In this part of the study the mechanism and molecular details underlying ribosomal interaction of the specialized Hsp70 chaperone Ssb were elucidated. Besides its contacts with the ribosome-associated co-chaperone RAC and the nascent polypeptide, binding of Ssb to the ribosome is multilayered involving direct interactions with the translation machinery. These interactions are mediated by two Ssb-specific regions characterized by positively charged amino acid side chains. The key contact to ribosomes is made by 13 amino acid residues (601-13) of the CTD, a second contact with lower affinity is provided by a KRR motif within the SBD of Ssb (Fig. 28).
Figure 28: Model of multilayered interactions of the Hsp70 Ssb at the ribosomal exit site. Ssb possesses two intrinsic ribosome-binding sites: basic amino acid side chains within residues 601-13 of the C-terminal domain (CTD) mediate a strong key contact (black/orange) to the ribosome, KRR (428-30) of the substrate-binding domain represents a second, low affinity attachment point which might serve for correct positioning of Ssb at the ribosomal exit site. Further indirect ribosomal contacts of Ssb are mediated by binding of the nascent polypeptide (brown) and by a transient J-domain interaction with Zuotin (Zuo) that forms the RAC complex together with Ssz.
Findings of this study resolve long-standing open questions, as the issue of ribosome binding of Ssb has been addressed several times. Results of earlier studies were puzzling rather than providing a molecular understanding of Ssb’s ribosomal interaction. Previous analyses revealed only the necessity of at least two out of three domains (NBD, SBD, CTD) of Ssb for ribosome binding. Ssa-Ssb domain chimeras were used to demonstrate that constructs expressing two domains of Ssb have residual ribosome-binding capacity; the presence of the CTD of Ssb had the strongest impact on migration of chimeras with polysomes (JAMES et al., 1997). However, a mechanistic and detailed understanding of this phenomenon was lacking. Moreover, this result was curious given the fact that other ribosome-associated chaperones such as Trigger Factor in bacteria or the yeast Hsp40s Zuo1 and Jjj1 involve discrete ribosome-binding domains (HESTERKAMP et al., 1997; KASCHNER et al., 2015).
Since a direct interaction of Ssb with different kinds of nascent polypeptides could already be shown (PFUND
et al., 1998; GAUTSCHI et al., 2002; HUNDLEY et al., 2002; WILLMUND et al., 2013), it was unexpected that the ribosomal interaction of Ssb is not sensitive to ATP treatment (PFUND et al., 1998). Findings of this study now provide an explanation of all these findings, identifying two conserved modules specifically found in Ssb that are involved in a direct ribosomal interaction. The deletion or substitution of the positively charged Ssb residues 601-13 of the CTD showed the strongest impact on ribosomal interaction qualifying this region as a major contact site. The additional attachment via the KRR428-30 region within the SBD, which showed mild effects
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upon mutation, expands this interaction and likely serves for the correct positioning of the substrate-binding pocket of Ssb in close proximity to the ribosomal tunnel exit. It could be shown that both ribosome-binding regions of Ssb involve conserved positively charged amino acid side chains which likely interact with the negatively charged ribosomal RNA. This is in high agreement with earlier studies showing that Ssb bound to non-translating ribosomes could be stripped off with high salt (PFUND et al., 1998; RAUE et al., 2007), suggesting electrostatic interactions between the chaperone and the translation machinery.
Interestingly, blocking the autonomous binding of Ssb to ribosomes negatively influenced growth, ribosome biogenesis and translation rates only if RAC was missing in addition. This suggests a transient interaction of Ssb with the ribosome-nascent chain complexes mediated by its cofactor RAC, in addition to its direct ribosome attachment. Earlier studies showing a salt-resistant interaction of Ssb with RNCs, in contrast to the salt sensitivity of Ssb bound to vacant ribosomes (PFUND et al., 1998), support this finding of multilayered ribosomal interaction of Ssb. Thus, it can be hypothesized that Ssb, even if it lost its intrinsic ribosome-binding capability, is recruited from the cytosol to the nascent polypeptide by the cofactor RAC. Thus, the ribosome-binding mutant Ssb1#601-13 pictures the scenario of higher eukaryotic cells where e.g. mammalian mRAC recruits cytosolic Hsp70 to promote co-translational protein folding (JAISWAL et al., 2011). This suggested Ssb recruitment is further supported by earlier studies showing that Ssa as a cytosolic Hsp70 can partially complement the ssb1,2!
phenotype. In the absence of Ssb, Ssa transiently interacts with ribosomes via the nascent chain (YAM et al., 2005) or by binding translation initiation factors (HORTON et al., 2001). The functionality of Ssa at the ribosome in an ssb1,2! strain can be improved if Ssa is directly bound to ribosomes as an Ssb chimera (JAMES et al., 1997), if Jjj1 (that stimulates ATP hydrolysis of Ssa but not of Ssb) is overexpressed to recruit Ssa more efficiently to the ribosome (MEYER et al., 2007), or if the mammalian Zuotin homologue Mpp11 (that functions together with Ssa but not with Ssb) is overexpressed in a zuo1! background (HUNDLEY et al., 2005; OTTO et al., 2005; JAISWALet al., 2011). Nevertheless, Ssb is not just an Ssa variant with the additional ability to bind autonomously to ribosomes, as the specific interaction with cofactors, especially with RAC, plays an important role in the differentiation of the two Hps70 families. For that reason, recruitment of Ssa to the ribosome only partially complements the ssb1,2! phenotype, because a functional cooperation with yeast RAC is impossible in the case of Ssa or any mammalian Hsp70 (JAISWAL et al., 2011). Interestingly, mammalian RAC can partially complement an ssz1!zuo1! yeast strain, but complementation is even better if Ssb is missing in addition, indicating a more efficient interaction of mRAC with the cytosolic Hsp70 Ssa (JAISWAL et al., 2011). This proves that in principle another Hsp70-RAC or Hsp70/40 system can take over the function of the yeast Ssb-RAC triad.
Still, the question remains why the interaction of Ssb with the ribosome is not essential, especially as it was shown that other ribosome-tethered chaperones such as Trigger Factor or NAC are only functional when ribosome binding is maintained (KRAMER et al., 2002; KOPLIN et al., 2010; GAMERDINGER et al., 2015; OTT
et al., 2015). The same holds true for the Ssb cofactor Zuotin which critically needs ribosome attachment for in vivo functionality (YAN et al., 1998; PEISKER et al., 2008; ZHANG et al., 2014). Experiments of this study unambiguously proved that ribosome binding per se is not crucial for the overall function of Ssb. Moreover, it seems that this specialization was lost during evolution as mammalian mRAC interacts with cytosolic Hsp70.
Thus, it can only be speculated that for fungi like yeast with a high translation rate and fast cell division it seems to be beneficial to optimize its co-translational protein folding capacity by tethering Ssb directly to the ribosome.
Discussion & Outlook (A)
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Another important result of this study is the in vitro interaction of Ssb with several kinds of model peptide substrates observed for the first time. This interaction is characterized by a very low affinity, at least in the absence of cofactors, but similar substrate specificity compared to other Hsp70 chaperones. So far it was not understood why Ssb does not show any association with potential substrates in solution (e.g. PFUND et al., 2001) but binds to nascent polypeptides in the context of the ribosome (GAUTSCHI et al., 2002; HUNDLEY et al., 2002;
WILLMUND et al., 2013). Studies using Ssa-Ssb domain chimeras revealed that differences within the SBD are not required for any of the specific functions, suggesting that substrate specificity plays little or no role in determining the in vivo functional differences of the two Hsp70 families (JAMES et al., 1997). In addition, in silico MD simulations that were part of a cooperation of this study confirmed that binding of Ssb to canonical Hsp70 model substrates should be feasible in general. In this study, binding of Ssb to several canonic Hsp70 substrate peptides could be detected in vitro with different experimental approaches, although binding was characterized by a very low affinity and a binding curve that was not saturable. This might be the reason why earlier studies failed to monitor this interaction as either too low protein or peptide concentrations were used or the affinity of Ssb for peptides was compared with that of other Hsp70s and overestimated. However, the high concentration of unfolded proteins at the ribosomal tunnel exit might not need a high substrate affinity of Ssb. In contrast, strong binding of the nascent chain might even be counterproductive for the downstream acting cytosolic chaperone systems to which Ssb might hand over the unfolded polypeptide. Another reason for this low affinity binding to substrates could be a holdase function of Ssb for newly synthesized proteins, similar to the well-characterized Trigger Factor (KRAMER et al., 2004). In fact, expression of Trigger Factor could partially alleviate the aminoglycoside sensitivity of an Ssb-RAC! strain, indicating overlapping functions of the two chaperone systems (RAUCH et al., 2005). Low affinity for substrates might prevent strong and unintended binding of ribosome-detached Ssb to cytosolic substrates, e.g. upon release of a newly synthesized polypeptide.
In turn, efficient substrate interaction might only be possible in the environment at the ribosomal tunnel exit providing a high substrate concentration and the presence of RAC. This is underlined by the fact that Sse1, which promotes substrate release of Ssb (YAM et al., 2005), associates with the Hsp70 predominantly off the ribosome (WILLMUND et al., 2013), suggesting that Ssb releases substrates upon ribosome detachment.
In conclusion, low substrate affinity could represent a means to turn on efficient polypeptide binding of Ssb only in the context of the ribosome and to allow substrate release or hand-over to downstream chaperone systems upon dissociation of Ssb from the translation machinery.
Based on the findings in this part of the study, important open questions remain that should be addressed in the future:
(i) Which are the direct contact sites of Ssb at the ribosome? Ssb likely binds in close proximity to the cofactor RAC and the nascent chain since interactions with both could be observed (PFUNDet al., 1998; PFUND et al., 2001; GAUTSCHI et al., 2002). A specific crosslinking approach of the two ribosome-binding regions of Ssb might help to identify the ribosomal contact sites. This kind of analyses could also resolve the structure of ribosome-attached Ssb, e.g. with cryo-EM or X-ray crystallography which was, however, difficult up to now (Nenad Ban lab, ETH Zürich, personal communication). Particularly interesting in that context would be the comparison of ribosome-attached to free Ssb to understand possible differences of both states.
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(ii) How and when does Ssb detach from the ribosome? It is tempting to speculate that substrate binding and ATP hydrolysis induce conformational changes of Ssb that promote detachment from the ribosome, e.g. by movement of the lid towards the substrate binding pocket. A similar mechanism is described for Hsp70s of cellular organelles like the ER (BiP, Kar2 in yeast) or mitochondria (mtHsp70, Ssc1 in yeast). In both cases Hsp70s interact with the translocation machinery and detach from their partners in these complexes upon nascent polypeptide interaction and in dependency of their specific cofactors (MATLACK et al., 1999; LIU et al., 2003). It can be suggested that a similar mechanism leads to ribosomal detachment of Ssb upon nascent polypeptide interaction and RAC-stimulated ATP hydrolysis.
(iii) Why is the fusion of the CTD of Ssb to GFP or FLAG not sufficient for their targeting to ribosomes? It could be shown in this study that the very C-terminus of Ssb mediates the key contact to ribosomes. However, ribosome binding seems to be multilayered and involves at least a second attachment point in the SBD as well as indirect ribosome binding via the nascent chain and RAC. Therefore, it might not be sufficient to just fuse the high affinity contact site of Ssb to a non-ribosomal protein for ribosome targeting. Further experiments could focus on the cytosolic Ssa and mutate not only the C-terminus but also introduce the KRR motif into this Hsp70.
Although it is not clear whether the resulting chaperone mutant would be able to cooperate with RAC, efficient ribosome binding might be detectable in this scenario.
(iv) Does Ssb contribute to folding of its substrates in the context of the ribosome? Although it has been shown that Ssb interacts with canonical Hsp70 model substrates, the question remains whether it represents a chaperone that promotes nascent chain folding. As an ATP- and cofactor-dependent protein it is rather unlikely that Ssb functions as a holdase, however, the low substrate affinity makes also this consideration conceivable. A combination of in vivo and in vitro experiments monitoring correct folding of model substrates like luciferase or other enzymes could help to finally prove a chaperoning function of Ssb. Furthermore, this approach could be used to monitor whether Ssb-promoted folding occurs in the context of the ribosome or also after ribosome detachment. The question of substrate release in the cytosol or within other cellular compartments could be addressed in combination with the open issue whether cytosolic substrate binding occurs.
(v) What is the exact interplay of Ssb and its cofactor RAC at the ribosome? It is known that RAC stimulates ATP hydrolysis of Ssb (HUANG et al., 2005) and regulates its substrate specificity for nascent chains (WILLMUND et al., 2013). However, the molecular mechanism underlying this interplay is unclear so far. It might be possible that ribosome attachment and interaction with Zuotin changes the low affinity of Ssb for its substrates to a more efficient one, likely initiated by structural rearrangements upon cofactor interaction. Further experiments need to be done to understand the functional interplay between Ssb and RAC and the molecular basis of this interaction.
Results (B)
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4 Results, discussion and outlook (part B)
An important role of the yeast Hsp70 Ssb during nuclear maturation of ribosomal particles
Key results of the following chapter will be submitted as HANEBUTH et al. manuscript (see manuscript II;
appendix) and therefore contain data that were generated in cooperation with coworkers. However, the vast majority of results presented are part of this thesis. This includes creation of plasmids or strains as well as purification of proteins, pulldown of protein complexes or experimental design, data analysis, discussion of the results and manuscript writing.
Coworkers and contributions:
- Sandra Fries, Department of Biology, Molecular Microbiology, University of Konstanz (parts of microscopy) - Florian Stengel, Department of Biology, Cellular Proteostasis and MS, University of Konstanz (XL-MS)