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

Kolplin A., Preissler S., Ilina Y., Koch M., Scior A., Erhardt M., Deuerling E.

J Cell Biol. 2010 Apr 5;189(1):57-68.

Contributions

1. Quantitative analysis of polysome profiles

2. Analysis of association of biotin-labeled ribosomes with aggregates 3. Analysis of aggregate ubiquitination

4. Discussion of experiments and the manuscript

5. Contributions to manuscript writing and figure preparation

Abbreviations: RAC, ribosome-associated complex; NAC, nascent polypeptide-associated complex

4.6.1. Objective

In yeast, Ssb-RAC and the nascent polypeptide-associated complex (NAC) bind dynamically to ribosomes and contact nascent polypeptide chains (see introduction and Figure 8).

Whereas only Ssb interacts directly with nascent chains, Ssz and Zuo form the heterodimeric ribosome-associated complex (RAC), which acts as a co-chaperone for Ssb by stimulating its ATPase activity (Huang et al, 2005). This suggests a function of the Ssb-RAC system in the folding of newly synthesized proteins. In contrast, the role of NAC is very little understood.

NAC is a highly conserved complex, consisting of an α- and a β-NAC subunit, which do not belong to any of the classical chaperone families. Although both subunits contact nascent chains, only β-NAC interacts physically with the ribosome (Beatrix et al, 2000; Wiedmann et al, 1994). However, genetic deletion of NAC in yeast causes no obvious phenotype, making it difficult to investigate its in vivo function. Several studies proposed that NAC contributes to the regulation of protein translocation into the endoplasmatic reticulum or mitochondria (Del Alamo et al, 2011; George et al, 1998; Wiedmann et al, 1994). Based on the ability to interact with nascent polypeptides, a chaperone-like function of NAC is also conceivable, yet there is only little experimental support available for this hypothesis. We thus intended to analyze whether NAC is functionally connected to Ssb-RAC and the cytosolic chaperone network of yeast cells.

4.6.2. Summary of the experimental data

To investigate the functional relationship between the two ribosome-associated chaperone systems NAC and Ssb-RAC, yeast knockout strains lacking both systems individually (nacΔ and ssbΔ) or in combination (nacΔssbΔ) were generated and subjected to phenotypic analyses (Figure 26A). The nacΔssbΔ cells showed a significant synergistic growth defect and were hypersensitive to drugs, which affect translation or protein folding. These data indicate a genetic interaction between NAC and Ssb, and suggest that both systems act together in protein biogenesis. Interestingly, the nacΔssbΔ phenotypes could be suppressed by the expression of wt NAC from a plasmid, whereas expression of a ribosome-binding deficient NAC variant (NAC-RRK/AAA) had no curative effect (Figure 26A). This indicates that the ability of NAC to associate with ribosomes is crucial for its function in collaborating with the Ssb-RAC system.

Next, we focused on the role of NAC and Ssb-RAC in de novo protein folding. We indeed could show that newly made proteins aggregated specifically in ssbΔ cells and this effect was even stronger in nacΔssbΔ mutants. Moreover, when we isolated the insoluble proteins out of yeast cells using a quantitative assay, we observed severe accumulation of aggregated proteins in cells lacking Ssb or both, Ssb and NAC (Figure 26B). In either case the aggregated species were very similar, although aggregation was again more pronounced in nacΔssbΔ cells. Analysis of the aggregated proteins by mass spectrometry identified predominantly ribosomal proteins and ribosome biogenesis factors, suggesting that these proteins were especially prone to aggregate in the absence of ribosome-associated chaperones. Importantly, control experiments showed that ribosomal particles did not cosediment nonspecifically with aggregates formed in the knockout cells, showing that ribosomal proteins are specific components of the insoluble fraction. We also tested whether ribosomal proteins aggregate when the functionality of other chaperone systems is impaired.

Therefore, we analyzed protein aggregation in cells lacking Sse1, which acts as a nucleotide exchange factor for several Hsp70s, such as Ssb and Ssa1-4, to reduce the overall folding capacity of the cytosolic Hsp70 network. Deletion of the SSE1 gene resulted in aggregation of a different set of proteins, which was enhanced by the additional loss of NAC (Figure 26B).

From this we conclude that NAC has a function within the cytosolic Hsp70 system. In agreement with this finding, nacΔsse1Δ cells showed a synthetic growth defect. However, we identified mainly metabolic enzymes and only few ribosomal proteins in aggregates from nacΔsee1Δ mutants. In addition, a significant subpopulation of the insoluble proteins prepared from cells lacking Sse1 was polyubiquitinated, indicating that these proteins have

Results and discussion

polyubiquitination signals were detected in the aggregate fractions of ssbΔ or nacΔssbΔ mutants. From this we conclude that largely distinct sets of proteins aggregate in cells devoid of Ssb or Sse1, and that the aggregation of ribosomal proteins and ribosome biogenesis factors is specific for the loss of Ssb.

Figure 26: NAC cooperates with cytosolic chaperone systems. A) Growth analysis of mutant yeast cells.

Serial dilutions of cells were spotted on synthetic complete medium without uracil for plasmid selection. Drugs were added as indicated. When the cells were plated on the arginine analog L-canavanine, arginine was omitted from the medium. The cells were incubated at 30°C for three days. NAC-wt indicates cells expressing NAC from a plasmid under the control of its authentic promoter, whereas NAC-RRK/AAA indicates the expression a ribosome-binding deficient variant. As a control, cells were transformed with an empty plasmid (vector). B) Protein aggregates were isolated quantitatively from wt and chaperone mutant cells. The cells were grown to the exponential phase in YPD medium. Upon cell lysis, the insoluble proteins were extracted and separated by

SDS-ssb∆

lacking Sse1. Yeast cells were grown to the exponential phase and protein aggregates were isolated as in (B).

The proteins were separated by SDS-PAGE and polyubiquitinated proteins were detected by Western blotting using a primary α-ubiquitin antibody (abcam, P4G7). The accumulation of polyubiquitinated proteins (*) in the insoluble fraction is specific for cells lacking Sse1.

Importantly, the translation activity was significantly reduced in cells lacking Ssb, and even more pronounced in cells lacking Ssb and NAC. We also detected reduced amounts of ribosomal subunits in the mutant yeast strains and obtained signals for ribosomal half-mers, which are indicative for defects in the biogenesis of ribosomes. These data indicate that deletion of Ssb and NAC reduces the levels of ribosomal particles and thereby affects the translation activity of the cell. In line with this observation, the knockout combination of Ssb and Jjj1, a ribosome biogenesis factor, was synthetically lethal, supporting a function of Ssb in ribosome assembly. Although deletion of NAC alone did not result in any significant phenotype, our results demonstrate that NAC acts synergistically with Ssb-RAC and the cellular Hsp70 chaperone system. Furthermore, Ssb and NAC play an important role in ribosome biogenesis and thereby control the amount of actively translating ribosomes (Figure 27).

Figure 27: Model of how NAC and Ssb-RAC may 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 to assist de novo protein folding. Ribosome biogenesis factors and ribosomal proteins are among the potential proteins, which depend on NAC and Ssb to fold into their active conformations and to support the assembly of ribosomal subunits. Additionally, NAC and Ssb bind to ribosomes in a dynamic manner and may also contribute to the maturation of ribosomal subunits during ribosome biogenesis. The dual function of NAC and Ssb-RAC in chaperone-assisted protein folding and ribosome biogenesis aligns protein synthesis and ribosome production with the folding capacity of ribosome-associated chaperones.

Results and discussion