Genetic interactions of Jjj1 with Zuo1–Ssz1
The domain organization of Jjj1 bears striking similarities to that of Zuo1 (). Both proteins have an N-terminal J domain known to regulate the ATPase activity of Hsp70s (Mayer and Bukau, 2005
) and a K/R-rich, positively charged C-terminal domain, which, in the case of Jjj1, is flanked by two zinc fingers ( and Table S1). Alignment of Jjj1 and Zuo1 across species revealed an additional homology region that defines a novel conserved domain of unknown function that we called zuotin homology domain (ZHD; Fig. S1
). Notably, this domain is unique to Jjj1 and Zuo1 homologues, pointing to a shared and unique function for these J domain CLIPS.
A possible functional overlap between Zuo1 and Jjj1 was revealed by their genetic interactions (). Single deletion of either JJJ1
caused a slow growth phenotype, showing that both proteins are independently important for cell survival. The double-deletion Δzuo1
exhibited a strong synthetic growth phenotype, suggesting that these proteins also have partially redundant functions within the same process (; Forsburg, 2001
). Synthetic growth interactions were similarly observed between Jjj1 and Ssz1, the partner of Zuo1 in RAC (). Thus, the entire RAC interacts functionally with Jjj1, with expression of at least one of these J domain proteins critical for cell survival. Of note, the function of either protein requires the presence of a functional J domain (Fig. S2 A
), suggesting that their activity involves regulation of the downstream Hsp70s SSB (Huang et al., 2005
) and SSA (Meyer et al., 2007
). Indeed, the N terminus of Zuo1, containing its J domain (111–165) and the N-terminal extension upstream from the J domain (1–111; Fig. S3
), binds directly to Ssz1 and SSB. In contrast, the J domain of Jjj1 directly binds to and activates the ATPase of the related Hsp70 SSA (Fig. S3; Meyer et al., 2007
Although RAC and Jjj1 overlap functionally, these chaperones are not fully interchangeable. Δzuo1
, and Δssb1/2
cells are hypersensitive to hygromycin, whereas Δjjj1
cells are not (Fig. S2 C; Albanèse et al., 2006
). In addition, Zuo1 overexpression could not rescue the Δjjj1
phenotype (Fig. S2 C; Meyer et al., 2007
). Interestingly, although overexpression of Jjj1 could rescue the slow growth of Δzuo1
cells (Fig. S2 C), it could not suppress their hygromycin sensitivity phenotype (). Thus, Jjj1 and the RAC–SSB CLIPS have overlapping but distinct functions in a common pathway essential for cell survival.
Loss of either Jjj1 or RAC–SSB impairs 60S ribosome biogenesis
The genetic interaction between Jjj1 and the RAC–SSB complex was puzzling given their very different proposed functions. The RAC–SSB network is thought to function in the cytoplasm by binding nascent chains emerging from ribosomes (Pfund et al., 1998
; Gautschi et al., 2002
; Yam et al., 2005
). In contrast, Jjj1 is proposed to interact with the cytosolic protein Rei1 to assist the cytoplasmic recycling of ribosomal export factor Arx1 (Demoinet et al., 2007
; Meyer et al., 2007
). We next tried to elucidate how these CLIPS with apparently disparate functions may cooperate.
Insight into the shared function of Jjj1 and Zuo1 was provided by sucrose gradient analysis of doubly deleted Δjjj1Δzuo1
cells (). Loss of both chaperones led to a reduction in the levels of 80S ribosomes and translating polysomes (, top) as well as the appearance of a prominent extra peak between the 60S and the 80S ribosomal particles, which corresponded to the 66S particle, an intermediate in 60S ribosome biogenesis (, top). The 66S particle is normally nuclear and contains an immature 27S rRNA (for review see Tschochner and Hurt, 2003
). This precursor would normally complete its processing and maturation in the nucleus to yield the 60S ribosomal subunit that contains the 25S rRNA. The identity of the 66S extra peak was confirmed by Northern blot analysis of the sucrose gradient fractions for the wild-type (WT) and Δjjj1Δzuo1
cells using a probe specific for the 27S rRNA (, bottom). This analysis revealed the accumulation of immature 27S rRNA precursors in Δjjj1Δzuo1
cells compared with WT extracts, where the low levels of 27S rRNA were below the detection limit (, bottom). Notably, the 27S rRNA was also present in heavier fractions in the gradient, which is suggestive of association with polysomes or formation of higher order aggregated species. This experiment suggested that the shared function of Jjj1 and Zuo1 was in ribosome biogenesis.
Figure 2. Jjj1 and the CLIPS Zuo1, Ssz1, and SSB participate in ribosome biogenesis. (A) Joint deletion of JJJ1 and ZUO1 accumulates a 66S preribosomal particle containing the 27S rRNA precursor. (top) Lysates from WT and Δjjj1Δzuo1 cells were separated (more ...)
Previous sucrose gradient analysis of polysome profiles from Δjjj1
cells showed that they contain half-mers within the polysomes ( [arrows]; Demoinet et al., 2007
; Meyer et al., 2007
). These shoulders are hallmarks of defective 60S ribosomal subunit maturation: the lack of mature cytoplasmic 60S ribosomal subunits results in irreversible binding of abortive 48S preinitiation complexes on mRNAs (Demoinet et al., 2007
; Meyer et al., 2007
). Strikingly, the polysome profiles of both Δzuo1
cells also exhibited half-mers in the polysomal fractions (). These anomalies indicate that the absence of the RAC–SSB chaperones also lead to a defect in 60S biogenesis.
Role of Jjj1 and RAC–SSB CLIPS in 60S ribosome maturation, export, and Arx1 recycling
The results in suggested that Jjj1 and RAC–SSB are both required for normal 60S ribosome biogenesis. In eukaryotes, ribosome biogenesis begins in the nucleolus and proceeds through nuclear steps of rRNA processing and ribonucleoprotein assembly, each mediated by a different set of nucleolar and nuclear factors leading to the formation of mature 60S and 40S particles (, yellow and green; and Table S2
; for reviews see Fromont-Racine et al., 2003
; Tschochner and Hurt, 2003
; Strunk and Karbstein, 2009
). These ribosomal subunits are exported to the cytoplasm with the help of shuttling factors, such as Arx1 for the 60S subunit (, blue), and the final cytoplasmic maturation is mediated by several factors, such as Rei1 for the 60S subunit (, red). We next examined how loss of Jjj1 and the RAC–SSB system affects three aspects of ribosome biogenesis, namely rRNA maturation (), ribosomal export from the nucleus (), and Arx1 recycling (). When compared with WT cells, we observed a strong accumulation of the 27S rRNA precursor in Δjjj1
strains as well as in Δssz1 and Δssb1/2
(). No accumulation was observed in strains deleted for other cytosolic chaperones, such as the CLIPS Gim2 or the stress-inducible Hsp104 (). Deletion of a single SSA
Hsp70 homologue, SSA1
, did not affect this process (unpublished data), most likely because of the presence of four redundant SSA
genes in yeast (Frydman, 2001
Figure 3. Distinct effects of Jjj1, Zuo1, and SSB on 60S ribosomal export and Arx1 recycling. (A) Schematic representation of ribosome biogenesis of the 60S ribosomal subunit. Nucleolar ribosomal biogenesis factors are represented in yellow, nuclear ones in green, (more ...)
Defects in 60S ribosomal biogenesis often impair the export of the 60S particles to the cytoplasm, leading to their accumulation in the nucleus (Hurt et al., 1999
). Thus, we examine whether 60S ribosomal export to the cytosol was affected in Δjjj1
, and Δssb1/2
cells using ribosomal protein Rpl25-GFP as a 60S marker (; Hurt et al., 1999
) and Sik1-RFP as a nucleolar marker (; Sung and Huh, 2007
). As expected, most 60S ribosomal particles in WT cells are cytoplasmic (). At 25°C, Δjjj1
cells accumulated Rpl25-GFP in the nucleus (, arrowheads; and note yellow overlap with the red nucleolar marker Sik1-RFP). The nuclear export defect in Δjjj1
cells was less marked at 30°C (unpublished data). Rpl25-GFP also accumulated in the nucleus in Δzuo1
cells (, arrowheads; yellow overlap in nucleus) and even more dramatically in Δssb1/2
cells (, bottom, arrowhead).
Jjj1 is proposed to bind to Rei1 and facilitate the recycling of Arx1 back to the nucleus (Demoinet et al., 2007
; Meyer et al., 2007
). Consistent with this role, we found that Arx1 accumulated in the cytoplasm of Δjjj1
cells (, arrowheads). Surprisingly, Arx1 recycling was also impaired in Δzuo1
cells (, arrowheads). The defective recycling of Arx1 in these cells was also detected biochemically, as failure to dislodge Arx1 from cytoplasmic 60S subunits caused Arx1 to migrate with polysomes in both Δzuo1
cells (unpublished data). Interestingly, Arx1 recycling appeared unaffected in Δssb1/2
cells (), indicating that SSB is not required for Arx1 recycling. Collectively, these results indicate that both Jjj1 and the RAC–SSB network participate in the maturation of the 60S ribosomal subunit and normal 60S ribosomal export.
Domain contribution of Jjj1 and Zuo1 to subcellular localization and ribosome association
To understand how Jjj1 acts during ribosome biogenesis, we explored the contribution of its various domains to its association with ribosomes () and its function ( and Table S3
). Jjj1 has three clearly identifiable domains: an N-terminal J domain, the ZHD, and a C-terminal–charged domain flanked by two zinc fingers (). We initially examined how the various Jjj1 domains contribute to its association with ribosomal fractions (). Full-length WT Jjj1 comigrates with the 60S and 80S ribosomal fractions and to a lesser extent with polysomes (, middle [low expression plasmid] and bottom [chromosomal-tagged copy]; and Fig. S4 A
, high copy plasmid). These interactions were largely abrogated by deletion of the charged domain (Jjj1-ΔC; , middle and bottom; and Fig. S4 A). A functional J domain, required for binding to SSA (Fig. S3), was not required for association with the 60S/80S ribosomal particles (Jjj1-Jm; and Fig. S4 A), although we noticed a consistent loss of association with polysomes even at high expression levels (Fig. S4 A). Notably, deletion of the ZHD domain had the opposite effect than the Jm mutation on Jjj1 sedimentation: Jjj1-ΔZHD was more weakly associated with the 60S and 80S fractions and enriched in the polysome fractions (Jjj1-ΔZHD; and Fig. S4). This suggests that the ZHD domain contributes to Jjj1 association with a subset of 60S ribosomal particles, whereas the C-terminal–charged domain mediates the stable association with ribosomes and polysomes. Collectively, our results reveal a complex pattern of interactions between Jjj1 and ribosomes, whereby different domains have distinct contributions to its interaction with ribosomes at different stages. A similar domain analysis for Zuo1 also indicated that the charged domain is required for polysome association (unpublished data) as previously reported (Yan et al., 1998
Figure 4. Jjj1 is a conserved modular protein with distinct functional domains. (A) Domain mutants of Jjj1. Jm, mutated in the canonical HPD motif of the J domain; ΔZHD, lacks the ZHD; ΔC, lacks the C-terminal K/R-rich domain. Asterisk indicates (more ...)
Figure 5. A nuclear form of Jjj1 that does not bind Rei1 suffices to restore its function in ribosome biogenesis and Arx1 recycling. (A) Complementation of Δjjj1 by Jjj1 mutants. Cells expressing the Jjj1 variants were grown overnight at 30°C, and (more ...)
We next examined the effect of the various Jjj1 domains on its subcellular localization (; and Fig. S4, B and D). As described previously, full-length Jjj1 is largely cytoplasmic (, top; Meyer et al., 2007
). Similarly, Jjj1-Jm and Jjj1-ΔZHD were also cytoplasmic (). Surprisingly, the Jjj1-ΔC mutant showed a strong accumulation in the nucleus (, arrowheads). Thus, we searched for nuclear export signals (NESs; la Cour et al., 2004
) and identified a canonical NES at the C terminus spanning amino acids 409–419 (LQALQAELAEI). Disruption of the NES by a single leucine to alanine mutation in the first leucine (Jjj1-NESm) led to the nuclear accumulation of Jjj1-NESm, which is similar to Jjj1-ΔC (, arrowheads). Thus, Jjj1 is largely cytosolic under steady-state conditions but shuttles between the cytoplasm and nucleus. Interestingly, Jjj1-ΔC was more tightly colocalized with the nucleolar Sik1 marker than Jjj1-NESm (, compare bottom merge panels), suggesting that Jjj1-ΔC may be concentrated in the nucleolus. Strikingly, similar results were obtained for Zuo1-ΔC, which also concentrated in the nucleolus (). Thus, Zuo1 and Jjj1 can localize to the nucleolus, which is the site of ribosome biogenesis.
Because Jjj1 is shown to associate with Rei1 through its zinc fingers (Meyer et al., 2010
), we next examined the domains that mediate this interaction in both Jjj1 (, left) and Rei1 (, right). Cells expressing the indicated epitope-tagged Rei1 and Jjj1 domain variants were subjected to immunoprecipitation against one of the proteins followed by immunoblotting against the other (). As expected, the C terminus of Jjj1 was indeed required for interaction with Rei1. For Rei1, we find that the N terminus of Rei1, encompassing the first of the three zinc fingers of Rei1, mediates the interaction with Jjj1 (, right).
Nuclear Jjj1 can rescue all ribosome biogenesis defects in Δjjj1 cells
We next assessed the contribution of the Jjj1 domains to the Δjjj1 phenotype (; and Fig. S4, B and C). As expected, the slow growth phenotype was rescued by expression of WT Jjj1 (). The J domain mutant (Jjj1-Jm), which no longer interacts with the Hsp70 SSA (Fig. S4), does not rescue the Δjjj1 phenotype. Similarly, the ZHD domain (Jjj1-ΔZHD) is also required for Jjj1 function. Surprisingly, the slow growth phenotype is fully rescued by Jjj1 lacking the entire C-terminal–charged domain (Jjj1-ΔC; ; and Fig. S4, B and D). This is independent of the expression levels, as WT growth rates are also observed upon deletion of the C-terminal domain or the Zf2 zinc finger from the chromosomal copy of JJJ1, where Jjj1 is expressed at endogenous low levels (Fig. S4 C). Thus, Jjj1 function requires both the J domain and the ZHD domain but, surprisingly, not the C-terminal–charged domain, which mediates association with polysomes and contains the Rei1-binding site. The Jjj1-NESm mutant was also able to rescue the slow growth phenotype of Δjjj1 ( and Fig. S4 B). This further suggests that a mostly nuclear form of Jjj1 can rescue its growth phenotype.
We next examined the contribution of various Jjj1 domains to the 40S/60S balance ( and Fig. S4 D). Expression of the full-length Jjj1 in Δjjj1 cells restored the normal polysome profiles (). Mutation of the J domain (Jm-Jjj1) or deletion of the ZHD domain could not restore the 40S/60S balance. In contrast, expression of Jjj1 lacking the C-terminal domain (Jjj1-ΔC) fully restored the normal polysome profile (). Importantly, restoration of the normal polysome profiles by Jjj1-ΔC was also observed at the low endogenous expression levels in cells carrying a deletion of the C-terminal domain or of the Zf2 zinc finger of the chromosomal copy of Jjj1 (Fig. S4 D). Furthermore, the Jjj1-NESm mutant also rescued the balance between 40S/60S ribosomal subunits (Fig. S4 E).
We next examined the role of Jjj1 domains in Arx1 recycling from the cytoplasm to the nucleus (). In cells expressing WT Jjj1, Arx1 was primarily in the nucleus (, Jjj1, yellow overlap with Sik1 marker), whereas Δjjj1
cells contained a significant pool of cytoplasmic Arx1 (; Demoinet et al., 2007
; Meyer et al., 2007
). The Arx1 recycling defect was not rescued by either Jjj1-Jm or Jjj1-ZHD (). Strikingly, Jjj1-ΔC fully restored Arx1 nuclear localization, despite lacking the entire Rei1 interaction domain (, arrowheads). This result does not support the idea that Jjj1 facilitates Arx1 recycling via recruitment of Rei1. The observation that Jjj1-ΔC does not bind to Rei1, yet rescues every Δjjj1
defect we tested, suggests that Jjj1 plays an important role in normal growth and ribosome biogenesis that does not involve cytoplasmic interaction with Rei1. Although it is in principle possible that a small subpopulation of Jjj1-ΔC enters the cytosol, the predominantly nuclear localization of Jjj1-ΔC and Jjj1-NESm strongly argues for a nuclear function for Jjj1, such as assisting the correct conformational maturation of the 60S ribosomal particles in the nucleus (see and ).
Figure 6. Jjj1 and Zuo1 play early and distinct roles in ribosome biogenesis. (A) Association of Jjj1 and Jjj1-ΔC with nuclear and cytoplasmic (Cyto) ribosomal precursor particles. Ribosome biogenesis intermediates containing the chromosomally TAP-tagged (more ...)
Figure 7. Jjj1 and Zuo1 play early and distinct roles in ribosome biogenesis. (A) Schematic representation of biogenesis of eukaryotic ribosomal rRNA maturation particles. The position of endonucleolytic cleavage steps and the subcellular localization of each step (more ...)
The predominantly nuclear form of Jjj1 overlaps functionally with the RAC–SSB system
We next examined the Jjj1 domains required to rescue the growth defect of Δzuo1 cells (). Inactivation of the J domain or deletion of the ZHD in Jjj1 abolished the rescue of Δzuo1 slow growth (, JJJ1-Jm and JJJ1-ΔZHD). In contrast, JJJ1-ΔC restored the normal growth of Δzuo1 cells (, JJJ1-ΔC). Thus, the nuclear Jjj1-ΔC variant contains the region that overlaps functionally with Zuo1. Because Zuo1 regulates the activity of SSB, which also appears important for ribosome biogenesis, we next examined whether Jjj1 or its variants interact genetically with SSB (). Remarkably, Jjj1 overexpression fully restored the slow growth phenotype of Δssb1/2 (). Similar to Δzuo1 cells, the growth phenotype of Δssb1/2 was also fully restored by the nuclear Jjj1-ΔC variant (). In sum, the predominantly nuclear Jjj1 variant that does not interact with Rei1 can nonetheless rescue the Δjjj1, Δzuo1, and Δssb1/2 growth phenotypes. Collectively, our results suggest that the functional overlap between Jjj1 and the RAC–SSB chaperone system likely involves nuclear steps of ribosome biogenesis.
Jjj1 and Zuo1 interact with nuclear ribosome biogenesis intermediates
A possible nuclear function for Jjj1 and Zuo1 in ribosome biogenesis was further explored by assessing their interaction with ribosome assembly intermediates along the biogenesis pathway ( and Table S2). For Jjj1, ribosome biogenesis intermediates were isolated via chromosomally integrated tandem affinity purification (TAP)–tagged assembly factors in cells expressing Jjj1 or Jjj1-ΔC (). Immunoblot analysis for Jjj1 indicated that this chaperone indeed associates with nucleolar and nuclear 60S ribosome biogenesis intermediates as well as with later intermediates in the biogenesis pathway. Interestingly, the nuclear Jjj1-ΔC interacted much more strongly than full-length Jjj1 with the very early 60S biogenesis factors Noc1 and Nsa3 (), perhaps because of the higher concentration of Jjj1-ΔC in the nucleus. A similar analysis for Zuo1 () also revealed its association with nuclear ribosome biogenesis intermediates, most notably with particles containing Ecm1 (). These results support the role of both Zuo1 and Jjj1 in nuclear steps of ribosome biogenesis.
Jjj1 and RAC–SSB affect 35S and 27S rRNA maturation
Further insight into the function of these chaperones in ribosome biogenesis exploited the fact that eukaryotic ribosome biogenesis involves an ordered pathway of rRNA processing (; for review see Tschochner and Hurt, 2003
). Ribosome biogenesis starts in the nucleolus with the formation of a 90S preribosomal particle containing the 35S rRNA precursor ( and ). Subsequent rRNA processing steps lead to the formation of 66S and 43S particles. The 66S particle, containing the 27S rRNA, completes its processing and maturation in the nucleus to form the 60S ribosomal subunit containing the 25S rRNA. The 43S particle, containing the 20S rRNA, is processed into the 40S small ribosomal subunit, containing the 18S rRNA. As perturbations in the processing and maturation steps lead to the accumulation of rRNA precursors that can be identified by hybridization with probes specific for different rRNA regions (), we next examined whether loss of Jjj1 or Zuo1 function led to specific blocks in the rRNA processing pathway.
Publicly available genomic data on ribosome biogenesis provided insight into Jjj1 function (Peng et al., 2003
). A previous study had used oligonucleotide microarrays to examine defects in the biogenesis of noncoding RNAs in a large set of mutant strains (Peng et al., 2003
). The microarray allowed detection of accumulation or depletion of specific RNA processing intermediates in a given mutant strain, thereby revealing defects in particular steps of rRNA maturation and ribosome biogenesis. The gene for Jjj1 (then a gene with unknown function called YNL227c) was included in this study. Clustering analysis linked JJJ1 to a subset of ribosome biogenesis proteins and exosome-associated factors required for processing of the 3′ end of 5.8S rRNA within the 27S rRNA, as well as for an earlier processing step 3′ of the 18S rRNA (; Peng et al., 2003
). Nug1, but not Rei1 nor Arx1, clustered together with Jjj1 in this analysis (Peng et al., 2003
). Interestingly, we find that Jjj1 physically interacts with Nug1-containing particles ().
Because information on other chaperones was absent from the public domain, we next performed a similar microarray-based analysis to examine a possible role for Zuo1 and SSB in nuclear steps of ribosome biogenesis ( [probes schematically indicated]; Peng et al., 2003
). Our analysis of Δjjj1
yielded similar conclusions as those obtained previously (; Peng et al., 2003
), confirming that Jjj1 plays a role in 27S rRNA processing and probably also in 35S rRNA processing. Analysis of Δzuo1
cells indicated a block at very early 35S rRNA processing steps as well as a block in the 27S rRNA processing step, which is similar to that observed for Jjj1. The accumulation of 27S rRNA biogenesis intermediates in Δjjj1
, and Δssb1/2
cells was consistent with our previous Northern blot analyses and the polysome profiles ( and ). We further examined the rRNA processing defect in cells lacking RAC by pulse-chase analysis of rRNA maturation using [3
H]uracil (). This experiment further confirmed that loss of RAC function led to impaired rRNA maturation, which was very noticeable at the 35S rRNA cleavage (). Indeed, a function for Zuo1 and SSB in 35S rRNA maturation could explain the slight defect in 40S biogenesis observed in these cells (, low 40S peak; and not depicted).