevity.

Protein degradation and clearance

A key process for proteostasis maintenance is the selective removal of misfolded proteins by the ubiquitin-proteasome system (UPS). The Polyubiquitin tag is attached to misfolded proteins and acts as a selective marker for degradation by the ubiquitin-proteasome 98-100. Ubiqutilyation is a highly systematic process that mediates degradation of more than 80% of all proteins 101. For ubiquitylation, several enzymes function sequentially starting with ubiquitin activating enzyme (E1) followed by ubiquitin conjugating enzymes (E2) and finally ubiquitin ligating enzymes (E3) that attach the ubiquitin moieties on the target protein or the growing ubiquitin chain via isopeptide bonds involving lysine side chains 102. Interestingly, a ubiquitylation signal confers different messages to the cell depending on the length of the ubiquitin chains. Mono-ubiquitination of substrates has recently been reported to act like a degradation signal for the proteasome apart from its role in protein interaction, localization and activation 103,104. As a sequential step, further ubiquitination can occur at any of its seven lysine residues (lys6, lys11, lys27, lys29, lys33, lys48 and lys63). Generally, a ubiquitin chain of 4 four ubiquitin moieties functions as a degradation signal for the proteasome 105. The proteasome is a highly organized proteolytic complex with many different protein subunits functioning together. The degradation of ubiquitylated substrates starts with the

20 recognition by the 19S regulatory particle, de-ubiquitylation of the substrates and finally targeting the substrates to the central proteolytic chamber of the core particle 106. In the proteolytic chamber the polypeptides are proteolysed resulting in generation of smaller peptides that later are processed by endo and amino-peptidases to give rise to single amino acids 107. In summary, the UPS degradation system ensures degradation of potentially misfolded proteins thereby reducing the burden and toxicity associated with it.

In parallel to the UPS autophagy also ensures the degradation of misfolded proteins in the lysosome/vacuole 100. Autophagy clears substrates that have escaped the surveillance and degradation by the UPS. Here, the substrates are isolated in a double membrane structure that invaginate into the lysosome/vacuole and are degraded by the lysosomal hydrolases. 86,108. Depending on the mode of substrate delivery autophagy is divided into three pathways: microautophagy, macroautophagy and chaperone-mediated autophagy (CMA). Microautophagy is a process where small cytosolic fractions are degraded non-selectively 109. In macrophagy misfolded proteins in association with chaperones are autophagosized and delivered to the lysosome for degradation by lysosomal hydrolases 110,111. CMA on the other hand is a highly selective and organised pathway. Here, the substrates are recognised by Hsp70 (Hsc70) and are transported into the lysosome for degradation. The Hsc70 chaperone specifically identifies substrates with KFERQ signature and assist in CMA 112-114. Taken together, cells ensure clearance of the misfolded proteins with the dual degradation machinery, thereby lowering the proteotoxicity and restoring proteostasis.

21 3 Molecular chaperones and their regulation

Molecular chaperones

Molecular chaperones are a set of factors that assist in protein folding by interacting with the substrates without forming a part of their final structures 11. The first evidence of chaperones came in 1978 when Laskey and co- workers discovered that the nuclear protein nucleoplasmin prevented the aggregation of histones with DNA 115. Both the molecular chaperone function and many of the core chaperone proteins are conserved between bacteria, archea and eukaryotes 116. Apart from a direct involvement in de novo folding during protein biogenesis, chaperones are also involved in a wide variety of PQC functions including the degradation and reactivation of misfolded and aggregated proteins (Figure 3) 9,35,117,118. Moreover, chaperones are central players when translocating proteins over membranes, both to maintain the proteins unfolded and competent for movement through the pore structure and to generate a pulling force 83,119,120. Finally, chaperones functions to regulate the cell both when it comes to directly controlling signal transduction components, including transcription factors and to provide conformational alterations in important biological pathways. For example, during endocytosis, vesicles coated with a clathrin skeleton depend on the chaperone Hsp70 to destabilize the protein structure resulting in uncoating of the vesicles 121,122.

Even though chaperone function is diverse and not always well understood, chaperones typically perform their function by binding to the exposed hydrophobic residues of proteins that occupy non-native conformations 123- 125. During folding these residues are buried deep inside the native conformation explaining the chaperone affinity for non-native protein conformations. Binding of exposed hydrophobic residues enhances productive folding pathways and suppresses off-pathway conformational transitions that lead to misfolding and aggregation. The binding of chaperones to the substrate hinders both intermolecular and intramolecular interactions. Timely action of chaperones reduces non-native interactions

22 and releases the substrate for folding in an efficient manner. Paradoxically, chaperone affinity for amino acid residues that are exposed specifically in unfolded proteins may result in unfolding of proteins. For example bacterial Hsp70 (DnaK) binds misfolded firefly luciferase and unfolds it in an ATP dependent manner. This characteristic function of a chaperone is termed unfoldase and in a cellular environment a significant number of chaperones functions as unfoldases 120,126,127. The other characteristic function of chaperones is the holding function. Initially, the fundamental property of reducing the growth of an aggregate was called a holdase function. Not all chaperones function by preventing aggregation. For example Hsp100 chaperones are not able to hold proteins from aggregation but are an efficient disaggregase that disaggregates and reactivates proteins in a concerted manner together with Hsp70 and ATP. The term holdase is somewhat misleading since in biochemical terminology it suggests that the chaperone provides a catalytic activity, which clearly is lacking in this scenario. Therefore, Goloubinoff and colleagues suggested the term ‘holding chaperone’ over ‘holdase’ that is highly appropriate. Moreover, the terminology used provides functional characterization of the chaperones and does not follow the canonical terminology employed in enzymology. Independent of the terminology applied, chaperones impact on conformational transitions by interacting with specific residues or peptide stretches present in their substrates.

Molecular chaperones are further classified based on their ability to bind ATP and consume its energy by hydrolysis. Thus chaperones may be ATP dependent and ATP independent and display ATPase activity. Many of the chaperones that assist in de novo protein folding promote the process by repeated cycles of substrate binding and release controlled by their ATPase activity. The ATPase activity is in turn controlled by other cofactors or co- chaperones (discussed below in detail) that as an outcome regulate chaperone-substrate interactions. Principally, the ATPase-regulated interactions with substrate may be viewed as a timer of the interaction but also as harvesting energy for inducing conformational changes. For example GroEL/GroES, a bacterial cytosolic chaperone of the chaperonin type have been shown to promote folding of proteins post-translationally by pulling apart the misfolded proteins and enhancing folding powered by ATP hydrolysis 128,129. The chaperonins performs its folding function by isolating the misfolded proteins into a cage-like structure and stretching them apart, leading to unfolding of the substrate protein 128-131. In contrast, ATP- independent chaperones block aggregation in an energy-independent 23 manner. A vast majority of ATP-independent chaperones bind substrates and prevent aggregation, maintaining them in folding competent states but do not promote refolding 132. It is yet not clear, how these two different chaperone classes selectively identify their substrates and what intrinsic or extrinsic features mediate the specificity. In the simplest model they kinetically compete for overlapping and distinct binding sites. It is intriguing to find the coexistence of ATP-dependent and independent chaperones in nature and also to think about the evolutionary advantages offered by them. Clearly ATP-independent chaperones are important in compartments with reduced ATP levels, for example in the bacterial periplasm or the extracellular matrix of eukaryotic cells 132-134. Clusterin is a classical example of an ATP- independent chaperone found associated with amyloids and is involved in reducing the toxicity and finally enhancing proteostasis 135. Another function for ATP-independent chaperones can be attributed to proteostasis maintenance under stressful conditions where the ATP levels are reduced. Studies suggest that ATP-independent chaperones like the small Hsps (sHsps) are the first chaperones that encounter unfolded proteins and prevent their aggregation 136. Many ATP-independent chaperones form highly organized, regulated and dynamic oligomeric structures thereby increasing the local concentration of chaperones around the client proteins. The orchestrated action of these chaperones protects the cell from toxic accumulation of unguarded misfolded proteins.

24

Figure 3: Functions of chaperones in the proteostasis system. Proteostasis is maintained by chaperones involved in various functions including refolding, degradation and disaggregation of misfolded proteins. Pathway favouring folding, degradation and conformational maintenance are colour coded with green, red, and purple respectively 137.

A glimpse into the eukaryotic chaperone network

Proteins are constantly under the surveillance of chaperones and co- chaperones that ensures proteostasis. Starting from their ribosomal translation to the degradation and clearance by the proteasome proteins encounter a vast number and type of chaperones. Hsp70 associates with the translating ribosomes where Ssb (Stress seventy subfamily B) binds the nascent polypeptides and ensures efficient co-translational protein folding 10,31,36. Misfolded proteins are first recognized first by sHsps (Hsp42, Hsp26)

25 and are recruited to Hsp70 for quality control. Hsp70 in concert with other disaggregases belonging to the Hsp100 family including Hsp104 (in yeast) and ClpB (in bacteria) disaggregates the protein (discussed later in detail) 138,139. Finally, the damaged proteins are routed for degradation via the proteasomes 88,140. Another key player in proteostasis maintenance is Hsp90. Hsp90 is important for substrate maturation, substrates degradation and proper integrity of the proteasome 141-145. In conclusion, with the proper guidance of molecular chaperones and its associating partners cells achieve proteostasis.

The heat-shock response

The expression of many chaperones is induced by imbalances in protein homeostasis. Since heat shock was an experimental condition early on employed to trigger such stress, the induced proteins were originally called Heat Shock Proteins (Hsps) 146. The gene-regulatory program that triggers Hsps is termed the heat-shock response (HSR). In eukaryotic cells the HSR genes are under the control of an ancient transcription factor called heat shock factor (HSF) 147,148. The genome of large eukaryotes and plants encodes multiple HSFs. However, yeast possesses only the single Hsf1 117. HSF1 constitutes an essential gene in S. cerevisiae that is involved in both housekeeping and stress-induced transcriptional activation 149,150. A genome wide analysis has revealed almost 165 genes being associated directly with Hsf1 with diverse cellular functions, including chaperone function, ubiquitylation, transport, cell wall and cytoskeleton maintenance and signal transduction 151. Hsf1 architecture comprises an N-terminal activation domain followed by a DNA binding domain, three leucine zippers responsible for trimerization and a C-terminal transactivation domain 117. The promoters of Hsf1 target genes harbour the conserved sequence ‘nGAAn’ that form the basis for heat shock elements that Hsf1 binds to 117.

Hsf1 transactivation was initially thought to be controlled by two factors; the chaperone regulated and phosphorylation mediated 147. The chaperone- regulation model suggests that the Hsf1 rests latently in a complex with chaperones under non-stressful conditions and is activated during stressful conditions. Upon stress proteins misfold and titrate out the available chaperones that are recruited to the damaged proteins. As an outcome Hsf1 is released and subsequently activated, binds to HSEs in the promoters of its

26 target genes and activate transcription 117,152. Upon, recovery from stressful conditions chaperones rebinds to Hsf1 and down regulate Hsf1-dependent transcription. Since the HSR includes Hsf1-regulating chaperone genes it represent a proteostasis feedback system 152. The phosphorylation model suggests that upon stressful condition the released Hsf1 becomes phosphorylated and gain competence to activate transcription of chaperone genes 149. However, systematic mutational analysis have recently shown that the phosphorylation is not required for activation of Hsf1 transcription and may instead be involved in fine tuning gene expression during stress 152. Thus, the present understanding is that Hsf1 is regulated by chaperone titration and that at least in yeast the regulator is Hsp70 152.

In parallel to HSF1-mediated transcription of Hsp genes in yeast the two homologous transcription factors Msn2 and Msn4 (Msn2/4) also activate transcription in response to multiple stressors including heat shock 117. Msn2/4 recognise stress response elements (STREs) in the promoters of the target genes and activate transcription 153,154. Msn2/4 are 41% identical on amino acid level and simultaneous inactivation results in sensitivity towards osmotic and thermal stress 155,156. The combined action of Hsf1 and Msn2/4 explains most of the HSR observed in yeast. Yet Msn2/4 are downstream transcriptional effectors of the PKA-pathway and are only involved in proteostasis responses indirectly via metabolic signalling. Taken together, the proteome and chaperome of cells are highly regulated on the transcriptional level via Hsf1 and HSR in response to proteostasis perturbations.

27 4 Saccharomyces cerevisiae as a model organism

Model organisms are studied to increase our understanding of biological processes or in the medical context, to replace difficult or unethical studies on humans. The use of model organisms to study biological processes has accelerated scientific development tremendously. S. cerevisiae is a versatile, widely used model organism with several advantages for addressing cell biological questions. This includes rapid growth, low pathogenicity, a well defined genetic system, stable haploid and diploid growth forms and a highly efficient DNA transformation system 157,158. S. cerevisiae was the first eukaryote with a genome to be completely sequenced, which accelerated research efforts 159. The yeast genome is almost devoid of introns and extensive repetitive DNA, which together with the available genetic methodology make genetic manipulations easy and fast. Approximately 30% of genes involved in human disorders have orthologs in yeast 160. Importantly the fundamental setup of the proteostasis system is also conserved including the Hsp70 system with most of its cofactors. S. cerevisiae have been used to study protein homeostasis-associated processes including aging, PQC, gene expression, the cell cycle, metabolism, neurodegenerative disorders and apoptosis. The studies included in this thesis have used S. cerevisiae as the model organism to understand PQC mechanism and aging.

28 5 The Hsp70 system

Hsp70 chaperones

Heat shock protein 70 (Hsp70) is a chaperone that is conserved in bacteria, archea and eukaryotes. Hsp70 is involved in many functions in the cell, including co- and post-translational folding, translocation, refolding, protein reactivation, assembly and disassembly of proteins 3,123. Thus Hsp70 is critical to maintain proteostasis in the cell. The experimental contributions presented in this thesis focuses in depth on Hsp70-dependent cellular functions using the model organism budding yeast, S. cerevisiae.

Hsp70 is structurally composed of a 44 kDa nucleotide-binding domain (NBD), a ~ 25kDa substrate-binding domain (SBD) 161-165. Hsp70 exists in two conformations that are allosterically regulated by ATP and ADP binding. In the ATP-bound conformation Hsp70 populates an open conformation and binds to hydrophobic surfaces of client proteins with low affinity. ATP hydrolysis results in substrate trapping with high affinity to Hsp70-ADP. The rebinding of ATP to the NBD converts the Hsp70 to an open conformation 164,166-169. The repeated cycling between open and closed conformations of Hsp70 mediates protein folding by transiently interacting with its substrates and prevents aggregation (Figure 4). The Hsp70 chaperone cycle is mediated by several co-chaperones including Hsp40 and nucleotide exchange factors (NEFs) that mediate the conversion of ATP bound form to ADP and vice versa. Briefly, the binding of Hsp40 and the substrate protein accelerates ATP hydrolysis of Hsp70. ADP exchange for ATP is driven by interactions with NEFs.

Different mechanisms have been proposed for Hsp70 mediated folding. ‘Kinetic partitioning’ is a proposed mechanism by which Hsp70 binding to the substrates ensures reduced concentration of free substrate and thereby inhibits aggregation pathways and promotes folding of free molecules. In protein translocation Hsp70 have been proposed to perform its activity by ‘Brownian ratchet’ mechanism, where binding of Hsp70 to the translocating polypeptide ensures unidirectional movement and protects substrates from backsliding 170,171. This mechanism is incomplete since this model does not 29 explain how the well-folded substrates unfold in the first place to be translocated through the membrane pores 83. Another proposed mechanism is the ‘power-stroke’ where Hsp70 apply power to the bound substrate during the ATPase cycle thereby pulling the polypeptides into the matrix and subsequently unfolding the substrates in the cytosol 119,172. Goloubinoff and colleagues have combined all the above modes to a united action called ‘entropic pulling’. Here, the energy from the ATPase activity of Hsp70 unleashes a force capable for unfolding the substrates or to translocate across membranes 119. This mechanism is highly plausible as it explains how Hsp70 can participate in protein folding, translocation and disaggregation. However, it cannot be ruled out that Hsp70 in the cell may perform its activity by combining all the above mechanisms.

Figure 4: Hsp70 chaperone cycle. Iterative cycling between ATP and ADP bound conformations promote protein folding by Hsp70.

Allosteric chaperone mechanism of Hsp70

The SBD of Hsp70 comprises of a β-sheet and an α helical subdomain 165,173. The β-sheet subdomain has two twisted, four-stranded β-sheets possessing upward extending loops for enclosing the substrates. The α-helical subdomain has five helices (A-E). A and B helices are positioned adjacent to the β subdomains with B helix forming salt bridges and hydrogen bonds to the outer loops from the β sheet finally forming a lid for the bound substrates 173. The C-E helices form a structural part to the domain as deletion of this domain affects the structural stability of helix B 174,175. Hydrogen bonds and 30 hydrophobic interactions stabilizes the interaction between substrates and Hsp70 at the substrate-binding pocket 173. The stability of Hsp70-substrate complex is also contributed by the hydrophobic interactions between the substrates and the SBD residues (Met404, Ala429), making an arch over the bound substrate 173. Studies from independent labs have shown a highly dynamic nature of the Hsp70 depending on the nucleotide bound state 176-178. In the ATP bound state the SBDα and SBDβ are detached from each other and placed adjacent to the NBD. This leads to the stabilization of the open conformation with a broader substrate-binding cleft as compared to SBD bound with substrate 177 supporting low affinity and high ON-OFF rate of substrates.

The Hsp70 NBD comprises mainly two lobes with a deep cleft in between for nucleotide binding 173. The NBD is highly dynamic in nature and regulates substrate binding and release from the SBD based on its nucleotide bound status. In the ATP bound state the β-sheet and α-helix of the SBD are separated from each other resulting in low affinity towards the substrates, and in the ADP bound conformation the α-helix is in tight affinity to the β- sheet resulting in tight association with the substrates 176,179-184. The linker region between NBD and SBD is important in allosteric regulation and has been shown to be involved in inducing ATP hydrolysis at the NBD upon substrate binding at the SBD 185,186.

The allosteric regulation of Hsp70 depends on several factors and co- chaperones. Hsp70 binding to Hsp40 (a 40 kDa chaperone protein) and substrates accelerate ATP hydrolysis of the Hsp70 thereby increasing substrate affinity and reducing the substrate binding and release rates. The resulting tightly bound conformation with the substrate is terminated by the action of several NEFs. They interact with the NBD, accelerate the release of bound ADP and thereby accelerate the exchange to ATP. The ATP- induced allosteric conformational changes result in the release of bound substrate. Repeated chaperone binding and release cycles regulated by the co-chaperones ensures the function of Hsp70.

31 Hsp70 in yeast

In this section, I will present the Hsp70 system with a special emphasis on its layout in the model organism yeast. Hsp70 is ubiquitous in the cytosol, endoplasmic reticulum (ER) and mitochondria with high basal expression levels under non-stressful conditions and induced expression following stress. Table 1 highlights the Hsp70 forms expressed in yeast cells and their subcellular localization. While the cytosolic and ER Hsp70 are eukaryotic in origin the mitochondrial Hsp70 bears hallmark of its bacterial relative DnaK. I will from here on focus on the Hsp70 system that is present in the cytosol and nucleus of yeast cells, which is the main focus of the study.

Table 1: Hsp70 chaperones in S. cerevisae, their expression and localization.

Protein Localization Expression References Ssa1 Cytosol & Constitutively 187-189 Nuclear expressed at high levels and stress induced Ssa2 Cytosol & Constitutively 187,189 Nuclear expressed at high levels Ssa3 Cytosol/ Lowly expressed 187,189-193 Nucleus but the expression increases drastically during entry to stationary phase Ssa4 Cytosol & Lowly expressed 187,189,190,194 Nuclear but the expression increases during stress Ssb1 Cytosol Constitutively 187,188,193,195,196 expressed Ssb2 Cytosol Constitutively 187,193,195,196 expressed Kar2 Endoplasmic Constitutively 197-201 reticulum expressed, but

32 expression increases by stress Ssc1 Mitochondria Constitutively 202-205 expressed but expression increases by stress

Hsp40 in yeast

Hsp40 functions as an Hsp70 co-chaperone by recognizing non-natively misfolded proteins and recruiting them to Hsp70. Hsp40 stimulates the ATPase activity of Hsp70 and thereby is required for the basic function of Hsp70 3,131,206. While the human genome encodes 44 Hsp40s the yeast genome encodes 20 80,207,208. Thus the Hsp40s change more rapidly in evolution than the core Hsp70 chaperone suggesting that they diversify the function of the Hsp70 system. For example, the fate of the bound misfolded proteins to Hsp70 depends on the type of Hsp40 it interacts with. When human Hsp70 is paired with DnaJB12 and ubiquitin ligase CHIP the bound substrate is targeted to a degradation pathway 209-211. On the contrary yeast Hdj2 and Ydj1 promote folding of bound non-native substrates 207,212,213. In metazoans, disaggregation and reactivation activity is enhanced synergistically by the action of the two types of Hsp40 together with Hsp70 214. In S. cerevisiae, Sis1 a Type II Hsp40 has been shown to be involved in the degradation of misfolded proteins in cooperation with Hsp70 215. All these findings highlight how the core Hsp70 chaperone is modified by Hsp40 to perform specialized functions.

All identified Hsp40 proteins has two domains, a conserved N-terminal J- domain (named after DnaJ, the first identified Hsp40 in E. coli) that accelerates Hsp70 ATP hydrolysis 207,216 and a SBD. The J-domain region is highly conserved with a functionally important signature HPD motif containing a histidine, a proline and an aspartic acid residue 208. It was reported that Hsp70 interacts transiently with Hsp40 via the exposed residues including the HPD motif in the J-domain 208,217,218. Studies using atomic force microscopy have shown that the bacterial DnaJ first interacts with the substrates and transfers it to Hsp70 via an anchoring and docking mechanism 206,219. Interdomain communication is the key for Hsp40 powered Hsp70 function, first Hsp40’s J-domain interaction with Hsp70 is important

33 for the ATPase activity and second the substrate transfer to Hsp70 from Hsp40 triggers an interdomain communication between the SBD to the NTD of Hsp70. Recent studies demonstrate that the structural stability of the J- region is highly important for stimulating the Hsp70s ATPase activity 220. In summary, Hsp40 is a key regulator of Hsp70 chaperone system.

Hsp40s including the ones represented in yeast are divided into three different classes based on the J-domain organization 221 222. Type I and II Hsp40 proteins possess a J-domain at the N-terminus that is connected to the substrate binding via a G/F (Gly/Phe)-rich linker region. The central domain of Type I and Type II is structurally different. Type I possess zinc finger (ZF) motifs whereas Type II have G/M (Gly/Met) rich region and a polypeptide-binding site. A dimerization domain is observed in Type I and Type II Hsp40’s however not all Hsp40 functions as dimers 223. Type III in contrast, does not have zinc finger motifs and the J-domain is positioned anywhere in the protein. The J-domain of Type III forms dimers 224. The central cysteine rich region of Hsp40 binds the unfolded substrates and delivers it to the Hsp70. Figure 5 represents the different types of Hsp40 in the cell.

Figure 5: Classification of Hsp40 based on the J-domain location in the protein structure. G/F denotes glycine/phenylalanine rich region in the Type I and II Hsp40. ZF and G/M represents the zinc finger motif and glycine/methionine rich region in Type I and II respectively. CTD and DD corresponds to the c-terminal domain and dimerization domain in Type I and Type II respectively. Type III possess J domain and lacks other domains that are conserved in other Hsp40 Types. Adapted from 223. 34 Nucleotide exchange factors

Nucleotide exchange factors accelerate the exchange of the ADP for ATP in Hsp70 by facilitating the rate-limiting step of ADP release. Release of the bound ADP from Hsp70-NBD results in allosteric communication between the domains and leads to release of the bound substrate from SBD 9. The yeast cytosol and endoplasmic reticulum harbors different families of structurally unrelated NEFs namely Snl1 (Bag family), Fes1 (Armadillo family) and Sse1 and Sse2 (Hsp110 family). The endoplasmic reticulum harbours two different NEFs, Lhs1 (Grp170 family) and Sil1 (Armadillo family). Lhs1 is the sole Grp170 family in yeast 117,225,226 (summarized in Table 2). The existence of the different family of NEFs suggests that they possess specialised roles in the cell. In the following section I will discuss each family of cytosolic NEFs in terms of its abundance in the cell.

Table 2: NEFs of Hsp70, their localization and chaperone partners.

NEF Localization Chaperone Reference partner Snl1 Endoplasmic Ssa1-4, Ssb1-2 187,227,228 (Bag family) reticulum and nuclear membrane Fes1 Cytosolic Ssa1-4 187,229,230 (Armadillo family) Sse1 & Sse2 Cytosolic Ssa1-4, Ssb1-2 187,231,232 (Hsp110 family) Lhs1 Endoplasmic Kar2 187,225,233,234 (Grp170 reticulum family) lumen Sil1 Endoplasmic Kar2 234,235 (Armadillo reticulum family) Mge1 Mitochondria Ssc1 236-242 (GrpE family)

35 The BAG family-Snl1

Bag1 was first identified in the mammalian cytosol, where it interacts with an anti apoptotic protein Bcl2, thereby increasing cell survival 243. The Bag domain interacts with Hsp70’s ATPase domain 244 and was suggested to interact with the proteasome using the ubiquitin like domain suggesting a linking factor for Hsp70 and the proteasome 245. The Bag domain of Bag1 protein forms a 3 helix bundle, of which helix 2 and 3 interacts with IB and IIB of Hsc70 ATPase domain, causing a 14° rotation of IIB and affecting the amino acid residues orientation, finally resulting in the opening of nucleotide binding cleft exchanging ADP for ATP 244,246. A lysine rich region in the BAG domain has the potential to interact with the large subunit (60S) of the ribosome, although the biological significance is still unclear.

Snl1 is an integral membrane protein localized to the nuclear envelope and endoplasmic reticulum of yeast cells 227. Snl1 is tethered to the membrane with a single 20 amino acid residue transmembrane domain at the N- terminus and C-terminus facing the cytosol. Overexpression of Snl1 suppresses the lethal phenotype of nup116-c cells defective in nuclear pore complex function (termed as suppressor of nup116-C lethal) suggesting a role in protein folding at endoplasmic reticulum membrane 227. However strains lacking Snl1 do not exhibit any apparent phenotype. Snl1 is a functional NEF that interacts both with Ssa and Ssb class of Hsp70 228. It is the only yeast Hsp70 NEF that has a BAG domain 228. The Bag domain homology was found at the CTD of Snl1 oriented towards the cytosol in yeast cells 227. It has been shown that Hsp70 and ribosomes interact with Snl1 at different sites and associates in a non-competitive manner 229. The mechanism of NEF activity was also seen conserved in Snl1 as demonstrated by hydrogen-deuterium exchange experiments 246. With all the above- mentioned features Snl1 remains as a unique NEF of the Hsp70 chaperone system.

The Armadillo family- Fes1

Fes1 (Factor exchange for Ssa1), a cytosolic homologue of Sil1 (ER NEF for Hsp70 Kar2), is a highly conserved NEF structurally belonging to the armadillo family of NEFs. Fes1 is the homolog of mammalian HspBP1 (Hsp70 binding protein 1) 247,248. Fes1 shares 25% identity and 38% 36 similarity to human HspBP1 230. Structurally, Fes1 and HspBP1 share a conserved core domain (BP1C) that functions as a NEF and therefore stimulates the ATPase activity of Hsp70 in presence of Hsp40 249. BP1C has an elongated shape with α-helical repeats that resembles armadillo repeats. BP1C interacts with the Hsp70 NBD subdomain IIB (part of lobe II i.e., subdomain IIa and IIb) 249. Upon complex formation Hsp70 NBD lobe I becomes sensitive to proteolysis suggesting that the mechanism for nucleotide release involves destabilization of the NBD by distortion of lobe I together with separation and opening of lobe II results in lower affinity for nucleotides 249,250. Indeed, Fes1 binding to the Ssa1 NBD induced loss or tertiary structure of entire NBD with the exception of the binding site at subdomain IIB 246. In summary, Fes1 employs a unique mode of NEF activity on Hsp70.

Fes1 has been suggested to partner with both the Ssa and the Ssb class of cytosolic Hsp70s in yeast 247. Consistent with an interaction with the ribosome-associated Ssb class initial studies suggested that Fes1 functioned at the ribosome 247. However, immunoprecipitation experiments only detect Fes1 associated with the Ssa class and not with Ssb class of Hsp70s or ribosomes, suggesting Fes1 is not a main factor for translation-associated protein folding 229. Also, Fes1 is not required for Hsp70-dependent protein reactivation 251. A cell biological function for Fes1 has emerged from a recent study that revealed that Fes1 releases misfolded substrates from Hsp70 and targets them for ubiquitin-mediated degradation 252. Cells lacking Fes1 accumulated misfolded proteins associated with Hsp70 and failed to target misfolded proteins for ubiquitylation. Moreover, the cells were sensitive to conditions that accelerated protein misfolding and induced the HSR, presumably as a result of the accumulation of misfolded proteins 230,252. Study III and IV in this thesis elaborate on the biological function of Fes1 and its human homologue HspBP1 that may explain the role of this factor in the eukaryotic proteostasis system.

The Hsp110 family- Sse1 and Sse2

The major NEF of Hsp70 in the yeast cytosol belongs to the Hsp110 family, with isoforms Sse1 and Sse2. They are the homologs of human Hsp110 with 41% identity253. Sse1 and Sse2 are 76% identical and exhibit 70% similarity to the Hsp70 Ssa 254.

37

The Hsp110 family members are similar to Hsp70 in structure but harbour an extended SBD with an acidic loop between the β subdomain and the C- terminus 255. Hsp110 are the third most abundant heat shock protein in mammalian cells, with expression varying in different cell types. It was reported to be expressed the most in liver and brain and lowest in heart and skeletal muscles 256,257. Hsp110 accelerates nucleotide exchange in Hsp70 and this activity is conserved from yeast to humans 247,258,259.

The mode of NEF activity of Sse1 resembles Snl. In ADP bound form Sse1 is loosely folded and highly dynamic but upon ATP binding structural changes occurs in Sse1 leading to a highly compact structure that exhibits NEF activity 260. ATP bound Sse1 physically interacts with the NBD of Ssa1 resulting in release of bound ADP from Ssa1, thereby forming a transient Ssa1-Sse1 interaction 260,261. The dependence of nucleotide for NEF activity has been established for Sse1 and the structurally related NEF Lhs1 in the ER 262 (see Table 2). The lobe I of Hsp70 NBD interacts with lobe II of the Sse1 NBD resulting in a face-to-face orientation of the two molecules 179,246,253. In parallell, the α-helical SBD-like subdomain of Sse1 interacts with Hsp70 at the upper periphery of subdomain IIB. The interaction causes structural changes at the Hsp70 NBD causing structural incompatibility with nucleotide binding. The Hsp70-Sse1 interaction results in opening of the nucleotide-binding cleft by tilting lobe II. Interestingly, an analogous structural modulation of the Hsp70 NBD has been established for the Ssa1- Snl1 interaction, hence two structurally different NEFs employ similar mode of action for nucleotide exchange activity 246.

In yeast, Sse1 is the predominant isoform expressed during non-stressed growth. In contrast Sse2 is poorly expressed but is dramatically induced upon heat stress 254. SSE1 deletion reduces cell growth, whereas SSE2 deletion has no observable phenotype. The double deletion is lethal 254,263,264. Sse1 associates with Ssa1, Ssa2, Ssb1 and Ssb2 in an ATP dependent manner. On the contrary Sse2 associates with Ssa1 and not with Ssb1 261. The high abundance, evolutionary conservation, and existence of the isoforms suggest their importance in Hsp70 mediated PQC.

The biological significance of Hsp110 substrate binding is under debate. In vitro, Hsp110 has the ability to hold misfolded proteins in a folding competent state (holding chaperone). Specifically, Hsp110 protects luciferase from aggregating by directly binding to heat-denatured luciferase 38 265. It has been found that an equimolar concentration of Hsp110:Hsp70 effectively reactivates denatured firefly luciferase and an increase or decrease in Hsp110 had inhibitory effect 232. In metazoans, Hsp110 support the disaggregation and reactivation of proteins in an Hsp40-Hsp70 dependent manner. Depleting Hsp110 using RNAi in C. elegans leads to the accumulation of persistent insoluble protein aggregates after heat shock and reduced life span 266. In vitro reconsitution experiments have shown that Hsp40, Hsp70 and Hsp110 form a complex that is efficient in disaggregation 214,226,266-268.

The reactivation mediated by Hsp70-Hsp110 is also observed using reconstituted yeast proteins. A ratio of 5-10:1 of Sse1:luciferase has been reported to promote the reactivation of thermally denatured firefly luciferase 269 and a 2:1 ratio of Ssa1:Sse1 has been shown to effectively reactivate firefly luciferase 259. Sse1 enhances efficient firefly luciferase recovery as compared to Sse2 probably because of partial and reversible unfolding of Sse1, but not Sse2, at higher temperature 179,258. Thus Sse1 potentially have a more direct role in substrate binding and aggregate prevention even though it is unclear if the partial unfolding observed in vitro ever happens in the cell. Sse1 binds peptide substrates directly and exhibit a partially different recognition sequence compared to Hsp70 270. Sse1 prefers aromatic residues in contrast to the hydrophobic aliphatic residues preferred by Hsp70 271. The physical interaction of Sse1 and Ssa1 is important for the reactivation of misfolded proteins, however the role of the ATPase activity and putative substrate binding by Hsp110 is still under debate 179,259,268. Goloubinoff and colleagues have shown using human Hsp110 that apart from being a mere NEF of Hsp70 chaperone Hsp110 acts as an independent bona fide chaperone capable of reactivating misfolded proteins in an Hsp40 and ATP dependent manner 120,267. However, an in vivo evidence for the same is lacking.

Recent work by Morano and colleagues using a substrate-binding defective mutant of Sse1 found that substrate-binding activity of Sse1 is dispensable for its role in protein reactivation and Hsp70 mediated protein degradation 272,273. Yet the mutant still retained residual holding activity and exhibited phenotypes including an activated HSR and growth defects at elevated temperatures. Perhaps, in vivo the relevance of high abundance of Hsp110 can be attributed to be more than a NEF but also to switch Hsp70 to a potential disaggregase. This specialised dual role of Hsp110 may in turn reduce the burden of having high concentration of Hsp70 in the cell. 39 6 Protein disaggregation

Following protein aggregation cells employ several strategies to remove the aggregates either by disaggregation or by degradation. Protein aggregates are recognised by the chaperones that disentangles the aggregates leading to local unfolding followed by refolding to its native conformation through a series of step. Briefly, the aggregates are recognised by the Hsp40 that delivers it to Hsp70 that later recruit the major disaggregase Hsp104 to the aggregates followed by protein threading through the Hsp104 channel 274,275. The resulting polypeptides are subjected to PQC and are either refolded or targeted for degradation 274. The following section describes the disaggregation process in detail with special emphasis on Hsp104.

The Hsp104 disaggregase

Heat shock protein 104 (Hsp104), belongs to the family of AAA+ (ATPases Associated with diverse cellular Activities) protein superfamily and functions as an ATP-powered disaggregase 82,276. Bacteria harbour Hsp104 homologues including the E. coli ClpB that together with DnaK and the NEF GrpE performs ATP-dependent disaggregation. Hsp104/ClpB contains two AAA nucleotide binding domains (NBD) in head to tail orientation, an N-terminal domain and a middle domain forming a coiled structure (M- domain) 277 (Figure 6).

Figure 6: Domain representation of Hsp104 AAA+ ATPase. Adapted from 277

40 Structurally Hsp104 functions as a dynamic homo-hexamer with a narrow central pore 127,278-280. The NBD1 (AAA-1) offers a regulatory role by controlling the ATPase rate and substrate release rate and also confers substrate specificity 281. ATP binding to NBD1 inhibits ATP hydrolysis in other NBD1 domain and in contrast enhances ATP turnover in NBD2 (AAA-2). A conserved arginine residue (arginine finger) senses the nucleotide bound state of the adjacent protomer and mediates ATP binding and hydrolysis in an allosteric manner 282-285. NBD2 on the other hand is the main engine for the disaggregase that generates power for disaggregation and coordinates substrates binding 278,280,281

The working principle of Hsp104/ClpB has been elucidated using structural analysis. Hsp104/ClpB is divided into different functional segments as illustrated in Figure 6. The N-terminal domain (NTD) of ClpB interacts with the substrates via a binding groove in a nucleotide-independent manner 286. The NTD interacts both with the substrates and Hsp70 system 287. Truncation of N-terminal domain of Hsp104 (ΔN-Hsp104) has been shown to be defective in Hsp70 and Hsp40 interaction and dissolution of prions, and mutant cells exhibit reduced survival following heat stress 288. The N- terminal domain of ClpB prefers aliphatic residues in the substrate. The N- terminus also confers an inhibitory effect on ClpB by blocking the channel of the disaggregase and inhibits the engagement of unspecific substrates (partially folded or intrinsically disordered regions in a well folded protein). 283,289,290. A flexible N-domain appears to be the force-generating component for the disaggregase 277,291. Taken together NTD is crucial for cooperativity and plasticity of Hsp104/ClpB.

Central to Hsp104/ClpB regulation is the highly dynamic M-domain (MD) that dictates the activity of the disaggregase 292. It is the least conserved domain with 36% identity between Hsp104 and ClpB and are positioned adjacent to NBD1, facing the outside of the molecule 293-295. MD is important for NBD1-NBD2 communication and stabilization of the hexamer. MD interacts with the Hsp70-substrates and enables Hsp104/ClpB to dissolve aggregates 275,283,292-294,296-299. MD acts as a molecular toggle that negatively regulates Hsp104/ClpB. Thereby, MD exist in two states; (1) the repressed and (2) the activated state. In repressed state, MD associates tightly with NBD 1 in a horizontal confirmation and has low disaggregation rate, whereas in the activated state MD is tilted and results in low affinity with NBD 1 and also possess higher ATPase rate 277,300. In vivo repressed state is long-lived with no interaction with Hsp70 and activated state is short 41 lived 277. This regulation of Hsp104 via the MD ensures the efficient and controlled disaggregase activity.

The misfolded protein substrates are threaded through the central narrow pore of the hexameric Hsp104 in an ATP-driven process 301,302. A conserved tyrosine loop in the central pore channel acts as a clamp and prevents backsliding of the substrates 301,303-305. Hsp104 initiates disaggregation at the N, C-termini or at internal sites of its substrates and also support the simultaneous translocation of two polypeptides 306,307. The homo-hexamer structure of Hsp104 is highly dynamic and the monomers rearrange during disaggregation process thereby enhancing the efficiency and also preventing aggregated substrates blocking in the channel 295. Yokom and colleagues have reported on the existence of a left-handed spiral architecture of Hsp104 and have shown that substrate transfers between NBD 1 to NBD 2 of every protomer 280.

Till date three modes of action have been proposed for Hsp104 function: (1) probabilistic model, (2) subglobal connectivity and (3) global connectivity 277,295,308. In the probabilistic model, the subunits exist in a non-cooperative and independent state 309. Recent work has shown that Hsp104 uses probabilistic mode of action in translocating and disaggregating disordered aggregates 295. Subglobal connectivity model means that only some of the subunits collaborate as seen in case of less stable NM4 prions 295,310. Finally, in the global connectivity model all subunits cooperate and perform disaggregation in a collaborative manner. This mode of action is seen in case of highly stable prion structures for example NM25 prions 295,311. On the other hand ClpB is thought to perform highly cooperative ATPase activity to dissolve aggregates and thereby having increased efficiency in disaggregating substrates compared to Hsp104, however have limited activity on amyloids 277,295. The different mode of action enables Hsp104 to disaggregate a wide range of substrates depending on the circumstances. In summary, the highly dynamic working mode of Hsp104 offers a wide substrate range for the disaggregase and extends cellular fitness.

Bi-chaperone disaggregase machinery

Disaggregation is mediated by a bi-chaperone system comprised of Hsp104/ClpB and the Hsp40-Hsp70/DnaK-DnaJ-GrpE system 82,277,312,313.

42 Disaggregation is a highly controlled processed with orchestrated sequential steps involved starting with (1) binding of aggregated substrate proteins by Hsp40/DnaJ, (2) followed by recruitment of Hsp70/DnaK to the aggregates and (3), finally, delivery of the substrates to the AAA+ disaggregase Hsp104/ClpB 314,315. In vitro, weak physical interaction between Hsp40 and Hsp104 has been reported, and disaggregation rate is affected in the absence of either of them. Nevertheless, a combination of Hsp40 and Hsp104 does not enhance the refolding rate. However, addition of Hsp70 Ssa1 to Hsp40 and Hsp104 reactivates aggregated proteins efficiently 82. Genetic mutations and protein chimeras have revealed that helices 2 and 3 (motif 2) of the M- domain of Hsp104 interact with Hsp70, thereby enhancing the ATPase activity of Hsp104 275,316. Moreover, the inhibitory effect of ADP on Hsp104 is suppressed by the action Hsp70 317. In conclusion, substrates delivered by the Hsp70 system are efficiently disaggregated by the Hsp104/ClpB disaggregase.

Hsp70 NEFs play a role in the bichaperone disaggregation but likely not in a complex with Hsp70-Hsp104. Characterization of interaction sites of ClpB and GrpE on DnaK revealed that they share the same site suggesting NEFs are not directly involved in disaggregation process 277,318. Using in vitro reconstitution, it has shown that Hsp110 enhances the protein disaggregation by Hsp104, Hsp70 and Hsp40 266,268. Moreover, the protein disaggregation activity was observed only in Hsp110 (Sse1 and Sse2) but not with Fes1 and Snl1. In yeast cells, Sse1 has been reported to enhance protein reactivation following heat shock by an uncharacterized pathway since replacing the essential Sse1 and Sse2 with overexpressed Fes1 resulted in impaired protein reactivation following heat shock 259. In contrast, in recent work, Morano and colleagues systematically analysed NEF knocks and found that Sse1, Sse2, Fes1 and Snl1 all are dispensable for Hsp104-mediated protein reactivation 251. This ambiguity in the field is taken into consideration in Study I of the thesis were we in depth analysed the role of Sse1 and Sse2 in protein disaggregation. Mechanistically, Hsp70 and Hsp110 may also have important roles in folding of the extracted and translocated Hsp104 substrate. Yet formal evidence for such a function has not been published (Figure 7).

43

Figure 7: Mechanism of the Hsp104 disaggregase machinery working in concert with Hsp40 and Hsp70. Adapted from 274

44 Aims

The goal of this thesis is to provide understanding of the basic cellular function of Hsp70 NEFs of the Hsp110 and armadillo types in proteostasis and protein quality control. I have addressed the following aims:

Aim I: Elucidate the role of Hsp110 (Sse1/2) in Hsp104-dependent protein disaggregation. (Study I)

Aim II: Provide insight into the tuning of the proteostasis system by Hsp110 during stress and aging. (Study II)

Aim III: Characterize the splice isoforms of Fes1 to understand their function in protein quality control. (Study III)

Aim IV: Unravel the molecular mechanism that explains the cellular function of armadillo type NEFs using Fes1 as a model. (Study IV)

In parallel, I was also involved in developing methodology to study gene expression by bioluminescence in yeast. (Study V)

45 Summary of individual studies

Study I

Aim: Elucidate the role of Hsp110 (Sse1/2) in Hsp104-dependent protein disaggregation.

The study reveals the importance of the Hsp110 (Sse1/2) in Hsp104- dependent protein disaggregation and reactivation. Previously it was reported that Hsp70 NEFs are dispensable for reactivation of thermally denatured proteins in cells 251, yet Hsp110 has an accelerating effects on in vitro disaggregation reaction based on purified Hsp40, Hsp70 and Hsp104 214,226,266-268. Here, we have used the novel temperature-sensitive allele sse1- 200 that in the context of sse2Δ enables regulation of total cellular Hsp110 levels by temperature. Our results show that Hsp110 is an essential factor for disaggregation of protein aggregates induced by a transient heat-shock. Figure 8 represents a model for the study.

Results

Sse1 is important for reactivation of aggregated proteins in vitro

To assess the role of Hsp110 in disaggregation in vitro in the context of the entire chaperome, yeast cytosolic lysates were prepared from cells depleted of Sse1 and Sse2. The lysates were used to analyze the role of Sse1 in protein reactivation and were found to be competent of ATP- and Hsp104- dependent reactivation of aggregated firefly luciferase prepared from urea- denatured protein. Titration of recombinant Sse1 purified from E. coli to the depleted lysates accelerated the reactivation significantly until a point when the added Sse1 inhibited the reaction. Thus, consistent with previously published in vitro data Hsp110 is not strictly required for Hsp104-mediated disaggregation but greatly potentiates the system. High levels of Sse1 inhibit the disaggregation reaction, likely by forming unproductive complexes with apo-Hsp70.

46

In parallel and unpublished experiments we also attempted to reactivate firefly luciferase following heat-denaturation. Independently firefly luciferase was heat-denatured in buffer and in the cytosolic lysate. Under these conditions reactivation of the protein was generally poor and inconsistent. Thus the in vitro experiments were limited to investigating protein reactivation of species that are robustly reactivated, e.g. urea- denatured firefly luciferase.

Sse1 and Sse2 are essential for Hsp104-mediated protein disaggregation in cells

We performed analysis of the importance of the Sse1 and Sse2 for Hsp104- dependent protein disaggregation using sse1-200 sse2Δ cells subjected to transient heat shock. Analysis of fusions of GFP and firefly luciferase by activity measurements and microscopic evaluation of aggregation status revealed a strict requirement for Hsp110 in protein disaggregation. The function of Sse1 in Hsp104-mediated disaggregation depends on its interaction with Hsp70 as a NEF and it has to reside in the same compartment as the aggregated protein. Sse1 is important for the efficient recruitment of Hsp70 and Hsp104 to the protein aggregates. Taken the study together, we found that Sse1 and Sse2 are essential factors for Hsp70- Hsp104 mediated protein disaggregation and reactivation.

Figure 8: Model showing the role of Hsp110 in protein disaggregation. 47 Study II

Aim: Provide insight into the tuning of the proteostasis system by Hsp110 during stress and aging.

The regulation of the proteostasis system is central the physiology of the cell during stress and aging. In our on-going studies of Hsp110, we found that the nucleocytoplasmic partitioning of Sse1 fine-tunes the proteostasis system to enable improved growth under stressful conditions and longevity of cells that undergo chronological aging. The data suggest that the proteostasis system is modified by nuclear Sse1 to mobilize a latent pool of Hsp70 that is trapped by nuclear substrates. Our current understanding of the proteostasis regulation by the nuclear Sse1 is shown in Figure 9.

Results

Nuclear targeting of Sse1 by translational frameshift enhances proteostasis

We sought to learn more about the role of Hsp110 in the proteostasis system by mutagenesis of SSE1. Briefly, we employed a mutant strain that lacked the Hsp70 NEF Fes1 and the disaggregase Hsp104. The fes1Δ leads to impairment in protein degradation leading to accumulation of misfolded proteins and hsp104Δ abolishes efficient protein disaggregation. Together these mutations result in the massive accumulation of misfolded and aggregated proteins manifested as a non-growth phenotype at elevated temperatures. We isolated suppressing dominant SSE1 mutations, and found that cells counteracted the build-up of toxic misfolded proteins by nuclear targeting of Sse1 via frame-shifting of a cryptic NLS in the near-3' +1 reading frame. Analysis revealed that resulting nuclear Sse1 fortifies the proteostasis system as monitored by accelerated refolding of firefly luciferase in an Hsp104-independent manner. Interestingly, we found that translation of SSE1 in wild-type cells also involves near-3' translational frameshifting suggesting regulation of the nucleocytoplasmic pool of Hsp110. Nuclear Sse1 has the potential to impact on the proteostasis system by unleashing a latent pool of nuclear Hsp70 trapped with substrate and to relieve nuclear Hsf1 from Hsp70-dependent repression.

48 Nuclear Sse1 in association with Ssa1 improves longevity

Since the proteostasis system deteriorates during aging of cells, we analysed the impact of nuclear Sse1 on chronological life span (CLS). Interestingly, we found that nuclear targeting of Sse1 resulted in increased CLS. The induced longevity was dependent on Sse1 association with Ssa1. We envision that nuclear Sse1 increases the cellular pool of Hsp70-ATP by releasing nuclear Hsp70 from misfolded substrates. Importantly, we found the improved survival comes at the expense of hypersensitivity to misfolded Hsp70-substrates that are targeted to the nucleus. Taken together, nuclear Sse1 tunes the proteostasis system to tolerate stress and longevity but at the expense of sensitivity to misfolded nuclear proteins.

Figure 9: Model showing regulation of Hsp70 by nuclear Sse1 (Hsp110).

49 Study III

Aim: Characterize the splice isoforms of Fes1 to understand their function in protein quality control.

Misfolded proteins generated in the cytosol are targeted to the nucleus were they are subjected to degradation by the UPS. Fes1 has previously been shown to be important for such protein quality control but with nucleocytoplasmic spatial information lacking 252. Here, we found out that Fes1 undergoes alternative splicing to generate two functional isoforms with distinct subcellular localizations; Fes1S localizes to the cytosol and Fes1L localizes to the nucleus. Fes1L represents the first reported exclusively nuclear NEF. Fes1S was found to be the important species in protein quality control and enables the degradation of misfolded proteins that are associated with Hsp70. Figure 10 summarizes the major findings of the study.

Results

Alternative splicing of Fes1 and its compartmentalization

We identified two FES1 transcripts that arise as a result of competition between excision of an intron by splicing and alternative polyadenylation sites. Both transcripts express a functional isoform of Fes1. The longer isoform, Fes1L, carries a C-terminal NLS sequence while the shorter isoform lacks targeting signals. Fluroscent microscopy analysis revealed that Fes1L is targeted to the nucleus by the NLS while Fes1S localizes predominantly to the cytosol. The expression of Fes1S and not Fes1L is strongly induced by transient heat shock, suggesting a role of Fes1S in maintaining proteostasis during stressful conditions.

Functional analysis of the isoforms

Upon direct testing of the function of the two isoforms it was evident that Fes1S performs most of the previously characterized functions as removal of Fes1S resulted in known fes1Δ phenotypes including growth defects at elevated temperatures, upregulation of the HSR and impaired degradation of misfolded proteins. Neither Fes1S nor Fes1L is important for disaggregation and reactivation of heat denatured misfolded proteins. In conclusion, the data points to a functional role of Fes1S in maintaining proteostasis by

50 enabling Hsp70-mediated degradation of misfolded proteins. Fes1L constitutes the first described Hsp70 NEF targeted to the nucleus in yeast indicating that this isoform regulates Hsp70-substrate interactions in this compartment.

Figure 10: Model depicting the localization and role of Fes1 in Hsp70 mediated protein degradation.

51 Study IV

Aim: Unravel the molecular mechanism that explains the cellular function of armadillo type NEFs using Fes1 as a model

The study provides a mechanistic explanation of the specialized function of armadillo types NEFs and explains how Fes1 support proteostasis. Accordingly, in detailed analysis we found that Fes1 carries a flexible N- terminal Release Domain (RD) that regulates Hsp70 function by competing with the client substrates for chaperone binding. Consequently, the RD ensures that the substrate released from Hsp70 by Fes1 NEF binding and nucleotide exchange does not rebind. The mechanism ensures the efficient release of even persistent Hsp70 clients with multiple high-affinity Hsp70- binding sites such as misfolded proteins. The summary of the study is depicted as a model in Figure 11.

Results

Fes1 RD function is indispensable for proper functioning of Hsp70 system

We found that unlike other cytosolic NEFs, Fes1 carries an N-terminal extension that is crucial for its cellular function as a RD. Truncating RD resulted in a fes1 null phenotype with growth defects at stress conditions, impaired degradation of misfolded proteins and a constitutively induced HSR. Importantly, when the RD is transferred to the BAG domain of Snl1 the chimeric NEF gains the ability to complement fes1Δ phenotypes. Moreover, the function of the RD is conserved in the human Fes1 homologue HspBP1 since the N-terminal domain of HspBP1 can be successfully transplanted to replace the RD of Fes1. Moreover HspBP1 lacking its N-terminal domain is not functional in a p65 translocation assay for NFκB signalling. These observations highlight the importance of the RD for Fes1 function and in extension provide an explanation for the mechanistic role of armadillo type NEFs.

The RD contacts the SBDβ and prevents substrate binding

To gain in-depth understanding of the RD function we used a crosslinking strategy and found that the RD contacts SBDβ of Hsp70 Ssa1. To directly 52 test if the RD prevents substrate binding we took advantage of the fluorescently labelled Hsp70 substrate peptide dNR. Indeed Fes1 prevented binding of the substrate to Hsp70 dependent on the RD. The inhibitory effect of substrate binding was found to be ATP dependent, suggesting that the core NEF activity of the armadillo domain and RD functions in concert. In conclusion, we revealed the underlying mode of action of Fes1 in regulating Hsp70 mediated proteostasis.

Figure 11: Model showing the mechanistic action of Fes1 on Hsp70.

53 Study V

Bioluminescent reporter proteins have gained popularity due to their high sensitivity, large dynamic range during detection and ease of usability. Here, we adopted the recently developed luciferase Nanoluciferase (Nluc) for the expression in yeast from a codon-optimized gene (yNluc). The novel yNluc functions as a highly sensitive reporter for gene expression and protein stability in yeast.

Results

We have generated the codon-optimized yNluc and a destabilized version carrying a PEST sequence (yNlucPEST). Both reporter proteins are capable of faithfully reporting even the transient changes in gene expression during heat stress as shown by the induction of bioluminescence. As compared to the traditional LacZ, yNlucPEST senses changes in real-time in a fast and reliable manner. We have also employed Nluc as a tag to follow protein levels in the cell. Taken together this method is advantageous over traditional protein reporters in the following ways:

i) Ease of use ii) Reliable and fast read out iii) Higher sensitivity iv) Lower half-life, therefore can be used for measuring promoter activity.

54 Conclusion and Outlook

Since the discovery of molecular chaperones we have gained insight into many molecular mechanisms at play and their biological relevance. The studies included in this thesis highlight the importance of NEFs in regulating the Hsp70 family of molecular chaperones. The work sheds light on how cells employ several sets of structurally distinct factors in promoting PQC and enhancing cellular fitness via the proteostasis system. Mainly, the results here elucidate the function of two families of Hsp70 NEFs, the Hsp110 family (Sse1/Sse2) and the armadillo family (Fes1). Representatives of these two families are present in the cytosol, nucleus and ER of all eukaryotic cells suggesting that their combined action is key for proper function of Hsp70 in the proteostasis system. Even though they share activities as NEFs of Hsp70, they display fundamental structural differences and are involved in different proteostasis and PQC pathways. Thus the findings of this thesis regarding function of Sse1/Sse2 and Fes1 in the yeast cytosol and nucleus have the potential to more broadly explain the regulation of cytosolic and ER Hsp70 in eukaryotic cells.

PQC can be achieved via several routes where the Hsp110 family NEFs Sse1/Sse2 promote the reactivation of aggregated proteins together with Hsp40, Hsp70 and Hsp104. Different functions have been suggested to this protein family starting from acting as a bona fide chaperone to supplying NEF activity to the Hsp70 system. Even though both are plausible and in vitro evidence supports both functions the understanding of a potential chaperone function in the cell is yet not conclusive. Perhaps Hsp110 that is expressed at high levels in the cell functions in holding protein clients, presumably in a manner regulated via its interactions with Hsp70, but further studies are needed. Another curious aspect of Sse1 is the high affinity exhibited by the NEF to apo-Hsp70. The affinity stems from a large interaction interface where Sse1 embraces the NBD of Hsp70. This feature of the interaction suggests a functional adaptation of the NEF activity. Perhaps entropic pulling by Sse1 either directly on the substrate or via 55 Hsp70 that interacts with the substrate contributes to local unfolding or disentanglement of substrates. This fascinating scenario of dual action of Sse1 NEF activity, together with its potential substrate holding properties remain to be experimentally addressed in the future. More detailed understanding is needed to explain the underlying mechanistic differences in the function of the different NEFs.

The armadillo family NEF Fes1 acts on Hsp70 by exchanging ADP for ATP and simultaneously competes substrates off Hsp70 to ensure their efficient release. This mechanistic function is likely to be conserved in the ER- resident homologue Sil1. Specifically, like Fes1 Sil1 harbours a N-terminal extension that may function analogously as the Fes1 RD. Since inactivating Sil1 mutations cause Marinesco-Sjögren syndrome the mechanistic details elucidated in this thesis have implications in understanding proteostasis disorders. In analogy to the phenotypes exhibited by fes1Δ cells, the Marinesco-Sjögren syndrome may be a downstream manifestation of failure to maintain the ER Hsp70 system clear of persistent misfolded proteins. In conclusion, this thesis provides an in-depth understanding of Hsp70 regulation and proteostasis maintenance.

56 Acknowledgement

Successful research will not be possible without efficient communication and friendly colleagues. There are many people who have contributed to the work in this thesis by guiding me in the right track, giving advices, experimental guidance and moral support. I would like to thank each and every one from the deep of my heart.

First and foremost, I am very grateful to my supervisor Dr. Claes Andréasson for accepting me to his highly talented group and giving me a chance to work with them. The thesis would not have been made possible without his immense support and guidance. I am very fortunate to have you as my supervisor who encouraged me to take up challenges and also for your continuous support, motivation and patience from the day one until now. I learnt a lot from you regarding how to frame a question and how to work towards it, both theoretically and practically. I could not have imagined having a better supervisor for my PhD study.

I would also like to thank all past and present members of Claes Andréasson’s group Naveen Kumar Chandappa Gowda, Anna E. Masser, Ganapathi Kandasamy and Mats Holmberg for all the valuable discussion and the good times. I would like to thank Prof. Per Ljungdahl and group for the discussions and advice. I would also like to thank Kicki Ryman for all the advices, support and fun times. This study would not have been possible without the IFSU facility and I would like to thank Stina Höglund for the time, advices and suggestions in taking me through the microscopy world. I would also like to mention the positive vibe and research environment brought in by Dr. Sabrina Büttner and the group. I also enjoyed all the fruitful discussions and work with Lukas Habernig.

I would also like to thank our collaborators Prof. Marie Öhman and group for all the nice suggestions and comments. I like to thank Chammiran Daniel for her help with all the mammalian experiments and support. I would like to thank Prof. Martin Ött and group for all the valuable comments and suggestions. 57

Finally I would also like to thank Prof. Roger Karlsson, Prof. Eva Severinson, Prof. Ann-Kristin Östlund Farrants and Dr. Ann-Christin Lindås group for making the research environment great.

Again a special thanks to my previous and current lab mates Naveen, Mats, Anna and Ganapathi for all the good times we had together in the lab. Thank you Naveen for all the scientific discussions and I really enjoyed teaming up with you in all the fantastic projects. Thank you Anna for being patient enough to read through my manuscripts and thesis and Ganapathi for having critical discussions through out the studies. It was fun playing badminton with Naveen, Toni, Judit, Antonio, Yuan, Kicki, Anna, Mats, Ming, Ganapathi and making the research environment nice and friendly.

Finally, I would like to thank my family and friends for giving me the support and encouragement in all the decisions that I made in my life and for standing beside me. I would not have made it through with out the support of my family- late G.Mohanakrishnan, Jayasree Kaimal and Jayakrishnan M. Kaimal. Even if you are not with us I can feel you around and your blessings will always be with us. My wife, Jisha Raj has been the pillar during all the tough times that passed by during these years and I would like to thank her for all the support she have given me from the day she came to my life.

Last but not the least, I would also like to thank my friends Arun, Darsana, Praveen, Jiji, Siva, Sai, Visakh, Arya, Mansu and Srejith for all the fun, support and good times we had together.

58 References

1. Andreeva, A., Howorth, D., Chothia, C., Kulesha, E. & Murzin, A.G. SCOP2 prototype: a new approach to protein structure mining. Nucleic Acids Res 42, D310-4 (2014). 2. Anfinsen, C.B. Principles that govern the folding of protein chains. Science 181, 223-30 (1973). 3. Hartl, F.U. & Hayer-Hartl, M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852-8 (2002). 4. Dobson, C.M. Protein folding and misfolding. Nature 426, 884-90 (2003). 5. Itzhaki, L.S., Otzen, D.E. & Fersht, A.R. The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation- condensation mechanism for protein folding. J Mol Biol 254, 260-88 (1995). 6. Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J Mol Biol 277, 985-94 (1998). 7. Dinner, A.R., Sali, A., Smith, L.J., Dobson, C.M. & Karplus, M. Understanding protein folding via free-energy surfaces from theory and experiment. Trends Biochem Sci 25, 331-9 (2000). 8. Dobson, C.M. & Karplus, M. The fundamentals of protein folding: bringing together theory and experiment. Curr Opin Struct Biol 9, 92-101 (1999). 9. Hartl, F.U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324-32 (2011). 10. Frydman, J. Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem 70, 603-47 (2001). 11. Kim, Y.E., Hipp, M.S., Bracher, A., Hayer-Hartl, M. & Hartl, F.U. Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82, 323-55 (2013). 12. Schwartz, R., Istrail, S. & King, J. Frequencies of amino acid strings in globular protein sequences indicate suppression of blocks of consecutive hydrophobic residues. Protein Sci 10, 1023-31 (2001). 13. Ciryam, P., Tartaglia, G.G., Morimoto, R.I., Dobson, C.M. & Vendruscolo, M. Widespread aggregation and neurodegenerative diseases are associated with supersaturated proteins. Cell Rep 5, 781-90 (2013).

59 14. Gupta, R. et al. Firefly luciferase mutants as sensors of proteome stress. Nat Methods 8, 879-84 (2011). 15. Matsui, I. & Harata, K. Implication for buried polar contacts and ion pairs in hyperthermostable enzymes. FEBS J 274, 4012-22 (2007). 16. Fink, A.L. Protein aggregation: folding aggregates, inclusion bodies and amyloid. Fold Des 3, R9-23 (1998). 17. Chiti, F. & Dobson, C.M. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75, 333-66 (2006). 18. Stefani, M. Protein folding and misfolding on surfaces. Int J Mol Sci 9, 2515-42 (2008). 19. Bousset, L. et al. Structural characterization of the fibrillar form of the yeast Saccharomyces cerevisiae prion Ure2p. Biochemistry 43, 5022-32 (2004). 20. Munder, M.C. et al. A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy. Elife 5(2016). 21. Laporte, D., Salin, B., Daignan-Fornier, B. & Sagot, I. Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J Cell Biol 181, 737-45 (2008). 22. Narayanaswamy, R. et al. Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation. Proc Natl Acad Sci U S A 106, 10147-52 (2009). 23. Noree, C., Sato, B.K., Broyer, R.M. & Wilhelm, J.E. Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster. J Cell Biol 190, 541-51 (2010). 24. O'Connell, J.D., Zhao, A., Ellington, A.D. & Marcotte, E.M. Dynamic reorganization of metabolic enzymes into intracellular bodies. Annu Rev Cell Dev Biol 28, 89-111 (2012). 25. Petrovska, I. et al. Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation. Elife (2014). 26. Vermulst, M. et al. Transcription errors induce proteotoxic stress and shorten cellular lifespan. Nat Commun 6, 8065 (2015). 27. Kramer, E.B., Vallabhaneni, H., Mayer, L.M. & Farabaugh, P.J. A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae. RNA 16, 1797-808 (2010). 28. Drummond, D.A. & Wilke, C.O. The evolutionary consequences of erroneous protein synthesis. Nat Rev Genet 10, 715-24 (2009). 29. Ogle, J.M. & Ramakrishnan, V. Structural insights into translational fidelity. Annu Rev Biochem 74, 129-77 (2005). 30. Ibstedt, S., Sideri, T.C., Grant, C.M. & Tamas, M.J. Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress. Biol Open 3, 913-23 (2014). 31. Willmund, F. et al. The cotranslational function of ribosome- associated Hsp70 in eukaryotic protein homeostasis. Cell 152, 196- 209 (2013). 60 32. Ban, N., Nissen, P., Hansen, J., Moore, P.B. & Steitz, T.A. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289, 905-20 (2000). 33. Lu, J. & Deutsch, C. Folding zones inside the ribosomal exit tunnel. Nat Struct Mol Biol 12, 1123-9 (2005). 34. Woolhead, C.A., McCormick, P.J. & Johnson, A.E. Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell 116, 725-36 (2004). 35. Vabulas, R.M., Raychaudhuri, S., Hayer-Hartl, M. & Hartl, F.U. Protein folding in the cytoplasm and the heat shock response. Cold Spring Harb Perspect Biol 2, a004390 (2010). 36. Doring, K. et al. Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding. Cell 170, 298-311 e20 (2017). 37. Elcock, A.H. Molecular simulations of cotranslational protein folding: fragment stabilities, folding cooperativity, and trapping in the ribosome. PLoS Comput Biol 2, e98 (2006). 38. Stefani, M. Protein misfolding and aggregation: new examples in medicine and biology of the dark side of the protein world. Biochim Biophys Acta 1739, 5-25 (2004). 39. Stefani, M. & Dobson, C.M. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med (Berl) 81, 678-99 (2003). 40. Gsponer, J. & Babu, M.M. Cellular strategies for regulating functional and nonfunctional protein aggregation. Cell Rep 2, 1425- 37 (2012). 41. Maji, S.K. et al. Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science 325, 328-32 (2009). 42. Saad, S. et al. Reversible protein aggregation is a protective mechanism to ensure cell cycle restart after stress. Nat Cell Biol 19, 1202-1213 (2017). 43. Vidal, O. et al. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol 180, 2442-9 (1998). 44. Fowler, D.M., Koulov, A.V., Balch, W.E. & Kelly, J.W. Functional amyloid--from bacteria to humans. Trends Biochem Sci 32, 217-24 (2007). 45. Shorter, J. & Lindquist, S. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet 6, 435-50 (2005). 46. Wiltzius, J.J. et al. Molecular mechanisms for protein-encoded inheritance. Nat Struct Mol Biol 16, 973-8 (2009). 47. Heck, J.W., Cheung, S.K. & Hampton, R.Y. Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 61 ubiquitin ligases Ubr1 and San1. Proc Natl Acad Sci U S A 107, 1106-11 (2010). 48. Oling, D., Eisele, F., Kvint, K. & Nystrom, T. Opposing roles of Ubp3-dependent deubiquitination regulate replicative life span and heat resistance. EMBO J 33, 747-61 (2014). 49. Lim, K.L. et al. Parkin mediates nonclassical, proteasomal- independent ubiquitination of synphilin-1: implications for Lewy body formation. J Neurosci 25, 2002-9 (2005). 50. Devi, L., Raghavendran, V., Prabhu, B.M., Avadhani, N.G. & Anandatheerthavarada, H.K. Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem 283, 9089-100 (2008). 51. Di Maio, R. et al. alpha-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson's disease. Sci Transl Med 8, 342ra78 (2016). 52. Escusa-Toret, S., Vonk, W.I. & Frydman, J. Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nat Cell Biol 15, 1231-43 (2013). 53. Hill, S.M., Hanzen, S. & Nystrom, T. Restricted access: spatial sequestration of damaged proteins during stress and aging. EMBO Rep 18, 377-391 (2017). 54. Bucciantini, M. et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507-11 (2002). 55. Duennwald, M.L. Polyglutamine misfolding in yeast: toxic and protective aggregation. Prion 5, 285-90 (2011). 56. Verma, M., Vats, A. & Taneja, V. Toxic species in amyloid disorders: Oligomers or mature fibrils. Ann Indian Acad Neurol 18, 138-45 (2015). 57. Ko, H.S., Kim, S.W., Sriram, S.R., Dawson, V.L. & Dawson, T.M. Identification of far upstream element-binding protein-1 as an authentic Parkin substrate. J Biol Chem 281, 16193-6 (2006). 58. Ko, H.S. et al. Accumulation of the authentic parkin substrate aminoacyl-tRNA synthetase cofactor, p38/JTV-1, leads to catecholaminergic cell death. J Neurosci 25, 7968-78 (2005). 59. Cohen, F.E. & Kelly, J.W. Therapeutic approaches to protein- misfolding diseases. Nature 426, 905-9 (2003). 60. Qu, B.H., Strickland, E.H. & Thomas, P.J. Localization and suppression of a kinetic defect in cystic fibrosis transmembrane conductance regulator folding. J Biol Chem 272, 15739-44 (1997). 61. Cohen, E., Bieschke, J., Perciavalle, R.M., Kelly, J.W. & Dillin, A. Opposing activities protect against age-onset proteotoxicity. Science 313, 1604-10 (2006). 62. Morley, J.F., Brignull, H.R., Weyers, J.J. & Morimoto, R.I. The threshold for polyglutamine-expansion protein aggregation and 62 cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc Natl Acad Sci U S A 99, 10417-22 (2002). 63. Rakwalska, M. & Rospert, S. The ribosome-bound chaperones RAC and Ssb1/2p are required for accurate translation in Saccharomyces cerevisiae. Mol Cell Biol 24, 9186-97 (2004). 64. Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916-9 (2008). 65. Gidalevitz, T., Ben-Zvi, A., Ho, K.H., Brignull, H.R. & Morimoto, R.I. Progressive disruption of cellular protein folding in models of polyglutamine diseases. Science 311, 1471-4 (2006). 66. Bayir, H. & Kagan, V.E. Bench-to-bedside review: Mitochondrial injury, oxidative stress and apoptosis--there is nothing more practical than a good theory. Crit Care 12, 206 (2008). 67. Kwong, J.Q., Beal, M.F. & Manfredi, G. The role of mitochondria in inherited neurodegenerative diseases. J Neurochem 97, 1659-75 (2006). 68. Bence, N.F., Sampat, R.M. & Kopito, R.R. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292, 1552-5 (2001). 69. Labbadia, J. & Morimoto, R.I. The biology of proteostasis in aging and disease. Annu Rev Biochem 84, 435-64 (2015). 70. Powers, E.T., Morimoto, R.I., Dillin, A., Kelly, J.W. & Balch, W.E. Biological and chemical approaches to diseases of proteostasis deficiency. Annu Rev Biochem 78, 959-91 (2009). 71. Ben-Zvi, A., Miller, E.A. & Morimoto, R.I. Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proc Natl Acad Sci U S A 106, 14914-9 (2009). 72. David, D.C. et al. Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS Biol 8, e1000450 (2010). 73. Reis-Rodrigues, P. et al. Proteomic analysis of age-dependent changes in protein solubility identifies genes that modulate lifespan. Aging Cell 11, 120-7 (2012). 74. Chen, B., Retzlaff, M., Roos, T. & Frydman, J. Cellular strategies of protein quality control. Cold Spring Harb Perspect Biol 3, a004374 (2011). 75. McClellan, A.J., Tam, S., Kaganovich, D. & Frydman, J. Protein quality control: chaperones culling corrupt conformations. Nat Cell Biol 7, 736-41 (2005). 76. Bengtson, M.H. & Joazeiro, C.A. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467, 470-3 (2010). 77. Brandman, O. et al. A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell 151, 1042-54 (2012).

63 78. Dimitrova, L.N., Kuroha, K., Tatematsu, T. & Inada, T. Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome. J Biol Chem 284, 10343-52 (2009). 79. Wickner, S., Maurizi, M.R. & Gottesman, S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, 1888-93 (1999). 80. Buchberger, A., Bukau, B. & Sommer, T. Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms. Mol Cell 40, 238-52 (2010). 81. Diamant, S., Ben-Zvi, A.P., Bukau, B. & Goloubinoff, P. Size- dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery. J Biol Chem 275, 21107-13 (2000). 82. Glover, J.R. & Lindquist, S. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94, 73-82 (1998). 83. Goloubinoff, P. & De Los Rios, P. The mechanism of Hsp70 chaperones: (entropic) pulling the models together. Trends Biochem Sci 32, 372-80 (2007). 84. Gardner, R.G., Nelson, Z.W. & Gottschling, D.E. Degradation- mediated protein quality control in the nucleus. Cell 120, 803-15 (2005). 85. Rosenbaum, J.C. et al. Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates. Mol Cell 41, 93-106 (2011). 86. Mizushima, N. & Klionsky, D.J. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 27, 19-40 (2007). 87. Reggiori, F. & Klionsky, D.J. Autophagic processes in yeast: mechanism, machinery and regulation. Genetics 194, 341-61 (2013). 88. Nielsen, S.V., Poulsen, E.G., Rebula, C.A. & Hartmann-Petersen, R. Protein quality control in the nucleus. Biomolecules 4, 646-61 (2014). 89. Park, S.H. et al. PolyQ proteins interfere with nuclear degradation of cytosolic proteins by sequestering the Sis1p chaperone. Cell 154, 134-45 (2013). 90. Wilkinson, C.R. et al. Localization of the 26S proteasome during mitosis and meiosis in fission yeast. EMBO J 17, 6465-76 (1998). 91. Kaganovich, D., Kopito, R. & Frydman, J. Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088-95 (2008). 92. Miller, S.B. et al. Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition. EMBO J 34, 778-97 (2015). 93. Specht, S., Miller, S.B., Mogk, A. & Bukau, B. Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae. J Cell Biol 195, 617-29 (2011).

64 94. Zhou, C. et al. Organelle-based aggregation and retention of damaged proteins in asymmetrically dividing cells. Cell 159, 530-42 (2014). 95. Spokoini, R. et al. Confinement to organelle-associated inclusion structures mediates asymmetric inheritance of aggregated protein in budding yeast. Cell Rep 2, 738-47 (2012). 96. Malinovska, L., Kroschwald, S., Munder, M.C., Richter, D. & Alberti, S. Molecular chaperones and stress-inducible protein- sorting factors coordinate the spatiotemporal distribution of protein aggregates. Mol Biol Cell 23, 3041-56 (2012). 97. Saarikangas, J. & Barral, Y. Protein aggregates are associated with replicative aging without compromising protein quality control. Elife 4(2015). 98. Wolf, D.H. & Hilt, W. The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 1695, 19-31 (2004). 99. Ciechanover, A. The unravelling of the ubiquitin system. Nat Rev Mol Cell Biol 16, 322-4 (2015). 100. Ciechanover, A. & Kwon, Y.T. Protein Quality Control by Molecular Chaperones in Neurodegeneration. Front Neurosci 11, 185 (2017). 101. Wang, J. & Maldonado, M.A. The ubiquitin-proteasome system and its role in inflammatory and autoimmune diseases. Cell Mol Immunol 3, 255-61 (2006). 102. Pickart, C.M. Mechanisms underlying ubiquitination. Annu Rev Biochem 70, 503-33 (2001). 103. Kwon, Y.T. & Ciechanover, A. The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy. Trends Biochem Sci (2017). 104. Braten, O. et al. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination. Proc Natl Acad Sci U S A 113, E4639-47 (2016). 105. Thrower, J.S., Hoffman, L., Rechsteiner, M. & Pickart, C.M. Recognition of the polyubiquitin proteolytic signal. EMBO J 19, 94- 102 (2000). 106. Finley, D., Chen, X. & Walters, K.J. Gates, Channels, and Switches: Elements of the Proteasome Machine. Trends Biochem Sci 41, 77-93 (2016). 107. Tamura, N., Lottspeich, F., Baumeister, W. & Tamura, T. The role of tricorn protease and its aminopeptidase-interacting factors in cellular protein degradation. Cell 95, 637-48 (1998). 108. Cha-Molstad, H. et al. Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat Cell Biol 17, 917-29 (2015). 109. Kraft, C., Reggiori, F. & Peter, M. Selective types of autophagy in yeast. Biochim Biophys Acta 1793, 1404-12 (2009). 65 110. Lamark, T., Kirkin, V., Dikic, I. & Johansen, T. NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle 8, 1986-90 (2009). 111. Stolz, A., Ernst, A. & Dikic, I. Cargo recognition and trafficking in selective autophagy. Nat Cell Biol 16, 495-501 (2014). 112. Fuertes, G., Martin De Llano, J.J., Villarroya, A., Rivett, A.J. & Knecht, E. Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions. Biochem J 375, 75-86 (2003). 113. Kaushik, S. & Cuervo, A.M. Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol 22, 407- 17 (2012). 114. Massey, A.C., Kaushik, S., Sovak, G., Kiffin, R. & Cuervo, A.M. Consequences of the selective blockage of chaperone-mediated autophagy. Proc Natl Acad Sci U S A 103, 5805-10 (2006). 115. Laskey, R.A., Honda, B.M., Mills, A.D. & Finch, J.T. Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature 275, 416-20 (1978). 116. Hemmingsen, S.M. et al. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330-4 (1988). 117. Verghese, J., Abrams, J., Wang, Y. & Morano, K.A. Biology of the heat shock response and protein chaperones: budding yeast (Saccharomyces cerevisiae) as a model system. Microbiol Mol Biol Rev 76, 115-58 (2012). 118. Wong, E. & Cuervo, A.M. Integration of clearance mechanisms: the proteasome and autophagy. Cold Spring Harb Perspect Biol 2, a006734 (2010). 119. De Los Rios, P., Ben-Zvi, A., Slutsky, O., Azem, A. & Goloubinoff, P. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling. Proc Natl Acad Sci U S A 103, 6166-71 (2006). 120. Mattoo, R.U. & Goloubinoff, P. Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins. Cell Mol Life Sci 71, 3311-25 (2014). 121. Sousa, R. Structural mechanisms of chaperone mediated protein disaggregation. Front Mol Biosci 1, 12 (2014). 122. Sousa, R. & Lafer, E.M. The role of molecular chaperones in clathrin mediated vesicular trafficking. Front Mol Biosci 2, 26 (2015). 123. Mayer, M.P. & Bukau, B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62, 670-84 (2005). 124. Hoffmann, A., Bukau, B. & Kramer, G. Structure and function of the molecular chaperone Trigger Factor. Biochim Biophys Acta 1803, 650-61 (2010).

66 125. Hendrick, J.P. & Hartl, F.U. The role of molecular chaperones in protein folding. FASEB J 9, 1559-69 (1995). 126. Priya, S., Sharma, S.K. & Goloubinoff, P. Molecular chaperones as enzymes that catalytically unfold misfolded polypeptides. FEBS Lett 587, 1981-7 (2013). 127. Finka, A., Mattoo, R.U. & Goloubinoff, P. Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes. Annu Rev Biochem 85, 715-42 (2016). 128. Sharma, S. et al. Monitoring protein conformation along the pathway of chaperonin-assisted folding. Cell 133, 142-53 (2008). 129. Shtilerman, M., Lorimer, G.H. & Englander, S.W. Chaperonin function: folding by forced unfolding. Science 284, 822-5 (1999). 130. Kerner, M.J. et al. Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli. Cell 122, 209-20 (2005). 131. Langer, T. et al. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 356, 683-9 (1992). 132. Suss, O. & Reichmann, D. Protein plasticity underlines activation and function of ATP-independent chaperones. Front Mol Biosci 2, 43 (2015). 133. Goemans, C., Denoncin, K. & Collet, J.F. Folding mechanisms of periplasmic proteins. Biochim Biophys Acta 1843, 1517-28 (2014). 134. Quan, S. et al. Genetic selection designed to stabilize proteins uncovers a chaperone called Spy. Nat Struct Mol Biol 18, 262-9 (2011). 135. Narayan, P. et al. The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-beta(1-40) peptide. Nat Struct Mol Biol 19, 79-83 (2011). 136. Haslbeck, M. & Vierling, E. A first line of stress defense: small heat shock proteins and their function in protein homeostasis. J Mol Biol 427, 1537-48 (2015). 137. Hipp, M.S., Park, S.H. & Hartl, F.U. Proteostasis impairment in protein-misfolding and -aggregation diseases. Trends Cell Biol 24, 506-14 (2014). 138. Cashikar, A.G., Duennwald, M. & Lindquist, S.L. A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J Biol Chem 280, 23869-75 (2005). 139. Mogk, A. & Bukau, B. Role of sHsps in organizing cytosolic protein aggregation and disaggregation. Cell Stress Chaperones 22, 493-502 (2017). 140. Reeg, S. et al. The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome. Free Radic Biol Med 99, 153-166 (2016).

67 141. Nathan, D.F., Vos, M.H. & Lindquist, S. In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. Proc Natl Acad Sci U S A 94, 12949-56 (1997). 142. Alvira, S. et al. Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop. Nat Commun 5, 5484 (2014). 143. Li, J., Soroka, J. & Buchner, J. The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones. Biochim Biophys Acta 1823, 624-35 (2012). 144. McClellan, A.J., Scott, M.D. & Frydman, J. Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways. Cell 121, 739-48 (2005). 145. Imai, J., Maruya, M., Yashiroda, H., Yahara, I. & Tanaka, K. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J 22, 3557-67 (2003). 146. Gething, M.J. & Sambrook, J. Protein folding in the cell. Nature 355, 33-45 (1992). 147. Anckar, J. & Sistonen, L. Regulation of HSF1 function in the heat stress response: implications in aging and disease. Annu Rev Biochem 80, 1089-115 (2011). 148. Mager, W.H. & De Kruijff, A.J. Stress-induced transcriptional activation. Microbiol Rev 59, 506-31 (1995). 149. Sorger, P.K. & Pelham, H.R. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell 54, 855-64 (1988). 150. Wiederrecht, G., Seto, D. & Parker, C.S. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell 54, 841-53 (1988). 151. Hahn, J.S., Hu, Z., Thiele, D.J. & Iyer, V.R. Genome-wide analysis of the biology of stress responses through heat shock transcription factor. Mol Cell Biol 24, 5249-56 (2004). 152. Zheng, X. et al. Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation. Elife 5(2016). 153. Kobayashi, N. & McEntee, K. Evidence for a heat shock transcription factor-independent mechanism for heat shock induction of transcription in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 87, 6550-4 (1990). 154. Wieser, R. et al. Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae. J Biol Chem 266, 12406-11 (1991). 155. Estruch, F. & Carlson, M. Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae. Mol Cell Biol 13, 3872-81 (1993). 156. Martinez-Pastor, M.T. et al. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional

68 induction through the stress response element (STRE). EMBO J 15, 2227-35 (1996). 157. Gietz, R.D. & Woods, R.A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350, 87-96 (2002). 158. Sherman, F. Getting started with yeast. Methods Enzymol 350, 3-41 (2002). 159. Goffeau, A. et al. Life with 6000 genes. Science 274, 546, 563-7 (1996). 160. Foury, F. Human genetic diseases: a cross-talk between man and yeast. Gene 195, 1-10 (1997). 161. Flaherty, K.M., DeLuca-Flaherty, C. & McKay, D.B. Three- dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 346, 623-8 (1990). 162. Flaherty, K.M., McKay, D.B., Kabsch, W. & Holmes, K.C. Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. Proc Natl Acad Sci U S A 88, 5041-5 (1991). 163. Sharma, D. & Masison, D.C. Hsp70 structure, function, regulation and influence on yeast prions. Protein Pept Lett 16, 571-81 (2009). 164. Erbse, A., Mayer, M.P. & Bukau, B. Mechanism of substrate recognition by Hsp70 chaperones. Biochem Soc Trans 32, 617-21 (2004). 165. Zhu, J. An analysis of the social space structure of population in the Shanghai municipality. Chin J Popul Sci 8, 87-102 (1996). 166. Mayer, M.P. Hsp70 chaperone dynamics and molecular mechanism. Trends Biochem Sci 38, 507-14 (2013). 167. Mayer, M.P., Brehmer, D., Gassler, C.S. & Bukau, B. Hsp70 chaperone machines. Adv Protein Chem 59, 1-44 (2001). 168. Mayer, M.P. et al. Multistep mechanism of substrate binding determines chaperone activity of Hsp70. Nat Struct Biol 7, 586-93 (2000). 169. Zuiderweg, E.R. et al. Allostery in the Hsp70 chaperone proteins. Top Curr Chem 328, 99-153 (2013). 170. Neupert, W. & Brunner, M. The protein import motor of mitochondria. Nat Rev Mol Cell Biol 3, 555-65 (2002). 171. Okamoto, K. et al. The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation. EMBO J 21, 3659-71 (2002). 172. Matouschek, A., Pfanner, N. & Voos, W. Protein unfolding by mitochondria. The Hsp70 import motor. EMBO Rep 1, 404-10 (2000). 173. Mayer, M.P. Gymnastics of molecular chaperones. Mol Cell 39, 321-31 (2010).

69 174. Jiang, J., Prasad, K., Lafer, E.M. & Sousa, R. Structural basis of interdomain communication in the Hsc70 chaperone. Mol Cell 20, 513-24 (2005). 175. Wang, H. et al. NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone- protein interaction. Biochemistry 37, 7929-40 (1998). 176. Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol Cell 48, 863-74 (2012). 177. Mayer, M.P. & Kityk, R. Insights into the molecular mechanism of allostery in Hsp70s. Front Mol Biosci 2, 58 (2015). 178. Qi, R. et al. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nat Struct Mol Biol 20, 900-7 (2013). 179. Polier, S., Dragovic, Z., Hartl, F.U. & Bracher, A. Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding. Cell 133, 1068-79 (2008). 180. Schmid, D., Baici, A., Gehring, H. & Christen, P. Kinetics of molecular chaperone action. Science 263, 971-3 (1994). 181. Wei, J., Gaut, J.R. & Hendershot, L.M. In vitro dissociation of BiP- peptide complexes requires a conformational change in BiP after ATP binding but does not require ATP hydrolysis. J Biol Chem 270, 26677-82 (1995). 182. Bertelsen, E.B., Chang, L., Gestwicki, J.E. & Zuiderweg, E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc Natl Acad Sci U S A 106, 8471-6 (2009). 183. Swain, J.F., Schulz, E.G. & Gierasch, L.M. Direct comparison of a stable isolated Hsp70 substrate-binding domain in the empty and substrate-bound states. J Biol Chem 281, 1605-11 (2006). 184. Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606-14 (1996). 185. English, C.A., Sherman, W., Meng, W. & Gierasch, L.M. The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains. J Biol Chem 292, 14765-14774 (2017). 186. Vogel, M., Mayer, M.P. & Bukau, B. Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker. J Biol Chem 281, 38705-11 (2006). 187. Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686-91 (2003). 188. Shulga, N., James, P., Craig, E.A. & Goldfarb, D.S. A nuclear export signal prevents Saccharomyces cerevisiae Hsp70 Ssb1p from stimulating nuclear localization signal-directed nuclear transport. J Biol Chem 274, 16501-7 (1999).

70 189. Kabani, M. & Martineau, C.N. Multiple hsp70 isoforms in the eukaryotic cytosol: mere redundancy or functional specificity? Curr Genomics 9, 338-248 (2008). 190. Boorstein, W.R. & Craig, E.A. Structure and regulation of the SSA4 HSP70 gene of Saccharomyces cerevisiae. J Biol Chem 265, 18912- 21 (1990). 191. Boorstein, W.R. & Craig, E.A. Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae. Mol Cell Biol 10, 3262-7 (1990). 192. Werner-Washburne, M., Becker, J., Kosic-Smithers, J. & Craig, E.A. Yeast Hsp70 RNA levels vary in response to the physiological status of the cell. J Bacteriol 171, 2680-8 (1989). 193. Werner-Washburne, M. & Craig, E.A. Expression of members of the Saccharomyces cerevisiae hsp70 multigene family. Genome 31, 684-9 (1989). 194. Werner-Washburne, M., Stone, D.E. & Craig, E.A. Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae. Mol Cell Biol 7, 2568-77 (1987). 195. Bonner, J.J. et al. Complex regulation of the yeast heat shock transcription factor. Mol Biol Cell 11, 1739-51 (2000). 196. Craig, E.A. & Jacobsen, K. Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures. Mol Cell Biol 5, 3517-24 (1985). 197. Nakanishi, H., Suda, Y. & Neiman, A.M. Erv14 family cargo receptors are necessary for ER exit during sporulation in Saccharomyces cerevisiae. J Cell Sci 120, 908-16 (2007). 198. Mackenzie, R.J. et al. Absolute protein quantification of the yeast chaperome under conditions of heat shock. Proteomics 16, 2128-40 (2016). 199. Nishikawa, S.I., Fewell, S.W., Kato, Y., Brodsky, J.L. & Endo, T. Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J Cell Biol 153, 1061-70 (2001). 200. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.J. & Sambrook, J. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell 57, 1223-36 (1989). 201. Rose, M.D., Misra, L.M. & Vogel, J.P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell 57, 1211-21 (1989). 202. Liu, Q., Krzewska, J., Liberek, K. & Craig, E.A. Mitochondrial Hsp70 Ssc1: role in protein folding. J Biol Chem 276, 6112-8 (2001). 203. Craig, E.A., Kramer, J. & Kosic-Smithers, J. SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces 71 cerevisiae, is essential for growth. Proc Natl Acad Sci U S A 84, 4156-60 (1987). 204. Kawai, A., Nishikawa, S., Hirata, A. & Endo, T. Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells. J Cell Sci 114, 3565-74 (2001). 205. Nwaka, S., Mechler, B., von Ahsen, O. & Holzer, H. The heat shock factor and mitochondrial Hsp70 are necessary for survival of heat shock in Saccharomyces cerevisiae. FEBS Lett 399, 259-63 (1996). 206. Qian, X., Hou, W., Zhengang, L. & Sha, B. Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1. Biochem J 361, 27-34 (2002). 207. Cyr, D.M., Langer, T. & Douglas, M.G. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends Biochem Sci 19, 176-81 (1994). 208. Kampinga, H.H. & Craig, E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 11, 579-92 (2010). 209. Cyr, D.M., Hohfeld, J. & Patterson, C. Protein quality control: U- box-containing E3 ubiquitin ligases join the fold. Trends Biochem Sci 27, 368-75 (2002). 210. Grove, D.E., Fan, C.Y., Ren, H.Y. & Cyr, D.M. The endoplasmic reticulum-associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3-dependent degradation of nascent CFTRDeltaF508. Mol Biol Cell 22, 301-14 (2011). 211. Meacham, G.C., Patterson, C., Zhang, W., Younger, J.M. & Cyr, D.M. The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat Cell Biol 3, 100-5 (2001). 212. Fan, C.Y., Ren, H.Y., Lee, P., Caplan, A.J. & Cyr, D.M. The type I Hsp40 zinc finger-like region is required for Hsp70 to capture non- native polypeptides from Ydj1. J Biol Chem 280, 695-702 (2005). 213. Meacham, G.C. et al. Mutations in the yeast Hsp40 chaperone protein Ydj1 cause defects in Axl1 biogenesis and pro-a-factor processing. J Biol Chem 274, 34396-402 (1999). 214. Nillegoda, N.B. et al. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524, 247-51 (2015). 215. Summers, D.W., Wolfe, K.J., Ren, H.Y. & Cyr, D.M. The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein. PLoS One 8, e52099 (2013). 216. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C. & Zylicz, M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A 88, 2874-8 (1991).

72 217. Greene, M.K., Maskos, K. & Landry, S.J. Role of the J-domain in the cooperation of Hsp40 with Hsp70. Proc Natl Acad Sci U S A 95, 6108-13 (1998). 218. Jiang, J. et al. Structural basis of J cochaperone binding and regulation of Hsp70. Mol Cell 28, 422-33 (2007). 219. Szabo, A., Korszun, R., Hartl, F.U. & Flanagan, J. A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. EMBO J 15, 408-17 (1996). 220. Bascos, N.A.D., Mayer, M.P., Bukau, B. & Landry, S.J. The Hsp40 J-domain modulates Hsp70 conformation and ATPase activity with a semi-elliptical spring. Protein Sci 26, 1838-1851 (2017). 221. Hennessy, F., Cheetham, M.E., Dirr, H.W. & Blatch, G.L. Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins. Cell Stress Chaperones 5, 347-58 (2000). 222. Cheetham, M.E. & Caplan, A.J. Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell Stress Chaperones 3, 28-36 (1998). 223. Cyr, D.M. & Ramos, C.H. Specification of Hsp70 function by Type I and Type II Hsp40. Subcell Biochem 78, 91-102 (2015). 224. Mokranjac, D., Sichting, M., Neupert, W. & Hell, K. Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria. EMBO J 22, 4945-56 (2003). 225. Craven, R.A., Egerton, M. & Stirling, C.J. A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors. EMBO J 15, 2640-50 (1996). 226. Bracher, A. & Verghese, J. The nucleotide exchange factors of Hsp70 molecular chaperones. Front Mol Biosci 2, 10 (2015). 227. Ho, A.K., Raczniak, G.A., Ives, E.B. & Wente, S.R. The integral membrane protein snl1p is genetically linked to yeast nuclear pore complex function. Mol Biol Cell 9, 355-73 (1998). 228. Sondermann, H. et al. Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an Hsp70 co- chaperone in Saccharomyces cerevisiae. J Biol Chem 277, 33220-7 (2002). 229. Verghese, J. & Morano, K.A. A lysine-rich region within fungal BAG domain-containing proteins mediates a novel association with ribosomes. Eukaryot Cell 11, 1003-11 (2012). 230. Kabani, M., McLellan, C., Raynes, D.A., Guerriero, V. & Brodsky, J.L. HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor. FEBS Lett 531, 339-42 (2002). 231. Shaner, L., Wegele, H., Buchner, J. & Morano, K.A. The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb. J Biol Chem 280, 41262-9 (2005).

73 232. Dragovic, Z., Broadley, S.A., Shomura, Y., Bracher, A. & Hartl, F.U. Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s. EMBO J 25, 2519-28 (2006). 233. Steel, G.J., Fullerton, D.M., Tyson, J.R. & Stirling, C.J. Coordinated activation of Hsp70 chaperones. Science 303, 98-101 (2004). 234. Tyson, J.R. & Stirling, C.J. LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum. EMBO J 19, 6440-52 (2000). 235. Kabani, M., Beckerich, J.M. & Gaillardin, C. Sls1p stimulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum. Mol Cell Biol 20, 6923- 34 (2000). 236. Laloraya, S., Gambill, B.D. & Craig, E.A. A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation. Proc Natl Acad Sci U S A 91, 6481-5 (1994). 237. Bolliger, L. et al. A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability. EMBO J 13, 1998-2006 (1994). 238. Deloche, O. & Georgopoulos, C. Purification and biochemical properties of Saccharomyces cerevisiae's Mge1p, the mitochondrial cochaperone of Ssc1p. J Biol Chem 271, 23960-6 (1996). 239. Miao, B., Davis, J.E. & Craig, E.A. Mge1 functions as a nucleotide release factor for Ssc1, a mitochondrial Hsp70 of Saccharomyces cerevisiae. J Mol Biol 265, 541-52 (1997). 240. Schmidt, S., Strub, A., Rottgers, K., Zufall, N. & Voos, W. The two mitochondrial heat shock proteins 70, Ssc1 and Ssq1, compete for the cochaperone Mge1. J Mol Biol 313, 13-26 (2001). 241. Marada, A. et al. Mge1, a nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70 function. Mol Biol Cell 24, 692-703 (2013). 242. Moro, F. & Muga, A. Thermal adaptation of the yeast mitochondrial Hsp70 system is regulated by the reversible unfolding of its nucleotide exchange factor. J Mol Biol 358, 1367-77 (2006). 243. Takayama, S. et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell 80, 279-84 (1995). 244. Sondermann, H. et al. Structure of a Bag/Hsc70 complex: convergent functional evolution of Hsp70 nucleotide exchange factors. Science 291, 1553-7 (2001). 245. Luders, J., Demand, J. & Hohfeld, J. The ubiquitin-related BAG-1 provides a link between the molecular chaperones Hsc70/Hsp70 and the proteasome. J Biol Chem 275, 4613-7 (2000). 246. Andreasson, C., Fiaux, J., Rampelt, H., Druffel-Augustin, S. & Bukau, B. Insights into the structural dynamics of the Hsp110- Hsp70 interaction reveal the mechanism for nucleotide exchange activity. Proc Natl Acad Sci U S A 105, 16519-24 (2008). 74 247. Dragovic, Z., Shomura, Y., Tzvetkov, N., Hartl, F.U. & Bracher, A. Fes1p acts as a nucleotide exchange factor for the ribosome- associated molecular chaperone Ssb1p. Biol Chem 387, 1593-600 (2006). 248. Kabani, M., Beckerich, J.M. & Brodsky, J.L. Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p. Mol Cell Biol 22, 4677-89 (2002). 249. Shomura, Y. et al. Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange. Mol Cell 17, 367-79 (2005). 250. McLellan, C.A., Raynes, D.A. & Guerriero, V. HspBP1, an Hsp70 cochaperone, has two structural domains and is capable of altering the conformation of the Hsp70 ATPase domain. J Biol Chem 278, 19017-22 (2003). 251. Abrams, J.L., Verghese, J., Gibney, P.A. & Morano, K.A. Hierarchical functional specificity of cytosolic heat shock protein 70 (Hsp70) nucleotide exchange factors in yeast. J Biol Chem 289, 13155-67 (2014). 252. Gowda, N.K., Kandasamy, G., Froehlich, M.S., Dohmen, R.J. & Andreasson, C. Hsp70 nucleotide exchange factor Fes1 is essential for ubiquitin-dependent degradation of misfolded cytosolic proteins. Proc Natl Acad Sci U S A 110, 5975-80 (2013). 253. Schuermann, J.P. et al. Structure of the Hsp110:Hsc70 nucleotide exchange machine. Mol Cell 31, 232-43 (2008). 254. Mukai, H. et al. Isolation and characterization of SSE1 and SSE2, new members of the yeast HSP70 multigene family. Gene 132, 57- 66 (1993). 255. Easton, D.P., Kaneko, Y. & Subjeck, J.R. The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s. Cell Stress Chaperones 5, 276-90 (2000). 256. Lee-Yoon, D., Easton, D., Murawski, M., Burd, R. & Subjeck, J.R. Identification of a major subfamily of large hsp70-like proteins through the cloning of the mammalian 110-kDa heat shock protein. J Biol Chem 270, 15725-33 (1995). 257. Yasuda, K., Nakai, A., Hatayama, T. & Nagata, K. Cloning and expression of murine high molecular mass heat shock proteins, HSP105. J Biol Chem 270, 29718-23 (1995). 258. Raviol, H., Bukau, B. & Mayer, M.P. Human and yeast Hsp110 chaperones exhibit functional differences. FEBS Lett 580, 168-74 (2006). 259. Raviol, H., Sadlish, H., Rodriguez, F., Mayer, M.P. & Bukau, B. Chaperone network in the yeast cytosol: Hsp110 is revealed as an Hsp70 nucleotide exchange factor. EMBO J 25, 2510-8 (2006). 260. Andreasson, C., Fiaux, J., Rampelt, H., Mayer, M.P. & Bukau, B. Hsp110 is a nucleotide-activated exchange factor for Hsp70. J Biol Chem 283, 8877-84 (2008). 75 261. Shaner, L., Sousa, R. & Morano, K.A. Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1. Biochemistry 45, 15075-84 (2006). 262. Andreasson, C., Rampelt, H., Fiaux, J., Druffel-Augustin, S. & Bukau, B. The endoplasmic reticulum Grp170 acts as a nucleotide exchange factor of Hsp70 via a mechanism similar to that of the cytosolic Hsp110. J Biol Chem 285, 12445-53 (2010). 263. Liu, X.D., Morano, K.A. & Thiele, D.J. The yeast Hsp110 family member, Sse1, is an Hsp90 cochaperone. J Biol Chem 274, 26654- 60 (1999). 264. Shaner, L., Trott, A., Goeckeler, J.L., Brodsky, J.L. & Morano, K.A. The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family. J Biol Chem 279, 21992-2001 (2004). 265. Oh, H.J., Chen, X. & Subjeck, J.R. Hsp110 protects heat-denatured proteins and confers cellular thermoresistance. J Biol Chem 272, 31636-40 (1997). 266. Rampelt, H. et al. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO J 31, 4221-35 (2012). 267. Mattoo, R.U., Sharma, S.K., Priya, S., Finka, A. & Goloubinoff, P. Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with Hsp70 to solubilize protein aggregates. J Biol Chem 288, 21399-411 (2013). 268. Shorter, J. The mammalian disaggregase machinery: Hsp110 synergizes with Hsp70 and Hsp40 to catalyze protein disaggregation and reactivation in a cell-free system. PLoS One 6, e26319 (2011). 269. Goeckeler, J.L., Stephens, A., Lee, P., Caplan, A.J. & Brodsky, J.L. Overexpression of yeast Hsp110 homolog Sse1p suppresses ydj1- 151 thermosensitivity and restores Hsp90-dependent activity. Mol Biol Cell 13, 2760-70 (2002). 270. Goeckeler, J.L. et al. The yeast Hsp110, Sse1p, exhibits high- affinity peptide binding. FEBS Lett 582, 2393-6 (2008). 271. Xu, X. et al. Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity. J Biol Chem 287, 5661-72 (2012). 272. Garcia, V.M., Nillegoda, N.B., Bukau, B. & Morano, K.A. Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities. Mol Biol Cell 28, 2066-2075 (2017). 273. Mandal, A.K. et al. Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system. Mol Biol Cell 21, 1439-48 (2010). 274. Doyle, S.M., Genest, O. & Wickner, S. Protein rescue from aggregates by powerful molecular chaperone machines. Nat Rev Mol Cell Biol 14, 617-29 (2013).

76 275. Sielaff, B. & Tsai, F.T. The M-domain controls Hsp104 protein remodeling activity in an Hsp70/Hsp40-dependent manner. J Mol Biol 402, 30-7 (2010). 276. Neuwald, A.F., Aravind, L., Spouge, J.L. & Koonin, E.V. AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 9, 27-43 (1999). 277. Mogk, A., Kummer, E. & Bukau, B. Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation. Front Mol Biosci 2, 22 (2015). 278. Franzmann, T.M., Czekalla, A. & Walter, S.G. Regulatory circuits of the AAA+ disaggregase Hsp104. J Biol Chem 286, 17992-8001 (2011). 279. Duran, E.C., Weaver, C.L. & Lucius, A.L. Comparative Analysis of the Structure and Function of AAA+ Motors ClpA, ClpB, and Hsp104: Common Threads and Disparate Functions. Front Mol Biosci 4, 54 (2017). 280. Yokom, A.L. et al. Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation. Nat Struct Mol Biol 23, 830-7 (2016). 281. Johnston, D.M., Miot, M., Hoskins, J.R., Wickner, S. & Doyle, S.M. Substrate Discrimination by ClpB and Hsp104. Front Mol Biosci 4, 36 (2017). 282. Biter, A.B., Lee, S., Sung, N. & Tsai, F.T. Structural basis for intersubunit signaling in a protein disaggregating machine. Proc Natl Acad Sci U S A 109, 12515-20 (2012). 283. Mogk, A. et al. Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity. J Biol Chem 278, 17615-24 (2003). 284. Yamasaki, T., Nakazaki, Y., Yoshida, M. & Watanabe, Y.H. Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus. FEBS J 278, 2395- 403 (2011). 285. Zeymer, C., Barends, T.R., Werbeck, N.D., Schlichting, I. & Reinstein, J. Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor. Acta Crystallogr D Biol Crystallogr 70, 582-95 (2014). 286. Rosenzweig, R. et al. ClpB N-terminal domain plays a regulatory role in protein disaggregation. Proc Natl Acad Sci U S A 112, E6872-81 (2015). 287. Lee, J. et al. Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation. Sci Rep 7, 11184 (2017). 288. Sweeny, E.A. et al. The Hsp104 N-terminal domain enables disaggregase plasticity and potentiation. Mol Cell 57, 836-49 (2015). 77 289. Beinker, P., Schlee, S., Groemping, Y., Seidel, R. & Reinstein, J. The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity. J Biol Chem 277, 47160-6 (2002). 290. Liu, Z., Tek, V., Akoev, V. & Zolkiewski, M. Conserved amino acid residues within the amino-terminal domain of ClpB are essential for the chaperone activity. J Mol Biol 321, 111-20 (2002). 291. Mizuno, S., Nakazaki, Y., Yoshida, M. & Watanabe, Y.H. Orientation of the amino-terminal domain of ClpB affects the disaggregation of the protein. FEBS J 279, 1474-84 (2012). 292. Haslberger, T. et al. M domains couple the ClpB threading motor with the DnaK chaperone activity. Mol Cell 25, 247-60 (2007). 293. Lee, S. et al. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell 115, 229-40 (2003). 294. Mogk, A. & Bukau, B. Molecular chaperones: structure of a protein disaggregase. Curr Biol 14, R78-80 (2004). 295. DeSantis, M.E. et al. Operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients. Cell 151, 778- 93 (2012). 296. Cashikar, A.G. et al. Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein. Mol Cell 9, 751-60 (2002). 297. Doyle, S.M. et al. Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat Struct Mol Biol 14, 114-22 (2007). 298. Schirmer, E.C., Homann, O.R., Kowal, A.S. & Lindquist, S. Dominant gain-of-function mutations in Hsp104p reveal crucial roles for the middle region. Mol Biol Cell 15, 2061-72 (2004). 299. Wendler, P. et al. Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. Cell 131, 1366-77 (2007). 300. Lipinska, N. et al. Disruption of ionic interactions between the nucleotide binding domain 1 (NBD1) and middle (M) domain in Hsp100 disaggregase unleashes toxic hyperactivity and partial independence from Hsp70. J Biol Chem 288, 2857-69 (2013). 301. Weibezahn, J. et al. Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB. Cell 119, 653-65 (2004). 302. Tessarz, P., Mogk, A. & Bukau, B. Substrate threading through the central pore of the Hsp104 chaperone as a common mechanism for protein disaggregation and prion propagation. Mol Microbiol 68, 87- 97 (2008). 303. Doyle, S.M., Hoskins, J.R. & Wickner, S. DnaK chaperone- dependent disaggregation by caseinolytic peptidase B (ClpB) mutants reveals functional overlap in the N-terminal domain and nucleotide-binding domain-1 pore tyrosine. J Biol Chem 287, 28470-9 (2012). 78 304. Gates, S.N. et al. Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104. Science 357, 273-279 (2017). 305. Glynn, S.E., Martin, A., Nager, A.R., Baker, T.A. & Sauer, R.T. Structures of asymmetric ClpX hexamers reveal nucleotide- dependent motions in a AAA+ protein-unfolding machine. Cell 139, 744-56 (2009). 306. Burton, R.E., Siddiqui, S.M., Kim, Y.I., Baker, T.A. & Sauer, R.T. Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine. EMBO J 20, 3092- 100 (2001). 307. Haslberger, T. et al. Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments. Nat Struct Mol Biol 15, 641-50 (2008). 308. Murray, A.N. & Kelly, J.W. Hsp104 gives clients the individual attention they need. Cell 151, 695-7 (2012). 309. Martin, A., Baker, T.A. & Sauer, R.T. Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115-20 (2005). 310. Moreau, M.J., McGeoch, A.T., Lowe, A.R., Itzhaki, L.S. & Bell, S.D. ATPase site architecture and helicase mechanism of an archaeal MCM. Mol Cell 28, 304-14 (2007). 311. Lyubimov, A.Y., Strycharska, M. & Berger, J.M. The nuts and bolts of ring-translocase structure and mechanism. Curr Opin Struct Biol 21, 240-8 (2011). 312. Goloubinoff, P., Mogk, A., Zvi, A.P.B., Tomoyasu, T. & Bukau, B. Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proceedings of the National Academy of Sciences 96, 13732-13737 (1999). 313. Parsell, D.A., Kowal, A.S., Singer, M.A. & Lindquist, S. Protein disaggregation mediated by heat-shock protein Hspl04. Nature 372, 475-478 (1994). 314. Acebron, S.P., Martin, I., del Castillo, U., Moro, F. & Muga, A. DnaK-mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface. FEBS Lett 583, 2991- 6 (2009). 315. Winkler, J., Tyedmers, J., Bukau, B. & Mogk, A. Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation. J Cell Biol 198, 387-404 (2012). 316. Miot, M. et al. Species-specific collaboration of heat shock proteins (Hsp) 70 and 100 in thermotolerance and protein disaggregation. Proc Natl Acad Sci U S A 108, 6915-20 (2011). 317. Klosowska, A., Chamera, T. & Liberek, K. Adenosine diphosphate restricts the protein remodeling activity of the Hsp104 chaperone to Hsp70 assisted disaggregation. Elife 5(2016).

79 318. Rosenzweig, R., Moradi, S., Zarrine-Afsar, A., Glover, J.R. & Kay, L.E. Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction. Science 339, 1080-3 (2013).

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