Our results provide the first in vivo evidence that NAC is functionally connected to the Hsp70 chaperone network. NAC genetically interacts and functionally collaborates with SSB–RAC and Sse1 in the folding of newly synthesized proteins. Moreover, we discovered a second and novel function of SSB–RAC and NAC in regulating the abundance of ribosomal particles, suggesting an important role for these ribosome-associated systems in the biogenesis of ribosomes.
The finding that NAC associates with ribosomes and interacts with the nascent chain previously evoked the hypothesis that this complex might be involved in cotranslational folding processes (Bukau et al., 2000
; Frydman, 2001
; Hartl and Hayer-Hartl, 2002
). However, this assumption was contentious for quite some time because of the lack of any in vivo phenotype correlated with protein folding or chaperone networks. In this study, we now provide in vivo evidence that NAC is functionally connected to the chaperone network that assists the folding of newly synthesized proteins in the eukaryotic cytosol. Deletion of NAC alone has no detectable consequences for the growth of yeast cells or the de novo folding of newly synthesized proteins. In contrast, deletion of NAC in the absence of the Hsp70 chaperone SSB or any other member of this Hsp70/40 chaperone system impaired growth at 30°C and resulted in a severe drop of cell viability on plates containing hygromycin B or L-canavanine. Additionally, the double mutant showed enhanced aggregation of newly synthesized proteins (). Thus, the phenotype of NAC becomes apparent only in absence of SSB–RAC, suggesting that NAC and the Hsp70/40 system work in overlapping or parallel pathways during protein folding and are able to functionally collaborate with each other. The fact that only ribosome-associated NAC can complement the phenotype, but not a NAC mutant deficient in ribosomal attachment, demonstrates that NAC cooperates with SSB–RAC exclusively on ribosomes (). In the absence of NAC, the function of SSB–RAC is sufficient to serve as a potent backup system. The finding that NAC contributes to the chaperone network was further emphasized by the genetic interaction suggested by the deletions of genes coding for NAC and Sse1, an NEF regulating both the SSB and SSA types of Hsp70s in the yeast cytosol.
How may NAC contribute to the folding of newly synthesized proteins? According to independent cross-linking studies, it is known that NAC acts very early on nascent polypeptides with a length of ~17 amino acids in vitro, whereas SSB cross-links to nascent chains of a length of ~58 amino acids, suggesting that NAC may act first, albeit comparative cross-linking studies have not been performed yet (Wang et al., 1995
; Pfund et al., 2001
). Moreover, it has been demonstrated that NAC protects nascent chains against proteolytic attack (Wang et al., 1995
). Such a protective function as described for NAC may promote the efficient interaction of newly synthesized proteins with the ribosome-associated SSB–RAC chaperone system as well as with downstream-acting chaperones of the SSA type. Whether NAC itself displays a canonical chaperoning function with the capacity to prevent aggregation and to promote folding of unfolded proteins awaits further detailed biochemical analysis.
The most intriguing new finding of this study is that the loss of ribosome-associated chaperones SSB and NAC strongly affected the levels of ribosomal subunits and translating ribosomes, which is likely the result of a defect in ribosome biogenesis. Deletion of SSB alone already caused a pronounced reduction of ribosomal particles; however, this defect was enhanced in the absence of NAC, suggesting a collaborative action of both chaperones for this function as well. A role of SSB and NAC in ribosome biogenesis is supported by several different lines of evidence: (a) ribosomal proteins together with some ribosomal biogenesis factors and RNA are major constituents of the aggregates isolated from nacΔssbΔ
cells, (b) ribosomal profiling and Western blot analysis revealed significantly decreased levels of 60S and 40S subunits, the formation of ribosomal half-mers, and a severe drop in actively translating 80S moiety and polysomes. Moreover, (c) cells lacking SSB and NAC accumulate ribosomal L25-GFP in the nucleus, and finally, (d) the combined deletion of SSB and Jjj1, which is described to have an active role in late steps of ribosome biogenesis of the 60S subunit, caused cell death (Demoinet et al., 2007
; Meyer et al., 2007
). Interestingly, recent work by Sahi and Craig (2007)
showed that overexpression of Jjj1 can in part complement the phenotype of a zuoΔ
deletion strain. They also showed that Hsp40 Jjj1 does stimulate the ATPase activity of SSA but not of SSB, which suggests that the Jjj1–SSA and SSB–RAC chaperone systems operate as defined chaperone systems in ribosomal biogenesis.
What might be the mechanistic basis for the role of SSB and NAC in ribosome biogenesis? Different models are plausible, which are not mutually exclusive. Our finding that ribosomal biogenesis factors and ribosomal proteins, which are required for the biogenesis of the small and large subunits, are constituents of the aggregates that accumulate in nacΔssbΔ
cells suggests that these ribosome biogenesis factors and proteins are among the major clients of SSB and NAC (). SSB and NAC may bind to ribosomal client proteins immediately upon their synthesis to prevent misfolding and perhaps even accompany these aggregation-prone proteins until they are engaged in ribosome assembly. Loss of SSB and NAC hampers de novo folding and activity of ribosomal proteins and biogenesis factors, thereby leading to a deficiency in the production of ribosomal particles (). Thus, the function of SSB and NAC in chaperoning nascent chains of ribosomal proteins and biogenesis factors would directly translate into consequences for ribosome biogenesis. Such a scenario would explain all defects observed in nacΔssbΔ
cells, including the finding that the level of both subunits is decreased in these chaperone-deficient cells. Interestingly, a recent study suggests that the N-terminal ubiquitin moiety of the ribosomal protein Rps31 serves a role as chaperone to facilitate the correct folding and assembly of Rps31 into 40S particles (Lacombe et al., 2009
). Besides Rps31, another ribosomal protein, Rpl40, is fused to a ubiquitin moiety in yeast. Remarkably, both proteins were not found among the major aggregation-prone species identified in the aggregation analysis of nacΔssbΔ
cells. Moreover, a very similar finding was recently described for the ribosome-associated chaperone trigger factor in bacteria. Trigger factor was suggested to bind to newly synthesized ribosomal proteins and thereby to facilitate the biogenesis of ribosomal complexes (Martinez-Hackert and Hendrickson, 2009
Figure 6. Model indicating that NAC and SSB–RAC link chaperone-assisted de novo protein folding with the production of ribosomes. NAC (yellow/red) and SSB–RAC (pink/green) associate with ribosomes and contact nascent polypeptides during synthesis (more ...)
Another possibility is that SSB–RAC and NAC display prime functions in ribosome biogenesis distinct from their chaperone function for nascent polypeptides. It is assumed that SSB–RAC and NAC bind transiently to ribosomes and cycle on and off the translation machinery (). This would allow them to serve other functions in the cell as well, e.g., promoting a distinct step of ribosome assembly before the particles are activated for translation. Interestingly, the aggregates isolated from cells lacking SSB and NAC contain several nuclear ribosome biogenesis factors (e.g., Dbp3, Dbp8, and Nog1) and perhaps premature nuclear 35S and/or 27S rRNA, which indicates that some of the aggregates originate from the nucleus. This finding, together with the observation that Rpl25-GFP accumulates in the nucleus of nacΔssbΔ
, suggests that the loss of SSB and NAC causes defects already during early steps of ribosome biogenesis in the nucleolus. Indeed, a study in this issue by Albanèse et al.
provides evidence for a role of SSB–RAC in assisting ribosome biogenesis in the nucleus.
Finally, SSB and NAC might contribute to the regulation of ribosomal particles at the transcriptional level. During preparation of this manuscript, von Plehwe et al. (2009)
reported about the involvement of SSB in glucose signaling via the SNF1 kinase network. The authors suggested that SSB, by an unknown mechanism, keeps SNF1 kinase in a dephosphorylated state in the presence of high glucose levels. Because phosphorylated SNF1 kinase negatively regulates transcription of ribosomal components, the loss of SSB may cause enhanced SNF1 phosphorylation, thereby reducing ribosome production indirectly at the transcriptional level. However, we found no difference in SNF1 phosphorylation in ssbΔ
cells compared with wt cells, and all strains responded similarly well to low glucose levels leading to enhanced SNF1 phosphorylation (Fig. S1 B). Thus, the global defect in ribosome biogenesis in cells lacking SSB and NAC detected in this study is not primarily caused by a dysfunction of SNF1 dephosphorylation under the growth conditions used in our analysis. It may well be that the different yeast strain backgrounds used by von Plehwe et al. (2009)
and in our study may account for the variations in SNF1 signaling.
Cells lacking both ribosome-associated systems are viable under normal growth conditions but show slow growth accompanied by misfolding and aggregation of ~2% of newly synthesized protein. However, very low concentrations of drugs that interfere with protein synthesis or folding caused lethality in cells lacking SSB and NAC, emphasizing the profound impact of these ribosome-associated chaperones on cell physiology and cell viability.
Interestingly, the loss of SSB and NAC did not provoke the induction of a heat shock response at permissive temperature (Fig. S1 B). Loss of SSB and NAC does, however, cause a severe decrease in the amount of ribosomal particles and, consequently, a decrease in the level of translating ribosomes. Ribosome production is one of the most energy-consuming processes in cells and is assumed to be tightly regulated by multiple environmental and physiological cues. This study describes a new cellular link between ribosome production and the function of ribosome-associated chaperone systems (). This link puts forward the attractive concept that the level of ribosome production for protein synthesis can be matched with the protein-folding capacity of ribosome-associated chaperones. We speculate that the adjusted level of actively translating ribosomes in cells lacking SSB and NAC may allow cells to cope with the loss of chaperone function and thereby reestablish protein homeostasis unless additional stress is applied (e.g., by drugs interfering with protein folding). How SSB and NAC may act as sensors for the folding capacity of the cell and how they probe ribosome production accordingly awaits further analysis.