Insights into functions and mechanisms of ribosome-associated chaperones from Saccharomyces cerevisiae

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of ribosome-associated chaperones from Saccharomyces cerevisiae

Dissertation

zur Erlangung des akademischen Grades

des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Universität Konstanz

Fachbereich Biologie

vorgelegt von Ansgar Koplin

Tag der mündlichen Prüfung: 30.07.2009

1. Referentin: Prof. Dr. Elke Deuerling

2. Referentin: Prof. Dr. Iwona Adamska

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I.a SUMMARY

The folding of newly synthesized proteins into their native structures is a fundamental but failure prone process and therefore controlled by a network of molecular chaperones to assist and ensure correct protein folding events. Chaperones that guide the folding of newly synthesized proteins in the cytosol are classified into two groups: chaperones that are recruited to the ribosome are the first interaction partners of newborn proteins. They are assumed to protect nascent polypeptides against harmful conditions and to support initial co- translational folding steps, while cytosolic chaperones act subsequently on a subset of newly synthesized proteins to mainly promote post-translational folding steps.

Chaperones associating with the ribosome have been found in all kingdoms of life. While the Trigger Factor (TF) is the only known ribosome-associated chaperone in bacteria, two systems exist in yeast and higher eukaryotes. Both systems, the nascent chain-associated complex (NAC) and the yeast tripartite Ssb-system (Ssb/RAC) are unrelated to TF. In contrast to bacterial TF, the functions of ribosome-associated systems in yeast are still barely defined. The Hsp70/40-based Ssb/RAC-system is per definition a canonical chaperone system, however, its precise function and potential substrates are unknown. Moreover, it is unclear whether NAC contributes to the folding network for newly synthesized proteins since that far no function of NAC could be unambiguously assigned.

This work adds to the knowledge about ribosome-associated chaperones in yeast and focuses on the functions and mechanisms of NAC and the Ssb/RAC-system during de novo protein folding. Genetic surveys were combined with biochemical approaches in order to gain insights into the complex cytosolic chaperone network of eukaryotes.

The key-results are summarized below:

• The first main contribution of this study is the finding that the Ssb-system functionally cooperates with NAC in co-translational protein folding in vivo.

Simultaneous deletions of genes encoding NAC and Ssb caused a severe synthetic sickness of cells grown at 30°C and the loss of cell viability under protein folding stress conditions. Deprivation of Ssb impaired NAC association with translating ribosomes and provoked aggregation of newly synthesized proteins, which was enhanced by additional deletion of NAC. Further analysis discovered a second function of Ssb/RAC and NAC in regulating the amount of 60S and 40S ribosomal

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revealed the formation of ribosomal halfmers and a pronounced deficiency of ribosomal subunits accompanied by strongly reduced amounts of translating ribosomes. These data provide for the first time evidence that the two ribosome- associated systems NAC and Ssb/RAC are functionally interconnected and contribute to two major cellular processes: the folding of newly synthesized proteins and the production of actively translating ribosomes.

• The second main contribution of this study is the identification of the genetic and functional interaction of NAC and the Hsp110 Sse1. Sse1 functions as nucleotide exchange factor (NEF) for two types of Hsp70s, the cytosolic Ssa and the ribosome- associated Ssb. This study showed that NAC and Sse1 genetically interact and the simultaneous deletion caused a severe growth impairment at 30°C, a sensitivity against drugs inducing protein folding stress and a mild induction of the cellular heat shock response. The phenotype could be complemented by expressing NAC or by overexpression of Fes1, the second NEF for Ssa and Ssb-type of Hsp70s. Loss of Sse1 was accompanied by protein aggregation including polyubiquitinated proteins which was enhanced by additional NAC deletion. Mass spectrometry identified 13 potential Sse1 and NAC substrates including the enzyme Glucose-6-phophate-dehydrogenase.

These data further support the theory that NAC is a bonafide member of the cytosolic chaperone network.

• An additional achievement of this study is a contribution to the conformational characterization of the stable heterodimer RAC (ribosome-associated complex) formed by the Hsp40 Zuo and the Hsp70 Ssz. RAC is essential to drive the chaperone cycle of ribosome-associated Ssb by stimulating Ssb’s ATP-hydrolysis and tight substrate binding. The recombinant expression and purification of RAC provided the basis for subsequent collaborative Hydrogen/Deuterium exchange experiments by other lab members. The results revealed that the highly flexible N-terminus of Zuo is the structural element which is essential to bind Ssz for dimer formation. Ssz is a highly dynamic protein and gets strongly stabilized upon complex formation mainly involving its C-terminal substrate binding domain.

In summary, the findings of this work have led to a clearer picture of the mechanisms and functional interplay of ribosome-associated chaperones as decisive regulators at the birthplace of new proteins.

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I.b ZUSAMMENFASSUNG

Die Faltung von neu synthetisierten Proteinen in ihre native Struktur ist ein fundamentaler Prozess in allen Zellen. Dieser Prozess ist allerdings anfällig für Fehler, und wird daher von einem Netzwerk molekularer Chaperone unterstützt, welche die korrekte Proteinfaltung kontrollieren und unterstützen. Chaperone, die die Faltung neu synthetisierter Proteine im Zytosol assistieren, werden in zwei Gruppen unterteilt: Chaperone, welche an das Ribosom rekrutiert werden, sind die ersten Interaktionspartner neu gebildeter Proteine. Man geht davon aus, dass sie wachsende Polypeptide schützen und co-translationale Faltungsschritte initiieren können. Zytosolische Chaperone hingegen sind an späteren Faltungsprozessen einiger neuer Proteine beteiligt und fördern vor allem post-translationale Faltungsschritte.

Ribosom-assoziierte Chaperone findet man in allen Reichen des Lebens. Der Trigger Factor (TF) ist bis heute das einzige bekannte Ribosom-assoziierte Chaperon in Bakterien. In Hefe hingegen binden zwei Systeme an das Ribosom: der ‚nascent chain-associated complex’

(NAC) und das aus drei Proteinen bestehende Ssb-System (Ssb/RAC). Beide Systeme weisen jedoch keinerlei Ähnlichkeit mit dem TF auf. Im Gegensatz zum TF in Bakterien sind die Funktionen der Ribosom-assoziierten Systeme in Hefe kaum bekannt. Alle Komponenten des Ssb/RAC-Systems sind Hsp70- bzw. Hsp40-Proteine und daher per Definition klassische Chaperone. Allerdings konnten die genaue Funktion oder potentielle Substrate noch nicht beschrieben werden. Unklar ist außerdem, ob NAC an der Faltung von neu synthetisierten Proteinen beteiligt ist, da NAC bisher keine eindeutige Funktion zugesprochen werden konnte.

Die vorliegende Arbeit trägt zu einem besseren Verständnis der Ribosom-assoziierten Chaperone in Hefe bei und legt den Focus dabei auf die Funktionen und Mechanismen von NAC und dem Ssb/RAC-System während der de novo-Proteinfaltung. Um neue Einblicke in die Komplexität des zytosolischen Chaperon-Netzwerks in Eukaryoten zu gewinnen wurden genetische mit biochemischen Methoden kombiniert.

Die Hauptresultate dieser Arbeit sind im Folgenden zusammengefasst.

• Als erster zentraler Aspekt dieser Arbeit konnte gezeigt werden, dass das Ssb-System und NAC während der co-translationalen Proteinfaltung in vivo kooperieren. Die gleichzeitige Deletion der NAC und Ssb kodierenden Gene führte in Zellen bei 30°C zum synthetischen Defekt und hatte eine verminderte Lebensfähigkeit unter Protein- Faltungsstress-Bedingungen zur Folge. In Abwesenheit von Ssb war die Assoziation

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von NAC an translatierende Ribosomen vermindert und bewirkte die Aggregation von neu-synthetisierten Proteinen. Diese Effekte wurden durch die zusätzliche Deletion von NAC verstärkt. Weitere Experimente haben eine zweite Funktion von Ssb/RAC und NAC bei der Regulation der Konzentration von 60S und 40S ribosomalen Untereinheiten gezeigt, was auf eine Beteiligung an der Ribosom-Biogenese schließen lässt. In nac!ssb! Zellen konnte die Bildung von ribosomalen Halbmeren und eine dramatisch geringere Konzentration an ribosomalen Untereinheiten begleitet von einer reduzierten Anzahl translatierender Ribosomen beobachtet werden. Diese Daten lieferten zum ersten Mal einen Hinweis auf die funktionale Verbindung zwischen den beiden Ribosom-assoziierten Systemen NAC und Ssb/RAC und deren Beteiligung an zwei wichtigen zellulären Prozessen: der Faltung neu synthetisierter Proteine und der Bildung aktiv-translatierender Ribosomen.

• Der zweite entscheidende Beitrag dieser Arbeit war die Identifizierung der genetischen und funktionalen Interaktion zwischen NAC und dem Hsp110 Sse1. Sse1 fungiert als Nukleotid-Austausch-Faktor für zwei Arten von Hsp70-Proteinen, dem zytosolischen Ssa und den Ribosom-assoziierten Ssb. In dieser Arbeit wurde die genetische Interaktion zwischen NAC und Sse1 gezeigt. Deren gleichzeitige Deletion führte zu einem Wachstumsdefekt bei 30°C, einer Sensitivität gegenüber Faltungsstress-induzierenden Drogen und zur Induktion einer schwachen Hitzeschock Antwort. Dieser Phänotyp konnte durch die Expression von NAC oder der Überexpression von Fes1, dem zweiten Nukleotid-Austausch-Faktor für Ssa und Ssb, komplementiert werden. Der Verlust von Sse1 führte zur Aggregation von poly- ubiquitinierten Proteinen. Dieser Effekt wurde durch die zusätzliche Deletion der NAC kodierenden Gene verstärkt. Mit Hilfe von Massenspektrometrie konnten 13 potentielle Sse1 und NAC Substrate, darunter das Enzym Glukose-6-Phosphat- Dehydrogenase, identifiziert werden. Diese Daten bestätigen, dass NAC eine wichtige Komponente im zytosolischen Chaperon-Netzwerk darstellt.

• Im Rahmen dieser Arbeit wurde ein zusätzlicher Beitrag zur strukturellen Charakterisierung des stabilen RAC-Komplexes (ribosome-associated complex), bestehend aus dem Hsp40-Chaperon Zuo und dem Hsp70-Chaperon Ssz, geleistet.

Dieser Komplex ist essentiell, um den Chaperonzyklus von Ssb durch die Stimulation seiner ATP-Hydrolyse und Substratbindung anzutreiben. Die rekombinante Expression und Aufreinigung von RAC waren die Grundlage für die darauf folgenden Wasserstoff-/Deuterium-Austausch Experimente in Kollaboration mit anderen

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Gruppenmitgliedern. Diese Ergebnisse beschreiben den äußerst flexiblen N-Terminus von Zuo, als strukturelles Element mit essentieller Funktion für die Dimerbildung mit Ssz. Ssz ist ein sehr dynamisches Protein und wird durch die Komplexbildung stabilisiert, woran seine C-terminale Substratbindedomäne hauptsächlich beteiligt ist.

Zusammenfassend lässt sich sagen, dass die Ergebnisse dieser Arbeit zu einem besseren Verständnis der Mechanismen und der funktionalen Beziehungen von Ribosom-assoziierten Chaperonen als maßgebliche Regulatoren am Geburtsort neuer Proteine geführt haben.

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II. INTRODUCTION

A fundamental process in all cells is the conversion of genetic information into functional proteins that carry out the genetic program. Ribosomes play an essential role in this process, as they translate mRNA into the primary amino acid sequence. In the subsequent step, the newly synthesized polypeptide has to fold into its specific, three dimensional conformation, which is called the ‘native state’ of the protein. Molecular chaperones, proteins that help other protein to fold, are crucial for this final step, which results in the formation of functional proteins.

Already a bacterial cell is estimated to contain about 20.000 ribosomes, whilst a human cell has up to a few millions. Especially liver cells, that have high rates of protein synthesis, have a great number of ribosomes. The translation speed is remarkably high, with about 15-20 aa per second in bacteria and 5-7 aa per second in eukaryotes. This means, a rapidly growing E.

coli cell produces an estimated total of 30.000 polypeptides per minute (Bukau et al., 2000;

Wegrzyn and Deuerling, 2005).

This work focuses on the folding of newly translated proteins in the eukaryotic model organism Saccharomyces cerevisiae and the role of molecular chaperones which are associated with the ribosome to assist early co-translational folding steps of nascent polypeptides.

II.1 The ribosome

Translation of the genetic code into proteins is performed by a complex apparatus comprising ribosomes, mRNAs, tRNAs and accessory protein factors. The ribosome, a universal dynamic cellular ribonucleoprotein complex, is the key player in this process. Ribosomes comprise two subunits that associate to form a functional machinery. Both subunits consist of large ribosomal RNAs (rRNA), within many ribosomal proteins (RPs) are entangled (Table 1).

Table 1: RNA and protein composition of ribosomes.

Despite the size difference, ribosomes from all kingdoms of life are functionally conserved and the highest level of sequence conservation can be found in the functional domains.

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During evolution, both eukaryotic subunits have increased in complexity, resulting in an overall gain of molecular weight (Spahn et al., 2001).

The translation process can be divided into three phases: initiation, elongation and termination. During initiation, the small ribosomal subunit, loaded with initiation factors and the initiator tRNA, associates with a mRNA and moves along the mRNA searching for the start codon. After encountering the start codon, initiation factors dissociate and the large subunit binds to assemble the translation competent ribosome, ready for elongation of the protein. After termination or already during translation, as soon as the nascent chain leaves the ribosome through a tunnel in the large subunit to encounter the cytosol, several factors determine the fate of the newly made proteins which will be presented later.

II.1.1 The homeostasis of ribosomes

Ribosome biogenesis and protein translation are among the most energy-consuming cellular processes, and it is therefore not surprising that these pathways are tightly controlled upon nutrient limitation. As a major consumer of the cell’s resources, ribosome biosynthesis plays a key role in cell growth, especially because new ribosomes represent an investment in new

‘machines’ for protein biosynthesis. Moreover, the regulation of ribosome biosynthesis has to ensure that the ribosomal components are available in equimolar amounts.

Under nutrient-rich conditions, eukaryotic cells assemble more than 2,000 ribosomal subunits every minute, resulting in about 200.000 ribosomes accounting for almost 50% of all cellular proteins in growing cells (Warner, 1999b). 60% of total transcription is devoted to ribosomal RNA (by Pol I), and 50% of RNA polymerase II (Pol II) transcription is devoted to ribosomal proteins. Coordinate regulation of the ~150 rRNA genes and 137 genes of ribosomal proteins (RP) that make such prodigious use of resources is essential for the economy of the cell. This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes.

The ‘target of rapamycin’ (TOR) pathway of protein kinases and phosphatases has been implicated in transducing the availability of nutrients or growth factors into growth (Thomas and Hall, 1997). In S. cerevisiae, inhibition of the TOR pathway by addition of rapamycin leads to a rapid repression of transcription of both rRNA and RP genes and prevents the activation of ribosome synthesis in response to added growth stimuli (Powers and Walter, 1999; Zaragoza et al., 1998).

The ras–cAMP–protein kinase A (PKA) pathway has also been implicated in the detection of

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changes in the source and availability of carbon and nitrogen. Constitutively active PKA leads to doubling of the amount of several RP mRNAs, whereas constitutively inactive PKA leads to reduced RP mRNA and to the cells’ inability to induce RP transcription in response to a carbon source upshift (Klein and Struhl, 1994). Depletion of cAMP leads to cessation of growth and repression of RP mRNA levels (Neuman-Silberberg et al., 1995).

Mature ribosomes are very stable, with an estimated half-life of several days. Starving cells perform a selective degradation of ribosomes, which is required for survival. Amino acids produced by this autophagy process, called ribophagy, are essential for synthesis of proteins under conditions of starvation (Kraft et al., 2008; Onodera and Ohsumi, 2005).

II.1.2 Ribosome structure

In the past 10 years remarkable progress in characterizing the machinery of protein biosynthesis has been made. Several structures of ribosomal particles have been determined, which include the large subunits of the archaeon Haloarcula marismortui (Ban et al., 2000) and the eubacterium Deinococcus radiodurans (Wimberly et al., 2000). Moreover, the structures of the small subunit and the entire ribosome from the eubacterium Thermus thermophilus could be resolved (Schluenzen et al., 2000; Yusupov et al., 2001). Since 2005, also the structure of the 70S ribosome of Escherichia coli is available (Schuwirth et al., 2005). Although, a crystal structure of a eukaryotic ribosome has not yet been published, a comparative study was performed where the cryo-electron microscopy (EM) structure of the yeast ribosome was used to extrapolate a detailed model of the eukaryotic ribosome using the existing atomic maps of the H. marismortui and T. thermophilus ribosomes (Spahn et al., 2001).

The decoding or translation process takes place at the interface between both ribosomal subunits. While elongation proceeds, both subunits operate cooperatively. The small subunit provides the mRNA-binding machinery, the path along which the mRNA progresses, the decoding centre and most of the components that control translational fidelity. The large subunit performs the main ribosomal catalytic function, namely amino acid (aa) polymerization and provides the protein exit tunnel, which the nascent polypeptide chains traverses to enter the cytosol. tRNAs, the molecules that decode the genetic information and carry the amino acids to be incorporated in the growing polypeptide, are the non-ribosomal entities that join the two subunits at each of their three binding sites, A (aminoacyl), P (peptidyl) and E (exit) (Figure 1).

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Figure 1: Interface views of the 50S and 30S subunits of T. thermophilus. The different molecular components are coloured for identification. 50S: gray, 23S rRNA; light blue, 5S rRNA; magenta, 50S proteins.

30S: cyan, 16S rRNA; dark blue, 30S proteins. A, P, and E are the A-, P-, and E-site tRNAs (gold, orange, and red, respectively). Taken from (Yusupov et al., 2001).

The surface of the inter-subunit interface, the so-called peptidyl-transferase centre (PTC), is composed predominately of rRNA and all functional sites are located close to this interface (Figure 1). Hence, unlike typical polymerases, which are protein enzymes, RNA is the major player in ribosome activities.

Nascent polypeptides progress through the exit tunnel of the large ribosomal subunit. This universal feature starts adjacent to the PTC (Ban et al., 2000; Harms et al., 2001) and is lined primarily by rRNA with a few ribosomal proteins (Figure 2).

Figure 2: The ribosomal tunnel of the H.

marismotui large ribosomal subunit (side view, subunit is cut in half to show the interior of the 50S complex). A modelled peptide spans the distance from the transferase centre (PT) to the surface. The tunnel surface is shown with backbone atoms of the 23S rRNA colour coded by domains. Domain I (yellow), II (light blue), III (orange), IV (green), V (red), 5S rRNA (pink), and proteins are blue. Taken from (Nissen et al., 2000).

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The dimensions of the tunnel (~120 Å in length, with a varying diameter of 10-25 Å) are large enough to accommodate a growing polypeptide segment of 30-35 aa in length in an extended conformation (Hardesty and Kramer, 2001). It can provide space for co-translational transient folding to a limited extent, which was indicated by results obtained by methods including fluorescence resonance energy transfer (FRET) measurements (Woolhead et al., 2004) and computational analyses (Ziv et al., 2005). !-Helices can be formed inside the tunnel but no "- sheets or tertiary structures.

II.1.3 Ribosomal tunnel exit and ribosome-associated factors

The exit site of the ribosomal tunnel is encircled by rRNA and several ribosomal proteins.

Four of these proteins L22, L24, L23 and L29 are conserved among the three domains of life, whereas the L19, L31 and L39 proteins are not found on prokaryotic ribosomes (Wegrzyn and Deuerling, 2005). These proteins surround the tunnel exit and provide docking platforms for diverse ribosome-associated factors, which contact and act on the emerging nascent polypeptide.

Three main processes have to be integrated once the nascent chain emerges from the ribosomal tunnel exit. 1) The new protein may have to be transported to a compartment other than the cytosol, 2) it may have to be modified chemically to fulfil its function or 3) it may start to fold into the native structure, aided by molecular chaperones (Figure 3).

The ribosomal protein L23 serves as a central anchor point for the ubiquitous targeting factor

‘signal recognition particle’ (SRP), which scans nascent chains for the presence of translocation signal sequences and targets secretory proteins to the translocon at the ER (Gu et al., 2003; Halic et al., 2004; Lutcke, 1995).

In addition, ribosome-bound modifying enzymes act during early stages of protein biosynthesis on the growing polypeptide. The essential ‘methionine aminopeptidase’ (MAP) removes the N-terminal methionine from nascent chains in pro- and eukaryotes (Li and Chang, 1995) and was reported to interact with the conserved L24 protein (Addlagatta et al., 2005). In bacteria, protein synthesis is always initiated by formylated methionine, which has to be deformylated first by the essential ‘peptide deformylase’ (PDF) before MAP can cleave the methionine (Giglione et al., 2004). Recently, the ribosomal protein L17 was shown to be the ribosomal docking site for PDF (Bingel-Erlenmeyer et al., 2008). The ‘N-terminal acetyltransferase’ (NAT) of yeast and higher organisms modifies nascent chains as soon as they expose the N-termini to the cytosol. Around 50% of the yeast proteome is N-terminally

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Figure 3: Processes at the ribosomal tunnel exit. Newly translated proteins are targeted to translocation, they are chemically modified and/or start folding to the native conformation.

acetylated, which neutralize positive charges and may influence protein function and stability (Polevoda and Sherman, 2003).

Nascent polypeptide chains are also met by chaperones as they emerge from the ribosome promoting their folding. The best studied ribosome-associated chaperone is the ‘Trigger Factor’ (TF) in bacteria. TF binds to unfolded polypeptide substrates and docks onto the ribosomal protein L23, which is next to the polypeptide exit site of the large ribosome subunit (Blaha et al., 2003; Hesterkamp et al., 1997; Kramer et al., 2002). Since ribosome-associated molecular chaperones are within the main focus of this study, a later chapter will give a more detailed introduction to these factors.

For most of these factors there is more detailed information available about their ribosomal binding sites. But it is still unknown how these different enzymes, targeting factors and chaperones are coordinated in their order and mode of action to ensure the correct fate of a newly synthesized protein.

II.2 Protein folding

Unfolded and newly synthesized proteins must fold to unique three-dimensional structures. It is firmly established from in vitro refolding experiments, that the native fold of a protein is encoded in its amino acid sequence and is adopted spontaneously (Anfinsen, 1973). Refolding in vitro is generally efficient for small, single-domain proteins that bury exposed hydrophobic

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contrast, larger proteins composed out of multiple domains fold inefficiently, owing to the formation of partially folded intermediates, including misfolded states, that tend to aggregate.

Misfolding originates from intra-/intermolecular interactions between regions of the folding polypeptide chain that are inaccessible in the native state, which is driven by hydrophobic forces and interchain hydrogen bonding (Radford, 2000). These non-native states often expose hydrophobic amino acid residues and segments to the solvent and self-associate into disordered aggregates.

II.2.1 Protein folding in the cell

The situation in living cells is, in contrast to in vitro refolding conditions, much more complex. First, the cellular environment is extremely crowded with high concentrations of proteins, nucleic acids and other macromolecules, reaching a concentration of 200 to 300 mg/ml in an E. coli cell (Zimmerman and Trach, 1991). The resulting ‘excluded volume effect’ has several consequences for the physical properties of the intracellular environment, most important is that intermolecular interactions become strongly favored, which results in protein aggregation (Ellis, 2001). For an unfolded protein, this means that aggregation with other unfolded species can compete with its folding to the native state.

Second, about one third of newly synthesized proteins must be translocated across membranes to fulfill their functions. These proteins are targeted as nascent or loosely folded polypeptides to their translocation machinery, and only after reaching their intended destination they fold to their native states. Targeting is achieved primarily through the co-translational action of the trans-acting factor SRP (Driessen et al., 2001; Walter and Johnson, 1994), and through molecular chaperones like Hsp70 (Schatz and Dobberstein, 1996).

Third, all proteins are synthesized by the ribosome in a vectorial manner, from the N- to the C-terminus, implying that, as long as polypeptide synthesis proceeds, the folding information remains transient and incomplete. The exposure of extensive hydrophobic surfaces on the unfolded nascent chain would render them prone to intermolecular aggregation, particular considering the fact that numerous unfolded polypeptides are synthesized in close proximity to one another on ribosomes that are translating from a single mRNA (Brandt et al., 2009).

To cope with these dangers, cells have developed a sophisticated system of molecular chaperones, which assist folding of polypeptides already when they emerge from the ribosomal exit tunnel. They guide the folding of non-native proteins, usually through cycles of binding and release of hydrophobic segments.

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Along with molecular chaperones, nature has evolved folding helper enzymes, so called

‘folding catalysts’. Two classes of enzymes are responsible for catalysing slow steps in protein folding, namely the cis/trans conformational interconversion of certain peptide bonds of the polypeptide backbone by peptidyl-prolyl cis/trans-isomerases (PPIases) and thiol/disulfide exchange processes characterized by reshuffling of covalent bonds by the protein disulfide isomerases (PDIs). Due to accelerating slow refolding kinetics, by polypeptide restructuring, these folding helper enzymes avoid the accumulation of reaction intermediates that are prone to form non-native protein conformations (Schiene and Fischer, 2000).

II.2.2 De novo protein folding: co- vs. post-translational protein folding

Considering that newly translated proteins are synthesized in a vectorial manner, it raises the question of how and when folding of a polypeptide chain to its structure is achieved in living cells. Three different models can be envisioned, whereas they are not mutually exclusive.

The first model suggests that newly synthesized proteins may exclusively fold post- translationally after their release from the ribosome (Agashe et al., 2004). In this scenario the complete folding information is available but the prolonged exposure of numerous hydrophobic stretches of an unfolded intermediate, which have to be shielded by several chaperones, may enhance misfolding and aggregation.

The second model proposes that the formation of secondary and tertiary structure begins already co-translationally. In this folding mode, intermolecular off-pathway reactions are minimized, because hydrophobic side chains are separated from the cytosol immediately.

However, for some proteins with complicated folds that require long distance interactions within the polypeptide sequence, it might lead to unproductive folding events.

The third model suggests a stepwise co-translational folding, in which co-translational folding is initially delayed by chaperones, and is allowed to proceed only when sufficient sequence information is available for generation of a folded core or domain. This domain-wise folding could be advantageous for the folding of multi-domain proteins, because it reduces possible unproductive inter- and intramolecular interactions during early folding steps (Deuerling and Bukau, 2004; Netzer and Hartl, 1997).

Several in vitro and in vivo studies provide evidence that protein folding can start co- translationally during biosynthesis as soon as a polypeptide leaves the ribosomal exit tunnel (Frydman et al., 1999; Kolb et al., 2000). For example, the groups of Helenius and

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Glockshuber demonstrated co-translational folding in vivo by analyzing the folding of the Semliki Forest virus capsid protein (C protein) (Nicola et al., 1999; Sanchez et al., 2004). The C protein localized at the N-terminus of five proteins, which are translated as a polyprotein precursor. It contains a chymotrypsin-like domain, which, once folded to its native structure, acts in cis to cleave itself from the precursor. It was shown that this domain folds rapidly during translation, well before termination of polyprotein synthesis.

The absolute amount of proteins that fold co-translationally remains unclear. Two studies discuss fundamental differences that might exist between pro- and eukaryotic organisms in their capacity to fold proteins co-translationally, as shown for the artificial Ras-DHFR fusion protein (Netzer and Hartl, 1997) and firefly luciferase (Agashe et al., 2004). They propose that rapid and effective folding of multi-domain model proteins occurs co-translationally in the eukaryotic system, while the bacterial system lacks the capacity for folding these model proteins co-translationally.

There is a growing number of examples showing that the elongation speed by the ribosome affects co-translational protein folding. It can be envisioned that accelerated rates of translation allow a particular part of a polypeptide chain to appear earlier in time than usual, which leads to an interaction of this premature region with the preceding region that has not yet folded properly (Komar et al., 1999). Temporary pausing permit more efficient domain folding without complicating the peptide’s C-terminus. The importance of pausing has been demonstrated for the signal recognition particle receptor !-subunit. In this case, pausing facilitates targeting and binding of the receptor !-subunit to the endoplasmatic reticulum membrane (Young and Andrews, 1996).

It is conceivable that specific sequences in the mRNA may have co-evolved to cause transient translational arrest, as they can guide interactions with other proteins, regulate expression or facilitate membrane insertion (Murakami et al., 2004; Wolin and Walter, 1993).

Several molecular mechanisms have been reported to control the amounts of a particular protein and/or regulate its folding by affecting translation speed:

- Rare codons: asymmetric tRNA abundance can cause variation in the rate of translation of each single codon. The translation rate is maximized at codons with highly abundant cognate tRNA and minimized at codons read by rare tRNAs (Lavner and Kotlar, 2005; Zhang et al., 2009);

- mRNA secondary structures: stable secondary structures of the translated transcript have to be unwound by the active ribosome, which leads to translational slow down (Chaney and Morris, 1979);

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- Charge-specific interactions between the ribosomal exit tunnel and the nascent peptide: the electrostatic potential inside the tunnel is negative and varies in magnitude along the length of the tunnel. Positively charged arginine or lysine sequences produce transient pausing before the nascent peptide is fully elongated. The rate of conversion from transiently arrested to full-length nascent peptide is faster for peptides containing neutral or negatively charged residues than for those containing positively charged residues (Lu and Deutsch, 2008; Lu et al., 2007).

Since molecular chaperones which mainly support protein folding in the cell, are the main topic of this work, the next chapter will give an broader overview of this protein family.

II.2.3 Molecular chaperones

The in vivo conditions are not beneficial for many proteins to support their spontaneous folding. Therefore, all cells have a set of molecular chaperones. These proteins assist folding and prevent the aggregation of misfolded proteins during de novo protein synthesis as well as under stress conditions. The fact that the level of many chaperones is elevated under heat shock conditions lead to the term ‘heat shock protein’ (Hsp) (Ellis, 1987). Heat shock proteins exist in several evolutionary conserved families, which are named according to their apparent molecular weight of a typical member, e.g. Hsp110, Hsp100, Hsp90, Hsp70, Hsp60 or Hsp40.

The Hsps can be grouped into three functional categories: holder chaperones, folder chaperones and disaggregases.

The holder chaperones prevent unfolded proteins from interacting with each other by binding to them and, hence, counteract protein aggregation. Typical examples for this functional group are the small heat shock proteins (e.g. IbpA and IbpB in E. coli, Hsp26 in human) (McHaourab et al., 2009), the Hsp90 family members (Buchner, 1999) or members of the J- domain/Hsp40 protein family (e.g. DnaJ in E. coli, Hdj-2 in mammals, Ydj1 in S. cerevisiae).

The folder chaperones have the unique feature to actively assist folding processes. Members of this group typically possess an ATPase domain because this function requires energy. Two well studied examples are the Hsp60 chaperones, also called chaperonins (e.g. GroEL in E.

coli, TRiC in eukaryotes), and the Hsp70 protein family (e.g. DnaK in E. coli; Hsp70, Hsc70 and BiP in mammals; Ssa and Ssb in S. cerevisiae) (Bukau and Horwich, 1998).

The chaperones with disaggregating activity are able to dissolve already formed protein aggregates to release polypeptide chains for refolding or degradation. Examples for this

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They are able to resolubilize protein aggregates in cooperation with members of the Hsp70 family (Glover and Lindquist, 1998; Mogk et al., 1999).

During the last 25 years much insight into the biochemical mechanisms of chaperones from different structural families has been gained. Furthermore, it is becoming increasingly clear that the different chaperone proteins cooperate with each other in living cells, providing a well-developed network of chaperone pathways that can handle polypeptides at all stages of folding.

The importance of chaperones in promoting and maintaining native conformation of cellular proteins is underscored by the toxic consequences of protein misfolding and aggregation. In several neurodegenerative disorders, including Parkinson’s and Huntington’s disease, the accumulation of protein aggregates accompanies neuronal death in specific brain regions, which leads to irreversible neurological symptoms of these disorders (Dobson, 2003;

Gregersen et al., 2006).

In the following, the Hsp70 chaperone family and its regulators will be described in more detail, because they are involved in nearly all cellular processes.

The Hsp70 family

The Hsp70 family is a ubiquitous and versatile group of molecular chaperones. Hsp70 homologues are found in almost every organism. Certain functional and structural hallmarks characterize this group of proteins:

- Hsp70 proteins consist of an N-terminal ATPase or nucleotide binding domain (NBD) and a C-terminal substrate binding domain (SBD);

- Hsp70 proteins bind to short hydrophobic stretches of their substrate proteins;

- Hsp70 proteins bind and hydrolyze ATP, which is directly coupled to their affinity for substrates;

- Hsp70 proteins interact with Hsp40 family members and nucleotide exchange factors;

Hsp70 family members participate in a number of cellular processes under normal conditions as diverse as folding of newly synthesized proteins, assisting translocation through membranes, activity control of regulatory proteins, disassembly of protein complexes and facilitating proteolytic degradation of certain substrate proteins (Hartl, 1996; Morimoto et al., 1994).

Hsp70 proteins belong to the group of foldases because they assist the folding of their substrates actively by repeated cycles of binding and release (McCarty et al., 1995; Schröder

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et al., 1993; Szabo et al., 1994). During this cycle they exist in two functional relevant states:

The ATP- and the ADP-state (Bukau and Horwich, 1998) (Figure 4).

Figure 4: Schematic illustration of the functional cycle of Hsp70 chaperones of the yeast cytosol.

In the ATP-bound state the affinity for substrates is low and the kon and koff for substrates are high. ATP hydrolysis changes the affinity for the substrate: The koff is decreased by three, the kon by only two orders of magnitude and the Hsp70 protein has a high affinity for substrates.

In general, the basal or substrate stimulated ATP hydrolysis rate is too low to be physiological relevant (Flynn et al., 1989; Jordan and McMacken, 1995; McCarty et al., 1995). Therefore, the chaperone cycle of Hsp70 proteins is modulated by co-factors that affect either ATP hydrolysis or exchange of ADP for ATP. The hydrolysis is stimulated by J-domain-harboring co-chaperones, whereas nucleotide exchange factors (NEFs) accelerate nucleotide release.

Rebinding of ATP and the concomitant dissociation of the NEF complete the exchange reaction (Figure 4).

The cytosol of S. cerevisiae contains two predominant classes of Hsp70s, Ssa (Ssa1-4) and Ssb (Ssb1-2). The Ssa chaperones are required for viability and are involved in housekeeping functions as well as stress-related protein folding processes (Werner et al., 1987). Ssb chaperones are dispensable for viability and appear to facilitate nascent polypeptide folding at the ribosome (Albanese et al., 2006; Pfund et al., 1998).

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J-protein Hsp40s

The J-domain (Hsp40) protein family is as ubiquitous as Hsp70 proteins but the family is much less conserved (Laufen et al., 1998; Sahi and Craig, 2007). All J-proteins stimulate the ATPase activity of their partner Hsp70s. The 65-aa J-domain, the defining feature of all J- proteins, is responsible for the stimulation. The J-domain is characterized by a highly conserved histidine–proline–aspartic acid (HPD) tripeptide signature motif that is important for J-domains’ stimulatory activity.

Hsp40 proteins act in the first step of the functional cycle of Hsp70s. They transfer substrates to Hsp70 and concurrently stimulate its ATP hydrolysis, resulting in a high affinity Hsp70 substrate complex. Presence of the substrate alone stimulates the basal ATPase rate of Hsp70 proteins only by two- to ten-fold, presence of substrate in combination with sub- stoichiometric amounts of Hsp40 stimulates by more than 100-fold (Laufen et al., 1999).

Despite the omnipresent J-domain, J-proteins are strikingly dissimilar, varying significantly in their domain organization and localization within the cytosol (Walsh et al., 2004).

In most cellular compartments, an Hsp70 works with multiple, structurally divergent J- proteins. The functional specificity of J-proteins and the complexity of the Hsp70:J-protein network is underscored by the fact, that in the yeast cytosol are about 13 different J-proteins, with the most abundant one named Ydj1 (Sahi and Craig, 2007).

Nucleotide exchange factors

Nucleotide exchange factors (NEF) exchange ADP for ATP. They promote the release of polypeptide, the last step in the functional Hsp70 cycle, by associating with specific conformations of the Hsp70 NBD that exhibit low affinity for ADP and ATP (Bukau and Horwich, 1998). Whereas Hsp40 proteins share a common J-domain as an interaction interface with the Hsp70 ATPase domain, the NEFs are evolutionary as well as structurally unrelated and it was shown that each NEF utilizes a unique mode of binding. They are classified into four groups:

- Homologues of the E. coli protein GrpE, which exist in eubacteria and organells of prokaryotic origin (Harrison et al., 1997);

- Eukaryotic BAG domain proteins (BAG, Bcl2-associated athanogene) (Höhfeld and Jentsch, 1997);

- HspBP1/Fes1 homologues found in eukaryotes (Dragovic et al., 2006b; Shomura et al., 2005);

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- Eukaryotic Hsp110/Grp170 family proteins (Shaner and Morano, 2007);

Interestingly, Hsp110s are themselves Hsp70-related chaperones (Easton et al., 2000).

Recently, it became clear that these members of the Hsp70 superfamily function as NEFs for canonical Hsp70 chaperones. The Hsp110 share their overall structure with canonical Hsp70s but differ by an extended SBD with an insertion of an acidic loop and an extended flexible C- terminus (Easton et al., 2000; Liu and Hendrickson, 2007). The crystal structure of the yeast Hsp110 Sse1 lends further support to the close relationship between Hsp70 and Hsp110 (Liu and Hendrickson, 2007). However, despite this similarity, Hsp110 proteins do not appear to be effective folding chaperones but rather act as NEFs for the cytosolic Hsp70 machinery. In vitro, Sse as well as Fes1 was shown to accelerate nucleotide exchange of the two yeast canonical Hsp70 chaperones Ssa and Ssb (Dragovic et al., 2006a; Dragovic et al., 2006b;

Raviol et al., 2006). S. cerevisiae harbors two highly homologous members of the Hsp110 family, the abundant Sse1 and the less expressed Sse2. SSE1 and SSE2 constitute an essential gene pair (Trott et al., 2005), but viability of null mutant cells can be restored by overexpression of the yeast HspBP1 homolog Fes1 or the Bag-domain of yeast Snl1 (Raviol et al., 2006; Sadlish et al., 2008). The complementation by well known NEFs and the fact that mutations abolishing Sse’s NEF activity are lethal, define nucleotide exchange as an essential function of Sse. Sse forms high affinity complexes with Ssa and Ssb, by binding to their NBDs, whereas the meaning of these stable complexes in the cell is still unclear (Polier et al., 2008).

II.3 Ribosome associated chaperones

Chaperones that assist the folding of cytosolic proteins can be classified into two subgroups according to their localization (Bukau et al., 2000; Frydman, 2001; Hartl and Hayer-Hartl, 2002; Wegrzyn and Deuerling, 2005). The first class is composed of soluble cytosolic chaperones that associate co- and post-translationally with newly synthesized proteins. They predominantly include members of the Hsp60 and Hsp70 families. As described before, the second class of chaperones assemble directly at the ribosomal exit tunnel. The main characteristics of the second category of chaperones are their attachment to the translation machinery and their interaction with the nascent polypeptide during protein biosynthesis. The coupling of protein biosynthesis with protein folding via ribosome-associated chaperones

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utilized by different organisms in this process are seemingly unrelated.

II.3.1 Ribosome-associated chaperones and de novo folding in the E. coli cytosol

In E. coli a network of molecular chaperones facilitates de novo protein folding (Figure 5).

The first chaperone interacting with the nascent chain is Trigger Factor, the only ribosome- associated chaperone of bacteria. Upon interaction with TF, some proteins fold without assistance from other chaperones, whereas many proteins, however, are aided by the downstream chaperone-systems DnaK and GroEL. DnaK, with its J-type co-chaperone DnaJ and NEF GrpE, is the most important member of the Hsp70 chaperone family in E. coli.

GroEL and its co-chaperone GroES belong to the Hsp60 and Hsp10 chaperone families, respectively. GroEL encloses substrates within a folding chamber in an ATP-dependent mechanism, offering a protected folding environment (Bukau and Horwich, 1998) (Figure 5).

Trigger Factor associates with ribosomes in an apparent 1:1 stoichiometry and, in contrast to DnaK and GroEL, TF is an ATP-independent chaperone but displays additionally peptidyl- prolyl cis-trans isomerase activity (Guthrie and Wickner, 1990; Lill et al., 1988; Stoller et al., 1995). This chaperone is not essential. However, simultaneous deletion of genes encoding TF (tig) and DnaK (dnaK) in E. coli leads to synthetic lethality at temperatures #30°C and to massive protein aggregation which suggests an overlapping function of both chaperone systems in de novo protein folding (Deuerling et al., 1999; Teter et al., 1999). The comparison of TF and DnaK substrate specificity demonstrated that both chaperones recognize similar hydrophobic stretches within unfolded proteins and DnaK was subsequently shown to associate with nascent chains in !tig cells (Patzelt et al., 2001; Schaffitzel et al., 2001). The fact that the overproduction of the GroEL/ES chaperone system complements !tig!dnaK induced phenotypes clearly demonstrated the robust chaperone network for efficient folding of newly synthesized polypeptides (Vorderwulbecke et al., 2004).

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Figure 5: De novo protein folding in the E. coli cytosol.

Trigger Factor is the first chaperone interacting with nascent chains. Some polypeptides fold spontaneously, whereas others are further assisted by the cytosolic Hsp70 (DnaK, DnaJ, GrpE) and Hsp60 (GroEL/S) chaperone systems.

Figure 6: Structure of Trigger Factor bound to the 50S ribosomal subunit. Full-length Trigger Factor, depicted as a ribbon model, positioned by superimposition onto the ribosome-bound fragment Trigger Factor 1–144 is shown together with a slice of 50S along the peptide exit tunnel with a modelled nascent chain in magenta, extending from the peptidyl transferase centre (PT). Taken from (Ferbitz et al., 2004).

Publication of the full-length E. coli Trigger Factor structure and of its ribosome-binding N- terminal domain in complex with the ribosome have opened the possibility to understand TF function on a molecular level (Ferbitz et al., 2004). The structure confirmed already existing data that L23 serves as ribosomal docking site of TF (Patzelt et al., 2002). Moreover, the structure revealed that TF hunches over the ribosomal exit tunnel and thereby orients its hydrophobic inner surface towards the emerging nascent polypeptide (Figure 6).

Based on this structural achievement, it was hypothesized that TF promotes co-translational folding of domains by providing a shielding environment at the tunnel exit. TF could

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accommodate a domain up to the size of 14kDa inside a defined folding space and would thus be supportive for productive co-translational de novo folding.

A recent study described the TF action on translating ribosomes as a dynamic reaction cycle involving certain structural rearrangements within the TF molecule (Kaiser et al., 2006).

These data support the idea, that TF functions by binding hydrophobic stretches of emerging nascent chains to prevent their aggregation but delays folding in doing so. Despite the already detailed insights of TF’s chaperone mechanism, it is still unclear weather TF promotes co- translational folding of nascent chains or rather keeps them in an extended conformation to delay folding.

II.3.2 Ribosome-associated chaperones and de novo folding in the yeast cytosol

Like in E. coli, eukaryotes locate molecular chaperones to the site of protein synthesis.

However, none of the proteins involved displays sequence homology to TF. It was therefore concluded that bacteria and eukaryotes have independently evolved chaperone systems for nascent chains (Craig et al., 2003).

The chaperone network in the model eukaryote S. cerevisiae is thoroughly studied and several proteins are known to bind to ribosomes and proposed to have a function in de novo protein folding. Two different systems have evolved, NAC (nascent chain-associated complex) and a Hsp70/40-based system, which is termed the Ssb/Ssz/Zuo chaperone triad in yeast. Both systems locate near the ribosomal tunnel exit and contact the emerging nascent chain (Figure 7).

Downstream chaperone systems of the Hsp70-family (Ssa) are also important in chaperoning nascent polypeptide chains and continue to assist in the folding of newly synthesized proteins together with the multimeric chaperonin TRiC (TCP1 ring complex, Hsp60-family).

Eukaryotic GimC (prefoldin-family), which is not found in bacteria, binds to a subset of nascent chains and newly synthesized proteins and cooperates with TRiC in their folding (Frydman et al., 1994; Hansen et al., 1999b; McCallum et al., 2000; Siegers et al., 2003;

Siegers et al., 1999; Yam et al., 2005).

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The nascent chain-associated complex (NAC)

NAC is highly conserved among eukaryotes and is a heterodimer composed of one !- and one

"-subunit (Figure 7), (Reimann et al., 1999; Rospert et al., 2002). Both subunits contain one central NAC-domain, with substantial homology to each other. Based on crystallographic data, raised from an archaeal !-NAC homodimer, it is believed that hetreodimerization occurs via the NAC domains (Spreter et al., 2005). NAC associates with ribosomes and nascent chains in an apparent 1:1 stoichiometry (Fünfschilling and Rospert, 1999; Raue et al., 2007;

Wiedmann et al., 1994). The "-subunit binds via a conserved RRK(X)nKK ribosome binding motif to the ribosomal protein L25 (ribosomal protein family L23) and attaches the entire complex to the ribosome (Beatrix et al., 2000; Wegrzyn et al., 2006). In yeast, NAC is encoded by three genes: !-NAC by EGD2 and the "-NAC subunit twice by EGD1 and BTT1, although BTT1 is significantly lower expressed than EGD1 (George et al., 1998; Reimann et al., 1999). NAC is not essential in yeast and deletion of NAC has no phenotypes. Only the presence of the !-subunit without its "-subunit is reported to show a mild growth defect at high temperatures in some strain backgrounds (Reimann et al., 1999).

Multiple potential functions of NAC are discussed in the literature. The !-subunit includes a

Figure 7: De novo protein folding network of yeast. NAC and/or Ssb, assisted by Ssz/Zuo, interact with nascent chains as ribosome-associated factors at the tunnel exit of the 60S ribosomal subunit. While some proteins fold spontaneously upon release from the ribosome, some require further assistance from downstream chaperones like Ssa (Hsp70), GimC (prefoldin) and/or TRiC (Hsp60).

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able to associate with ubiquitin chains is unclear. Due to existing data of co-translational degradation, it can be envisioned that NAC plays a role in co-translational protein quality control via the ubiquitin-proteasome pathway (Adachi et al., 2004; Chen and Madura, 2005;

Turner and Varshavsky, 2000). Based on in vitro data, a role for NAC in targeting new proteins into mitochondria and the ER has been suggested, which may propose a chaperone- like function of NAC (Beatrix et al., 2000; Fünfschilling and Rospert, 1999; George et al., 1998; Möller et al., 1998; Wiedmann and Prehn, 1999; Wiedmann et al., 1994). In humans, the intracellular levels of NAC vary dramatically in the context of severe diseases such as Alzheimers’s disease, Trisomy 21, AIDS and malignant brain tumors. A role for NAC in apoptosis was also proposed (Bloss et al., 2003; Kim et al., 2002; Kroes et al., 2000;

Scheuring et al., 1998). Based on the finding that NAC associates with translating ribosomes a function of NAC in de novo folding was proposed. However, due to the lack of in vivo data, NACs function in the cell remains obscure and a potential functional integration into the existing chaperone network could not described yet.

NACs function is probably diverse. Due to the fact that NAC is highly conserved among eukaryotes and mutations lead to embryonic lethality in mice, nematods and fruit flies its general significance is beyond controversy (Bloss et al., 2003; Deng and Behringer, 1995;

Markesich et al., 2000).

The Ssb/Ssz/Zuo chaperone triad

The second ribosome-associated, Hsp70/40-based chaperone system in yeast is composed of two Hsp70 members, namely Ssb and Ssz, and the Hsp40 co-chaperone Zuotin (Zuo) (Figure 7). Ssb is encoded by two genes (ssb1, ssb2), which are more than 99% identical (hereafter referred to as Ssb). Ssz and Zuo form the stable ribosome-associated complex termed RAC (Gautschi et al., 2001). Together, all these proteins form the so-called yeast chaperone triad.

The members of the yeast triad have been found to stably bind to ribosomes and thus been implicated in the folding of newly synthesized proteins (Gautschi et al., 2001; Nelson et al., 1992). Like NAC, Ssb shows an intrinsic affinity to ribosomes and can be crosslinked to short arrested nascent chains in vitro (Hundley et al., 2002; Pfund et al., 1998). The RAC complex is tethered to the ribosome by Zuo, which acts, with the assistance of Ssz, as an ATPase activator for Ssb (Huang et al., 2005). RAC stimulates ATP hydrolysis of Ssb thereby allowing Ssb to associate efficiently with the nascent chain.

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Unlike classical Hsp40/Hsp70 interactions, which are weak and transient, Zuo and Ssz exist in a highly stable complex. Interestingly, Ssz lacking its peptide-binding domain fully complements ssz! mutants, suggesting that, despite close sequence homology to canonical Hsp70s, Ssz does not function as a typical Hsp70 chaperone (Hundley et al., 2002). Instead, Ssz appears to modulate Zuo function in RAC through an unknown mechanism and this is required to stimulate Ssbs ATPase activity.

An importance of the triad for de novo protein folding has not been demonstrated directly, although some aspects support that the triad functions as a bona fide chaperone system:

- All triad members belong to classical chaperone families and thus they are by definition a chaperone system;

- Ssb interacts with nascent chains with its SBD, this is dependent on the stimulation by the J-domain of Zuo in RAC;

- The prokaryotic ribosome associated chaperone TF, when expressed in yeast, can partially substitute in vivo for the functions of the chaperone triad suggesting overlapping functions of these two systems (Rauch et al., 2005);

In keeping with the idea that all three proteins operate within one functional unit, deletion of each gene individually results in the identical set of phenotypes: slow growth, cold sensitivity and profound sensitivity to high salt conditions and to cationic aminoglycoside protein synthesis inhibitors, such as hygromycin B and paromomycin, that block protein synthesis and impair translational fidelity (Gautschi et al., 2002; Hundley et al., 2002). A possible explanation for the molecular basis for the observed phenotypes was presented by the Craig group. They showed that cells lacking components of the chaperone triad have an increased intracellular concentration of cations, including cationic aminoglycosides. Based on the fact, that strains having mutations in major K⁺-transporters share similar growth phenotypes, they conclude, that the major cause of the aminoglycoside sensitivity of cells lacking ribosome- associated molecular chaperones is a general increase in cation influx, perhaps due to altered maturation of membrane proteins (Kim and Craig, 2005).

For several years it was speculated, that fungi contain a specialized group of ribosome- associated chaperones that is not conserved in higher eukaryotes. But in 2005, human homologous of Zuo (Mpp11) and Ssz (Hsp70L1) could be identified, and complementation experiments indicate that the mammalian ribosome-associated complex is functional in yeast (Hundley et al., 2005; Otto et al., 2005), demonstrating the conservation of the entire RAC complex. It is interesting that mammalian RAC does not cooperate with ribosome-bound Ssb but works in concert with the cytosolic Hsp70-Ssa and may target cytosolic Hsp70s to nascent

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chains in higher eukaryotes (Hundley et al., 2005). These data are consistent with the fact, that no ribosome-bound Hsp70-Ssb ortholog has been detected in mammals yet.

Although, a modified system of ribosome-based chaperones exists in metazoans, the general principle is maintained from yeast to human: a ribosome-based Hsp40/70 system works on nascent chains during biosynthesis.

II.4 Aims of this work

Ribosome-associated chaperones form the interface between ribosomal protein synthesis and chaperone assisted protein folding. There is a need for early chaperone guidance, since nature evolved several molecular chaperones located at the ribosome assisting de novo protein folding from bacteria to humans. The bacterial Trigger Factor is the only and the best studied ribosome-associated chaperone. In eukaryotes the situation is more complex due to the existence of several different chaperones and systems positioned at the ribosomal tunnel exit to support early folding steps.

Therefore, this work aimed to better understand ribosome-associated chaperones in the model eukaryote Saccharomyces cerevisiae. In particular to get better insights into the complexity and robustness of the involved cellular chaperone network. Especially, the following aspects were in the main focus of this work:

1. In vivo functions of NAC and the Ssb/RAC-system in yeast (see III.1)

Two systems exist, NAC and Ssb/RAC, which associate with ribosome nascent chains. An important question to address was whether the two different chaperone systems functionally cooperate with each other? In a genetic survey, we investigated whether combined knockout mutations of NAC and Ssb/RAC show a synthetic defect or lethality and if these knockouts affect de novo protein folding.

To this end a detailed characterization of ssb!, nac! and ssb!nac! strains was performed to investigate the loss of these ribosome-associated chaperones and factors on the molecular level and to test for the consequences on protein folding.

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2. Analysis of potential functional cooperations between NAC and cytosolic chaperones (see III.2)

To better understand a potential role of NAC in the chaperone network, NAC should be tested for genetic interaction with downstream acting chaperones including the Sse-protein that acts as a nucleotide exchange factor for Ssb but also for Ssa. We investigated whether combined knockout mutations of NAC and Sse1 show a synthetic defect or lethality and if these knockouts affect de novo protein folding.

We combined knock-out mutations of all three genes encoding NAC subunits (nac!) with deletion of the SSE1 gene and characterized genetically and functionally the role of NAC in the chaperone network of yeast.

3. Structural characterization of yeast RAC (see III.3)

Finally, additional contribution should be made to better understand the Ssb/RAC system mechanistically. What is the molecular basis for the unusual stable pairing of the Hsp40 Zuo with its Hsp70-partner Ssz? Do the binding partners undergo any conformational rearrangements during heterodimerization? Which structural elements are involved in complex formation?

Supportive experiments were performed allowing to pursue Hydrogen-Deuterium-Exchange (HDX)-experiments in order to investigate the overall stability and conformational changes of Ssz and Zuo individually and in the RAC complex.

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III. RESULTS AND DISCUSSION

III.1 The in vivo functions of NAC and the Ssb/RAC chaperone triad in yeast

Title of the publication:

A dual function for the chaperones Ssb/RAC and nascent polypeptide-associated complex NAC on ribosomes

Koplin, A., Erhardt, M., Deuerling, E.

Submitted

Contributions

1. Experimental design

2. Generation of deletion strains - phenotypic characterisation - growth analyses

3. Design and cloning of yeast vectors for complementation experiments 4. Isolation and analyses of protein aggregates

5. Polysome profiling experiments

6. Quantification of ribosomes by western blotting 7. Preparation of all figures

8. Contributions to the writing of the manuscript

Motivation

In S. cerevisiae emerging nascent polypeptides encounter two conserved ribosome-associated chaperone systems: the NAC complex, in which both subunits are known to contact the nascent chain and the Hsp70/40-based Ssb/RAC system, in which the Ssb protein binds to the emerging polypeptide. While the Ssb/RAC system is per definition a bonafide chaperone system that is thought to be involved in the guidance of initial folding events. It was unclear whether NAC contributes to the folding of nascent chains. Moreover, it was unknown whether NAC and Ssb/RAC are functionally interconnected.

We were able to demonstrate for the first time that knock-out mutations in genes encoding Ssb/RAC have negative consequences on folding of newly synthesized proteins in vivo in a

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NAC dependent manner, which strongly supports a chaperone-like function of NAC in the complex chaperone network of the eukaryotic cytosol. Moreover, we discovered a second function of Ssb/RAC and NAC in regulating the amount of 60S and 40S ribosomal particles suggesting a profound role in the biogenesis of ribosomes.

Synergistic growth defects of cells simultaneously lacking NAC and Ssb

In order to investigate whether the two ribosome-associated systems NAC and Ssb/RAC (Figure 7) are interconnected and cooperate in the chaperone network, we combined knock- out mutations of all three genes encoding NAC subunits (egd1!, btt1! and egd2! referred hereafter as nac!) with deletions of chaperone triad, the two Ssb genes (ssb1! and ssb2!

referred hereafter as ssb!), Zuo (zuo!) and Ssz (ssz!). Cells carrying the nac!ssb! knock-out combination were viable, however, they showed a severe synthetic sickness. At 30°C nac!ssb! cells grew significantly slower than nac! or ssb! cells as judged by their colony size on plates (Figure 8A) and their extended doubling time in liquid cultures (Figure 8B).

The induction of protein folding stress conditions by the application of low concentrations of drugs such as the arginine analog L-canavanine or the translation inhibitor hygromycin B, resulted in a severe drop of cell viability of nac!ssb! cells as compared to control cells of wt, nac! or ssb! (Figure 8A). Hygromycin B reduces the translational fidelity by causing misreading of the mRNA and the incorporation of L-canavanine instead of arginine into cellular proteins prevents the proper folding of newly synthesized proteins and causes protein misfolding. Both drugs generate a large amount of substrate for the chaperone and/or ubiquitin-proteasome system.

The expression of authentic controlled wt NAC fully complemented the phenotype of nac!ssb! back to the character of ssb! cells, while the expression of a NAC-RRK/AAA ribosome-binding mutant did not. This provided evidence that the observed phenotype is specific for NAC and critically depends on its ribosome association. To demonstrate that the phenotype of nac!ssb! cells is related to all members in the chaperone triad, nac! mutations were combined with a zuo! and a ssz! mutation. These knock-out combinations resulted in similar synthetic defects as compared with nac!ssb! cells (Figure 8A and data not shown).

To exclude clone or strain specific defects the observed synergistic phenotype was reproduced in another strain background (data from M. Erhardt).