We report here for the first time an interaction between the S. cerevisiae BAG domain protein Snl1 and the ribosome. In addition, we show that this novel interaction is conserved in the C. albicans Snl1/Bag-1 homolog, implying a conserved function. Snl1 is unique in being the only known membrane-anchored Hsp70 nucleotide exchange factor, and we found that ribosomes tightly associate with both the membrane-associated and soluble truncated Snl1ΔN isoforms. We therefore conclude that the attachment of Snl1 to the ER/nuclear membrane does not occlude the ribosome binding site and, as a corollary, that the localization of Snl1 at the membrane does not dictate ribosome binding. Using the same affinity purification approach, we determined that Snl1 was unique among the S. cerevisiae cytosolic NEFs in ribosome associations, as we did not detect binding with the Sse1, Sse2, or Fes1 protein. In contrast, all four factors readily associated with Hsp70, verifying that the addition of the FLAG affinity tag does not noticeably distort protein-protein interactions.
Fungi are unique in having a dedicated ribosome-associated Hsp70 (Ssb), in contrast to other cell types, which recruit a general cytosolic Hsp70 to assist in nascent-chain folding via an intermediate tether protein. However, our results suggest that the interaction of Snl1 with the ribosome is independent of its association with Hsp70 and its function as a NEF. First, we found that Snl1 binding to Hsp70 was salt resistant, whereas the binding to ribosomes was largely abolished at high salt concentrations (0.5 M KCl). Second, the genetic elimination of both Ssb1 and Ssb2 had no effect on the Snl1-ribosome interaction, and the addition of exogenous Ssb to purified Snl1-ribosome-Ssb complexes likewise did not reduce the Snl1-ribosome association. Third, we observed that a previously characterized mutant of SNL1
with amino acid substitutions shown to abolish Hsp70 interactions (E112A R141A) retained the ribosome association. Fourth, we identified the ribosome binding interface in Snl1 and localized it to residues 52 to 58 within the predicted helix 1 based on homology to mammalian Bag-1, distinct from the Hsp70-interacting residues located in helices 2 and 3. These interactions invite comparisons to the recruitment of Ssb by the ribosome-associated complex (RAC) composed of Ssz1 and Zuo1 in fungi and the recently demonstrated recruitment of cytosolic Hsp70 in mammals by the Zuo1 ortholog Mpp11 () (17
). In both cases, these ribosome-associated proteins serve two roles: (i) tethering an Hsp70 chaperone to the ribosome and (ii) stimulating Hsp70 cycling via the acceleration of its intrinsic ATPase activity. Ribosome-associated Snl1 would be predicted to fulfill both these roles, stimulating Hsp70 ATPase activity via its role as a potent nucleotide exchange factor. Genes encoding ribosomal or ribosome-associated proteins are frequently coregulated at the transcriptional level (23
). Using the Serial Pattern of Expression Levels Locator (SPELL) database, we found that SNL1
transcript levels correlated well with those of ribosomal protein genes when queried over all available data sets (20 of the top 50 gene hits) (see Table S3 in the supplemental material) (13
). These transcriptional data support our contention that Snl1 is a bona fide ribosome-associated factor and suggest that cells may regulate levels of Snl1 to match protein biosynthetic needs.
Fig 5 Hsp70 recruitment by ribosome-associated cofactors. Hsp70 protein chaperones are shown to be recruited to intact, translating ribosomes via interactions with ribosome-associated factors. Mpp11 and RAC (Zuo1) are J proteins, whereas Snl1 is a nucleotide (more ...)
Results from both mass spectrometry and immunoblot analyses indicate that Snl1 interacts with assembled ribosomes, as we detected associations with both large (60S)- and small (40S)-subunit proteins. The treatment of affinity-purified Snl1-ribosome complexes with the divalent cation chelator EDTA resulted in the release of the small subunit, while binding to the large subunit was maintained. We therefore favor a model wherein Snl1 associates directly with one or more large-subunit components via the lysine-rich region that we have identified. A surface-exposed motif composed of positively charged residues [RRK(X)n
KK] mediates the ribosome association by the β-subunits of the nascent-chain-associated complex (NAC) in yeast and mammals (41
). A similar charged interface is utilized for ribosome associations by the bacterial trigger factor (TF) (22
). Our identification of the lysine-rich region required for ribosome binding by Snl1 is thus consistent with previously described ribosome-associated chaperone systems. Based on homology to mammalian Bag-1, this region is predicted to form helix 1 of a three-helix bundle. In contrast, the ribosome binding sites within the NAC and TF lie within flexible loop domains. Structure determination of the Snl1 cytosolic domain will shed light on the precise geometry of the Snl1-ribosome interaction, which is of particular importance given the proximity of the Hsp70 binding domain within the BAG motif. In addition, sequence heterogeneity within the amino terminus of BAG domain-containing proteins may contribute to their functional diversification (37
). The stress-signaling kinase Raf-1 binds Bag-1 directly, and the binding sites for Raf-1 and Hsc70 on Bag-1 overlap, dictating that the two proteins interact with Bag-1 in a mutually exclusive manner (33
). Thus, the ability of a BAG domain to mediate binding to multiple partners is not without precedent.
How widespread is the interaction between BAG domain proteins and the ribosome? In this report, we demonstrate that the Snl1/Bag-1 homolog in the pathogenic fungus Candida albicans
is capable of binding the intact ribosome when expressed in S. cerevisiae
. Moreover, we verify that this homolog is membrane associated in C. albicans
via fluorescence microscopy utilizing a CaSnl1-GFP fusion. Interestingly, the genome of the fission yeast Schizosaccharomyces pombe
contains two predicted Bag domain-containing proteins, only one of which possesses a positively charged region. The human Bag-1 protein is expressed as different isoforms that arise from a single mRNA by alternative translation initiation (1
). The Bag-1 isoform used in our experiments was the 207-residue short isoform, Bag-1S. Sequence analysis of the medium and large isoforms, denoted Bag-1M and Bag-1L, respectively, revealed a short, positively charged sequence at their N termini that was absent from Bag-1S (see Fig. S1 in the supplemental material). It is therefore possible that cells may express distinct Bag-1 proteins with or without the capacity to associate with ribosomes.
What roles might Snl1 be playing as a ribosomal membrane tether? Given its location at the ER membrane, a role in the transport of proteins from the cytoplasm into the ER membrane or lumen can be envisioned. Because cotranslationally translocated proteins are recruited to the ER and the translocon machinery via the signal recognition particle (SRP) system, it seems unlikely that Snl1 would contribute to this pathway. In contrast, posttranslationally translocated proteins are released into the cytoplasm after synthesis and must be directed to the translocon for import. At least one posttranslationally translocated protein in S. cerevisiae
, pre-pro-α-factor (ppαF), also requires cytosolic Hsp70 (Ssa1) to be maintained in a translocation-competent state (4
). Therefore, the concurrent ER targeting of both ribosomes and Hsp70 via Snl1 would likely enhance translocation by increasing the local concentrations of both factors. In an analogous scenario, the mitochondrial inner membrane protein OxaI is required for the proper insertion of respiratory-chain complexes synthesized on matrix-localized ribosomes and interacts with these ribosomes via a positively charged carboxyl-terminal motif (19
). Thus, OxaI could likewise promote substrate protein maturation in a nonspecific manner via ribosome recruitment. The formal testing of such hypotheses is confounded by the lack of detectable functional phenotypes in cells lacking SNL1
. For example, a preliminary analysis of ppαF processing failed to demonstrate a major defect in snl1
Δ cells (data not shown). In addition, we were unable to detect synthetic genetic phenotypes when SNL1
was deleted in combination with RAC, NAC, SSB, or ER translocation mutants (see Fig. S2 and S3 in the supplemental material). Additionally, we were unable to recapitulate the synthetic phenotypes reported by a recently published high-throughput whole-genome screen (see Fig. S4 in the supplemental material) (3
). It is therefore likely that the yeast Bag-1 homolog plays a subtle or condition-specific role in protein biogenesis that is nevertheless important enough to be conserved across more than 250 million years of separation between S. cerevisiae
and C. albicans
. Revealing such a role may require unbiased genomewide and/or proteome-wide studies, as was recently done for the role of the NAC, which remained enigmatic for a number of years after its discovery and is still not completely resolved (7
). Snl1 has been linked to nuclear pore function through its identification as a high-copy-number suppressor; it is therefore possible that it may also be involved in ribosome biogenesis and export, a function recently attributed to Ssb and NAC proteins. Despite the intriguing discovery of the novel interaction between fungal Bag proteins and the ribosome, it is clear that significant additional work is required to understand the importance of this association. Nevertheless, our discovery of Snl1 as a possible bridge between translating ribosomes and Hsp70 represents an important step in an understanding of the biology of this family of proteins.