The Ssb-RAC triad of Saccharomyces cerevisiae

In yeast the ribosome-associated complex (RAC), a stable heterodimer of the Hsp40 Zuotin (Zuo) and an unusual Hsp70, Ssz, functionally acts together with another ribosome-bound Hsp70, Ssb (Fig. 12) (GAUTSCHI

et al., 2001, 2002; PREISSLER &DEUERLING, 2012). Ssb is present in two interchangeable versions, Ssb1 and Ssb2 (referred to as Ssb hereafter), differing in only four amino acids (Fig. 14) that show no effect if deleted individually (CRAIG &JACOBSEN, 1985). RAC acts as a co-chaperone for the highly abundant Ssb by stimulating its ATPase activity (HUANG et al., 2005) and its interaction with nascent polypeptides (GAUTSCHI et al., 2002).

The complex itself is tethered to the ribosome via Zuo (GAUTSCHI et al., 2001). Zuotin bridges two important functional centers, the PTC and the ribosomal exit (PEISKER et al., 2008; ZHANG et al., 2014), thereby spatially connecting protein translation with de novo folding. Yeast cells lacking parts of or the entire Ssb-RAC system display a similar pleiotropic phenotype including slow growth, sensitivity towards high salt concentrations, aminoglycosides, translation inhibitory drugs, protein folding stress and low temperatures, indicating that they act as a functional triad (CRAIG &JACOBSEN, 1985; NELSONet al., 1992; YAN et al., 1998; GAUTSCHI et al., 2002; HUNDLEY et al., 2002; KIM &CRAIG, 2005). In the absence of Ssb, and even more so upon co-deletion of NAC, several proteins become prone to aggregation, suggesting a general role of Ssb-RAC in de novo protein folding or in the prevention of aggregation (KOPLIN et al., 2010; WILLMUND et al., 2013).

According to its domain structure, Ssb represents a canonic Hsp70 with a highly conserved 42 kDa NBD, an 18 kDa SBD-beta and a 6 kDa flexible lid of the CTD (or SBD-alpha) (Fig. 14; PEISKER et al., 2010). The Ssb reaction cycle is driven by the co-chaperones RAC, which stimulates ATP hydrolysis and substrate specificity (GAUTSCHI et al., 2002; WILLMUND et al., 2013), and by NEFs like Sse1, Sln1 and Fes1 (SONDERMANN et al., 2002; SHANER et al., 2005; YAM et al., 2005; DRAGOVIC et al., 2006).

Figure 14: Domain structure of the yeast Ssb-RAC system and mammalian homologues. The yeast chaperone triad is formed by the Hsp70 Ssb1,2 (green), the Hsp70 Ssz1 (dark cyan) and the Hsp40 Zuo1 (light cyan), the latter two forming the ribosome-associated complex (RAC). Mpp11 (light cyan) is the mammalian Zuo1 homologue, Hsp70L1 (dark cyan) corresponds to Ssz1. Members of the Hsp70 family possess a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). Hsp40s are characterized by an N-terminal domain (N), a J-domain (J) required for stimulating the ATPase of Hsp70s, a charged region (CR) involved in ribosome binding and, in case of Mpp11, two SANT domains. Asterisks within Ssb indicate the four residues differing between Ssb1 and Ssb2; the nuclear export sequence (NES) (according to SHULGA

et al., 1999) is indicated.

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Ssb is expressed at the same level as ribosomal proteins (RAUE et al., 2007), migrates with translating ribosomes (NELSON et al., 1992), and binds in a RAC-dependent manner to 66 % of all ribosome-nascent chain complexes, indicating a rather broad impact on co-translational protein folding (WILLMUND et al., 2013). Ssb can be crosslinked to different kinds of nascent chains in vitro, even to short ones exposing only 12 residues outside the ribosomal tunnel exit (PFUND et al., 1998; GAUTSCHI et al., 2002; HUNDLEY et al., 2002; ALBANESE et al., 2006). This suggests binding of Ssb close to this region of the ribosome, however, all attempts to solve the structure of ribosome-bound Ssb failed so far. Surprisingly, Ssb did not bind to any classic Hsp70 substrate peptide in earlier in vitro studies (PFUND et al., 2001). In addition, there is currently no evidence for Ssb playing a role in co-translational or general protein folding. However, it was shown that more nascent chains get ubiquitinated in the absence of Ssb (ALBANESE et al., 2006), whereby translation fidelity is unaffected (KIM & CRAIG, 2005). A deeper insight into the ribosomal substrate interaction of Ssb has been obtained in a study in which mRNAs that are being translated by Ssb-bound ribosomes were sequenced (WILLMUND et al., 2013). Thereby it was shown that Ssb binds a large fraction of newly synthesized polypeptides and that RAC regulates the specificity of this interaction. Interestingly, Sse1, which promotes substrate release of Ssb (YAM et al., 2005), associates with the Hsp70 off the ribosome suggesting that substrate binding and release by Ssb is spatially regulated (WILLMUND et al., 2013). Ssb preferentially interacts with ribosomes translating nuclear or cytosolic proteins and, in contrast to NAC, Ssb does not bind SRP substrates, indicating early sorting of nascent chains (WILLMUND et al., 2013). This fits with an analysis of the RAC translatome that indicated a broad and Ssb-independent binding of the complex to translating ribosomes, but a reduced interaction with RNCs exposing a signal sequence (RAUE et al., 2007). The Ssb translatome represents polypeptides that are longer than average and show properties that significantly challenge folding, such as enhanced aggregation propensity, regions of intrinsic disorder and complex domain architectures (WILLMUND et al., 2013). In accordance with that, the absence of Ssb leads to misfolding and aggregation of a number of different proteins (KOPLIN et al., 2010; WILLMUND et al., 2013).

A global analysis of the cellular chaperone network revealed that, unlike to other cytosolic chaperones, Ssb-RAC is transcriptionally co-regulated with the translational apparatus (LOPEZ et al., 1999; ALBANESE et al., 2006). In contrast to NAC or Ssb, the ratio of RAC to ribosomes is substoichiometric (RAUE et al., 2007) and very low expression levels of Ssz and Zuo are sufficient for proper growth (HUNDLEY et al., 2002). As already described, deletion of each part of the Ssb-RAC system leads to a similar pleiotropic growth defect, without a strong additive effect upon deletion of more than one component (HUNDLEY et al., 2002). Interestingly, the growth defects of ssz1! cells can be partially complemented by Ssb overexpression (GAUTSCHI et al., 2002), with a crucial role of the SBD in this substitution (CONZ et al., 2007). Similar effects were observed for Zuo overexpression (GAUTSCHI et al., 2002). In contrast, overexpression of any component of the Ssb-RAC system in ssb1,2! or zuo1! strains was not able to restore growth (GAUTSCHI et al., 2002). Remarkably, the heterologous expression of TF in Ssb-RAC! cells partially rescues the aminoglycoside sensitivity of this strain, whereas the cold sensitivity remains unchanged (RAUCH et al., 2005). This might indicate overlapping functions of ribosome-associated factors of different kingdoms in de novo protein folding but also indicates additional functions of the Ssb-RAC system off the ribosome.

Ssz represents an unusual Hsp70 variant as it is essential for the activity of RAC in vivo and in vitro but does neither require its peptide binding domain (HUNDLEY et al., 2002) nor the ability to bind or hydrolyze ATP

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(CONZ et al., 2007). It shares only 26 % identity with Ssb (PFUND et al., 2001) and diverges markedly from other Hsp70s as it contains an unusually short SBD lacking the C-terminal lid (Fig. 14). Crucial catalytic residues in elements involved in ATP hydrolysis are mutated in Ssz but the environment of the bound adenosine is conserved, suggesting ATP binding and, thus, a constantly low affinity for potential substrates (LEIDIG et al., 2013). The in vivo function of Ssz remains therefore enigmatic, and it is speculated that Ssz functions as a NEF for Ssb or serves as a scaffold to keep Zuo in an active conformation. Alternatively, Ssz might act as a low-affinity holdase that guides the growing nascent chain from the exit tunnel towards Ssb.

The RAC complex is connected to the ribosome via Zuo, whereas Ssz shows no direct ribosomal interactions (GAUTSCHI et al., 2002). The complex is highly stable, insensitive to nucleotides or salt (GAUTSCHI et al., 2001) and its formation strongly decreases the conformational dynamics in the C-terminal domain of Ssz and the N-terminal segment of Zuo (FIAUX et al., 2010). Deletion of the highly flexible N-terminus of Zuo (Fig. 14) abolishes association with Ssz in vitro and causes a phenotype resembling the loss of Ssz in vivo. The N-domain of Zuo contacts the SBD of Ssz as well as parts of the NBD leading to their mutual stabilization within the RAC complex (FIAUX et al., 2010). Zuo is a class III Hsp40 chaperone which in addition to its N-domain also possesses a J-domain (Fig. 14) that is essential (GAUTSCHI et al., 2002) and required for the stimulation of ATP hydrolysis by Ssb (HUANG et al., 2005). A C-terminal charged region of Zuo is involved in ribosome binding (YAN et al., 1998; PEISKER et al., 2008), together with other regions of the protein. In contrast to other J-domain proteins no interaction of Zuo with unfolded polypeptides has been detected. Furthermore, Zuo requires a stable interaction with Ssz to efficiently act as a co-chaperone (GAUTSCHI et al., 2001; PEISKER et al., 2008).

RAC is rather elongated in solution, whereas it appears to be bent when bound to the ribosome (LEIDIG et al., 2013). On the ribosome, the rRNA expansion segment ES27 seems to control the conformational state of RAC at the tunnel exit as part of an allosteric mechanism. Structural analysis identified a four-helix bundle within the C-terminus of Zuo wherein helix 1 mediates the main ribosomal contact (LEIDIG et al., 2013). Another study further analyzed the ribosome dissociation of Zuo and unfolding of the C-terminal four-helix bundle, which leads to a nuclear activation of the Pdr1 transcription factor involved in pleiotropic drug resistance (DUCETT

et al., 2013). Ssz, when not ribosome-associated, is also able to activate Pdr1 (HALLSTROM et al., 1998). It is therefore likely that both RAC components act together during this activation (DUCETT et al., 2013).

A recent study revealed the cryo-EM structure of the yeast RAC-80S ribosomal complex (ZHANG et al., 2014).

It showed that Zuo has at least three different contact sites with the 60S and 40S subunit thereby crosslinking the ribosome via a long and essential alpha-helix localized in the charged region (CR) (Fig. 14). This bridging limits intersubunit rotation and ratcheting of the ribosome during elongation, which raises the possibility that RAC couples nascent chain folding with translational speed (ZHANG et al., 2014). Ribosomal contacts of Zuo include already described ribosomal proteins: contact 1 (C1) lays within the N-terminal domain and binds to eL22, a protein forming the tunnel constriction, and the rRNA helix H59 (similar to LEIDIG et al., 2013); C2 positioned within the CR mediates a second 60S subunit contact via eL31 (similar to PEISKER et al., 2008; LEIDIG et al., 2013) and rRNA helix H101, whereas C3, also located within the CR, bridges to the 40S subunit via binding of ES12 of the 20S rRNA (ZHANG et al., 2014). This rRNA segment is directly connected with the decoding center of the ribosome allowing crosstalk of the PTC and the exit site via RAC. Several data are consistent with the hypothesis that Ssb-RAC is crucial for maintaining translational fidelity and nascent chain quality: It could be shown that loss of the Ssb-RAC system enhances read through of stop codons, while the overexpression of Ssb

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allows for their efficient recognition (RAKWALSKA & ROSPERT, 2004; HATIN et al., 2007). Furthermore, Ssb-RAC was shown to repress the translation of arrested polylysine proteins by stabilizing corresponding RNCs (CHIABUDINI et al., 2012). Zuo contains an ubiquitin-binding domain which possibly recognizes poly-ubiquitinated nascent chains (RICHLY et al., 2010). Deletion of Ssb-RAC leads to inhibition of -1 programmed ribosomal frameshifts likely due to impaired chaperoning which provokes backup of the nascent chain within the tunnel and aa-tRNA mispositioning (MULDOON-JACOBS & DINMAN, 2006). Finally, investigations of mammalian cell systems indicate chaperone-dependent pausing of translation elongation upon proteotoxic stress or heat shock (LIU et al., 2013; SHALGI et al., 2013). In sum, it is likely that the structural composition of the Ssb-RAC system at the ribosome allows the connection of protein folding and quality control at the exit site with translational fidelity at the decoding center. Further analyses have to reveal whether this suggested crosstalk is present and possible in both directions and how exactly the Ssb-RAC system connects and regulates these processes.

The mammalian RAC complex (mRAC) consists of the Zuo homologue Mpp11 and the Ssz homologue Hsp70L1 (Fig. 12 and 14) (OTTO et al., 2005), while Ssb is restricted to fungi (PFUND et al., 2001). Mpp11 contains a C-terminal extension with two SANT domains. Phylogenetic analyses suggest that Zuo originates from an Mpp11-like ancestor which has lost these domains (BRAUN &GROTEWOLD, 2001). Whereas Hsp70L1 is active in combination with Zuo, Mpp11 function is not supported by Ssz but by Ssa1, the homologue of mammalian Hsc70, indicating similarities of Hsp70L1 rather with Ssa than with the unusual Ssz (HUNDLEY

et al., 2005; OTTO et al., 2005). Knockdown of Mpp11 in mammalian cells results in hampered growth and sensitivity towards aminoglycosides, similar to what is observed for yeast cells lacking RAC (JAISWAL et al., 2011). As none of the human Hsp70 homologues is bound to ribosomes, mRAC partners with cytosolic Hsp70 but not with its close homologue Hsc70 (JAISWAL et al., 2011), that can both act on nascent chains (BECKMANN

et al., 1990; THULASIRAMAN et al., 1999). In conclusion, yeast and mammalian cells have evolved different solutions to ensure that combinations of Hsp40 and Hsp70 chaperones can efficiently assist the biogenesis of newly synthesized polypeptides.

Ssb was identified more than 25 years ago (SLATER &CRAIG, 1989) and a role in de novo protein folding in addition to other cellular functions was suggested several times. Yet, only few studies describe in vivo functions of this Hsp70. Ssb is unable to complement the loss of Ssa proteins, implying distinct and non-overlapping functions of the two Hsp70 families (BOORSTEIN et al., 1994). Differences within the SBDs of Ssa and Ssb are not required for either of the specific functions, suggesting that substrate interaction plays only little or no role at all in determining the differences of the two (JAMES et al., 1997). These differences are rather defined by the interplay with cofactors and their localization within the cell, as e.g. only Ssb but not Ssa binds to ribosomes (JAMES et al., 1997). Zuo is the only Hsp40 that stimulates ATP hydrolysis by Ssb (CYR &DOUGLAS, 1994;

GAUTSCHI et al., 2002) whereas Sse1 functions as a NEF for both Ssa and Ssb (YAM et al., 2005). Interestingly, the level of Ssa interacting with nascent polypeptides increases in the absence of Ssb (YAM et al., 2005) implying again similar substrate pools. The NBD and the ATPase function of Ssa and Ssb have different kinetic properties, which are governed by the respective CTDs that differ mostly between Hsp70s (LOPEZ-BUESA et al., 1998). This is surprising, as the sequences of the NBDs are highly conserved, yet, Ssb has an unusually low

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steady-state affinity for ATP and a higher maximal velocity of ATP hydrolysis than Ssa or classic DnaK-like Hsp70s (LOPEZ-BUESA et al., 1998).

Analysis of Ssa-Ssb domain chimeras showed that any two of the three Ssb domains (NBD, SBD, CTD) are necessary and sufficient for binding to ribosomes (JAMES et al., 1997). The interaction of Ssb and ribosomes is enhanced by RAC, stabilized during active translation, reduced upon chain release, not affected by ATP, and only the binding to vacant ribosomes is salt-sensitive (PFUND et al., 1998; GAUTSCHI et al., 2002; HUANG et al., 2005). However, which exact regions of Ssb and the ribosome interact remains unclear, as well as whether this interaction is crucial for the in vivo function of Ssb. Investigation of these open questions will therefore be part of this study (see chapter 3).

Another open question is the suggested role of Ssb-RAC during biogenesis of ribosomes. Several studies indicate a function in particular for Ssb and Zuo off the ribosome during ribosome assembly. This is in line with that loss of either Ssb or Zuo results in the aggregation of mainly ribosomal proteins and biogenesis factors and causes deficiencies in the production of ribosomal particles and culminating a decline in the level of active translation (ALBANESE et al., 2010; KOPLIN et al., 2010). Ssb1,2! cells show reduced levels of ribosomal proteins and mature ribosomal subunits and an accumulation of unprocessed rRNAs in insoluble protein aggregates (NELSON et al., 1992; KOPLIN et al., 2010). Interestingly, Ssb-RAC is functionally connected to the second ribosome-associated system, NAC, as their combined deletion shows a synergistic growth defect and a severe enhancement of protein aggregation and defective ribogenesis (KOPLIN et al., 2010; OTT et al., 2015).

Furthermore, the expression of Ssb correlates with that of ribosomal proteins with an increase of synthesis upon carbon upshift (LOPEZ et al., 1999), suggesting a role of Ssb in ribogenesis. Although various data indicate a functional role of Ssb-RAC in ribosome assembly, the mechanism by which they control ribogenesis is still unknown. A recent analysis revealed the presence of Zuo on nuclear pre-60S particles and a hampered rRNA processing in zuo1! cells (ALBANESE et al., 2010). A genetic interaction of Zuo with the late biogenesis factor Jjj1 is described and the deletion of both causes decreased 60S levels, accumulation of ribosomal precursors and of 35S and 27S rRNA. As an Hsp40, Jjj1 stimulates ATP hydrolysis of Ssa (MEYER et al., 2007) but not of Ssb, however, it shows synthetic lethality also with the latter one indicating overlapping functions (KOPLIN et al., 2010). Overexpression of Jjj1 in zuo1! or ssb1,2! cells partially rescues the growth defect of these strains (MEYER et al., 2007; ALBANESE et al., 2010) and, interestingly, even a nuclear Jjj1 mutant partly substitutes for Zuo (ALBANESE et al., 2010). However, a contradicting study claims that the main function of Jjj1 and Zuo, which both contain a zuotin-homology domain involved in ribosome binding, is in the cytosol and on the ribosome (KASCHNER et al., 2015). In conclusion, further studies are needed to fully understand the role of Ssb-RAC during cytosolic and nuclear ribogenesis and open questions concerning this function will be addressed in this study (see chapter 4).

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2 Aims of this thesis

Synthesis and processing of new polypeptides involves a plethora of ribosome-associated proteins like molecular chaperones or targeting factors. Nascent polypeptides must fold into defined three-dimensional structures; others need to be transported to different cellular compartments like the nucleus or the endoplasmic reticulum. Proteins that are part of large macromolecular assemblies need to be kept in an unfolded conformation until their stable incorporation into protein complexes is achieved. In all kingdoms of life various ribosome-associated factors directly interact with nascent chains and guide their folding, stabilization or targeting. In the yeast S. cerevisiae two systems assemble at the ribosomal exit site, an Hsp70/40-based system, Ssb-RAC, and the nascent polypeptide-associated complex (NAC). Many aspects concerning the precise function or molecular mechanism of these ribosome-associated factors are still unknown. The major aim of this thesis was therefore to investigate key functions of ribosome-associated factors on and off the ribosome to elucidate their role in the cellular proteostasis network.

(A) Investigation of the ribosomal interaction and substrate binding of the S. cerevisiae Hsp70 Ssb

The yeast ribosome-associated Hsp70 Ssb was already object of many analyses that tried to unravel the cellular function of this chaperone, its substrate interaction and the molecular basis underlying its ribosomal interaction.

Former studies using domain chimeras of Ssb and the cytosolic Hsp70 Ssa tried to elucidate ribosome binding of Ssb and the properties of its substrate recognition. Results from these investigations only proved that any two of the three Ssb domains (NBD, SBD, CTD) are necessary and sufficient for ribosome binding, however, substrate interaction of this Hsp70 has never been observed. This is puzzling, as canonic Hsp70 chaperones possess a well-defined binding motif with which they recognize their substrates. Furthermore, most ribosome-associated factors of pro- and eukaryotes contact the ribosome via precisely identifiable binding regions. It was therefore a main goal of this part of the thesis to investigate ribosome binding of Ssb and its interaction with substrates in more detail to understand the discrepancies of former analyses on a molecular basis.

Important questions were:

Which domains and residues of Ssb are involved in ribosome binding? Is ribosome binding crucial for the in vivo function of Ssb? How important is the interplay with RAC? What is the reason for the lacking data of substrate interaction in the case of Ssb? What are the substrates of Ssb and how are they recognized?

To address these questions, potential ribosome-binding mutants of Ssb were generated based on sequence alignments with the non-ribosome-attached Ssa, and characterized in vivo and in vitro. Furthermore, different in silico modeling and in vitro binding approaches were used to investigate the interaction of Ssb with potential model substrates.

(B) Investigating the role of the S. cerevisiae Hsp70 Ssb during synthesis of new ribosomal particles

Experimental evidence indicates a function of yeast Ssb and its cofactor RAC off the translational apparatus and during ribosome assembly. Recent findings emphasize this role especially in the case of the Hsp40 Zuotin for

Aims of this thesis

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which an impact on nuclear synthesis of ribosomal RNA and ribosomal subunits was observed. This suggests a similar role for the functionally cooperating Hsp70 Ssb. It is known that in the absence of Ssb ribosomal proteins as well as ribosome biogenesis factors aggregate, which results in decreased amounts of ribosomal subunits and translation rates. It is unclear, however, whether these defects result from reduced amounts of mature and

which an impact on nuclear synthesis of ribosomal RNA and ribosomal subunits was observed. This suggests a similar role for the functionally cooperating Hsp70 Ssb. It is known that in the absence of Ssb ribosomal proteins as well as ribosome biogenesis factors aggregate, which results in decreased amounts of ribosomal subunits and translation rates. It is unclear, however, whether these defects result from reduced amounts of mature and