Author response: A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins (2016)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. https://doi.org/10.7554/eLife.19105.001 eLife digest Ribosomes are the molecular machines in cells that produce proteins. The ribosomes themselves are composed of almost 80 different proteins that are held together by scaffolds made from molecules of RNA. Each protein is present in one copy, and so equal numbers of all proteins are needed to assemble a ribosome. However, because it takes many steps to produce a protein and biological processes are inherently imprecise, it is essentially impossible for a cell to produce exactly the same number of copies of all the proteins in a ribosome. Much research suggests that, to overcome these issues, a cell will make more of certain ribosomal proteins than it needs, and then degrade the leftovers that are not used. However, it was not clear how this happens, nor was it known what are the consequences of failing to degrade the leftovers. Now, Sung et al. show that yeast cells use an enzyme named Tom1 to attach a protein-marker called ubiquitin to ribosomal proteins that are made in excess and not assembled into ribosomes. The ubiquitin serves as a tag that marks proteins for degradation, and yeast cell that lack Tom1 fail to degrade any excess ribosomal proteins. Consequently, the mutant yeast become sensitive to any factors that alter the balance of the protein and RNA building blocks used to assemble ribosomes. The human equivalent of Tom1 is known as Huwe1, and the data of Sung et al. suggest that this enzyme acts in a similar pathway. Further experiments are now needed to explore the role of Huwe1 in greater depth, and investigate if problems with this enzyme are associated with any human diseases. Finally, working out the exactly how Tom1 recognizes unassembled ribosomal proteins will be another important challenge for future studies. https://doi.org/10.7554/eLife.19105.002 Introduction Protein quality control (PQC) has emerged as a major mechanism for maintaining protein homeostasis and cellular fitness. Defects in the cellular machinery that governs PQC cause multiple human diseases including multisystem proteinopathy (Brandmeir et al., 2008; Watts et al., 2004) and Amyotrophic Lateral Sclerosis (ALS) (Johnson et al., 2010; Kabashi and Durham, 2006). Other diseases, such as cancer, can exhibit heightened dependency on PQC pathways, which underlies the hypersensitivity of multiple myeloma cells to proteasome inhibitors (Cenci et al., 2012; Deshaies, 2014). Therefore, a deeper understanding of PQC will advance our understanding of both normal physiology and pathological states, and may enable novel approaches to treat multiple diseases. Ribosome biogenesis is an intricate process involving many chaperones and assembly factors (Kressler et al., 2010; Warner, 1999). Ribosomal proteins made in excess over rRNA and other ribosomal proteins are among the most rapidly degraded proteins in eukaryotic cells (Abovich et al., 1985; Dephoure et al., 2014; Gorenstein and Warner, 1977; Torres et al., 2010, 2007; Warner, 1977), suggesting that proper coordination of synthesis and assembly is critical. Newly-synthesized human ribosomal proteins are subject to degradation by the proteasome in the nucleolus (Lam et al., 2007), and we recently found that overexpressed yeast ribosomal proteins that fail to assemble are conjugated with ubiquitin and degraded by the proteasome in the nucleus (Sung et al., 2016). Insoluble material that accumulates upon transient inhibition of the proteasome in yeast is strongly enriched for ribosomal proteins (Sung et al., 2016), pointing to PQC of unassembled ribosomal proteins as a major pathway of proteostasis. However, the PQC pathway that mediates ERISQ remains unknown – an important gap in our understanding of PQC that we set out to address. Results Identification of Ubc4/5 and Tom1 as the E2 and E3 for ERISQ We evaluated 115 mutant yeast strains, each lacking a different non-essential ubiquitin-proteasome system (UPS) gene, for those that accumulated non-essential ribosomal protein Rpl26a tagged with a FLAG epitope (Rpl26aFLAG) upon its overexpression from the GAL10 promoter. Accumulation of Rpl26aFLAG in most mutants was similar to wild type (WT) and well below the level detected in rpl26a∆rpl26b∆ (Figure 1—figure supplement 1A and B), which accumulated overexpressed Rpl26aFLAG due to lack of competition from endogenous Rpl26 (Sung et al., 2016). Notably, Rpl26aFLAG accumulated to high levels in tom1∆ and ubc4∆ cells (Figure 1A and Figure 1—figure supplement 1A and B). Figure 1 with 3 supplements see all Download asset Open asset Ubc4/5 and Tom1 are the E2 and E3 enzymes responsible for ERISQ. (A) Rpl26aFLAG accumulates in tom1∆ and ubc4∆. Accumulation of Rpl26aFLAG upon galactose induction in WT, tom1∆ and ubc4∆ cells was evaluated by SDS-PAGE and immunoblotting with the indicated antibodies. n = 3 biological replicates. (B) Rpl26aFLAG ubiquitination depends on Ubc4/Ubc5. Rpl26aFLAG was induced in cells of the indicated genotypes and cell lysates were prepared and subjected to pull-down with UBA domain resin. Input and bound proteins were evaluated as in (A). n = 3 biological replicates. (C) Rpl26aFLAG accumulates in tom1CA cells. As in (A) except that the Tom1 ligase-dead (tom1CA) mutant was used. n = 3 biological replicates. (D) Rpl26aFLAG ubiquitination depends on Tom1. As in (B) except that cells expressing WT Tom1 or Tom1CA were treated with bortezomib for 45 min after addition of galactose. n = 3 biological replicates. (E) Rpl26aFLAG binds 3xHATom1. Anti-HA immunoprecipitates from cells expressing 3×HATom1 and Rpl26aFLAG were immunoblotted with antibodies to HA, FLAG, and hexokinase. n = 3 biological replicates. (F) In vitro ubiquitination of Rpl26aFLAG by Tom1. Rpl26aFLAG retrieved in 3xHATom1CA immunoprecipitates was supplemented or not with E1/E2/ubiquitin/ATP (Ubi) and Tom1 retrieved from untagged (NT), 3xHATOM1 (WT), or 3xHATOM1CA (CA) cells, as indicated. See detailed methods in Material and methods. n = 3 biological replicates. https://doi.org/10.7554/eLife.19105.003 Ubc4 is an ubiquitin-conjugating enzyme (E2) that is paralogous to and functionally redundant with Ubc5 (Seufert and Jentsch, 1990). Thus, subsequent experiments were performed with ubc4∆ubc5∆ mutants. To test whether Ubc4/Ubc5 promoted ubiquitination of unassembled ribosomal proteins, we examined ubiquitin conjugates of overexpressed Rpl26aFLAG that accumulated in proteasome-deficient pre9∆ cells (Sung et al., 2016). Ubiquitinated Rpl26aFLAG was detected in pre9∆ but not in ubc4∆ubc5∆pre9∆ cells (Figure 1B), indicating that Ubc4/Ubc5 promote ubiquitination of excess Rpl26a. Tom1 is an E3 ubiquitin ligase of the HECT (homologous to E6AP C terminus) family. To investigate Tom1 function, we constructed tom1CA strains in which the endogenous TOM1 locus was mutated such that the catalytic cysteine3235 was changed to alanine (tom1CA). We also appended a 3×HA epitope sequence to the 5' end of both TOM1 and tom1CA, and confirmed that the 3xHATom1 and 3xHATom1CA proteins were expressed equivalently (Figure 1—figure supplement 2A) and the 3xHATom1 was functional (Figure 1—figure supplements 2B–D). Using these strains, we established that Tom1 E3 activity was required for repression (Figure 1C) and ubiquitination (Figure 1D and Figure 1—figure supplement 3A) of overexpressed Rpl26aFLAG. Rpl26aFLAG was co-immunoprecipitated with 3×HATom1 (Figure 1E). Upon addition of an ubiquitination cocktail, immunoprecipitations of wild type but not mutant 3×HATom1 ubiquitinated co-precipitated Rpl26aFLAG (Figure 1—figure supplement 3B). Importantly, the activity defect of the 3×HATom1CA immunoprecipitate was complemented by adding 3×HATom1 but not 3×HATom1CA prior to the ubiquitination reaction (Figure 1F and Figure 1—figure supplement 3C). To identify the population of Rpl26aFLAG targeted by Tom1, we performed sucrose gradient fractionation. Mutant tom1CA cells, like cells treated with the proteasome inhibitor bortezomib (Sung et al., 2016), accumulated unassembled Rpl26aFLAG that co-fractionated with 3×HATom1CA (Figure 2A; note that 3×HATom1 and 3×HATom1CA fractionated similarly). Co-immunoprecipitation of 3×HATom1 or 3×HATom1CA with Rpl26aFLAG was only detected in these low MW fractions (Figure 2B). Moreover, ubiquitinated Rpl26aFLAG detected in low MW fractions from bortezomib-treated cells was almost entirely lost from tom1CA cells (Figure 2B). Consistent with the reported localization of Tom1 (Huh et al., 2003), Rpl26aFLAG or Rpl26aGFP that accumulated upon their transient overexpression in tom1CA cells were found in the nucleus and nucleolus (Figure 2C). Taken together, these data provide strong evidence that overexpressed Rpl26a failed to assemble into ribosomes and was directly bound and ubiquitinated by Tom1 in the nuclear/nucleolar compartments. Figure 2 Download asset Open asset Tom1 functions in non-ribosomal fractions. (A) Sucrose gradient fractionation behavior of 3xHATom1 and Rpl26aFLAG upon galactose induction of Rpl26aFLAG in 3×HATOM1 or 3×HATOM1CA cells. T indicates total extract. n = 2 biological replicates. (B) Tom1 is required for ubiquitination of unassembled Rpl26aFLAG. Left: experimental scheme. Right: cells were treated with bortezomib for 30 min after induction of Rpl26aFLAG with galactose and then lysed and fractionated as in panel A prior to being processed as depicted in panel B. n = 2 biological replicates. (C) Rpl26a accumulates in the nucleus of tom1CA cells. Left: Subcellular fractionation of Rpl26aFLAG induced in WT and tom1CA cells. Histone H3 and Hexokinase were used as nuclear and cytoplasmic markers, respectively. CA refers to Tom1-Cys3235Ala. Right: Fluorescence microscopy of Rpl26aGFP induced in WT and tom1CA cells. Nop56-RFP marks nucleoli. Shown at far right is the percentage of GFP positive cells. n = 2 biological replicates. https://doi.org/10.7554/eLife.19105.007 Tom1 targets a broad range of ribosomal proteins To address whether Tom1 might have a broader role in promoting degradation of excess ribosomal proteins other than Rpl26a, we evaluated accumulation of a set of eight ectopically overexpressed ribosomal proteins in tom1∆ and WT cells. Similar to what we observed with bortezomib (Sung et al., 2016), deletion of TOM1 enabled increased accumulation of at least seven of them (Figure 3—figure supplement 1A). We next sought to test whether Tom1 promoted degradation of unassembled ribosomal proteins in cells in which they were not deliberately overexpressed. We reasoned that if this is the case, Tom1 should directly associate with ribosomal proteins. Mass spectrometry of 3xHATom1 immunoprecipitates from bortezomib-treated cells revealed enrichment for several ribosomal proteins, including Rpl26b (Figure 3—figure supplement 1B and Supplementary file 3A). Ribosomal proteins are commonly identified in purified ubiquitin conjugates (Mayor et al., 2007, 2005; Peng et al., 2003) or in ubiquitination site mapping experiments that rely on purification of the GlyGly dipeptide that remains attached to a lysine side chain following digestion of an ubiquitin conjugate with trypsin (Kim et al., 2011; Lesmantavicius et al., 2014; Porras-Yakushi and Hess, 2014; Porras-Yakushi et al., 2015; Sarraf et al., 2013; Swaney et al., 2013; Udeshi et al., 2013b; Wagner et al., 2011). Thus, we reasoned that if Tom1 plays a broad role in PQC of unassembled ribosomal proteins as suggested by the experiments shown in Figure 3—figure supplement 1A and B, perhaps it accounts for the frequent recovery of ribosomal proteins in prior global ubiquitin conjugate profiling efforts. To address this possibility, we performed quantitative GlyGly profiling of tom1∆ and TOM1 cells using SILAC (Figure 3—figure supplement 1C) to identify changes in the level of ubiquitination of specific lysines that occur upon loss of Tom1. Analysis of three biological replicates (Figure 3A and Figure 3—figure supplement 2A and Supplementary file 3B) revealed 1980 unique ubiquitination sites in 920 distinct proteins, of which 972 unique sites in 532 proteins were quantified. All three tom1∆ biological replicates exhibited lower overall ubiquitination than wild type, suggesting a major role for Tom1 in PQC. Of the 141 sites that exhibited a ≥two-fold decrease in ubiquitination in tom1∆, 51 (36%) were in ribosomal proteins (Figure 3—figure supplement 2B). Moreover, of the ubiquitinated peptides derived from ribosomal proteins, >50% (51 of 101) decreased in abundance in tom1∆. By comparison, of 837 non-ribosomal sites identified, only 11% decreased in abundance in tom1∆. SILAC analysis of the unfractionated cell lysates indicated that the reduction in ribosomal ubiquitin conjugates in tom1∆ was not due to reduction in total ribosomal protein levels (Figure 3B). Gene ontology analysis of the GlyGly profiling data confirmed that ubiquitination of ribosomal proteins (Figure 3C), particularly those of the large (60S) subunit (Figure 3D), was disproportionately impacted by loss of Tom1. These trends are clearly evident from a plot of the top 25 Tom1-dependent modification sites within all proteins (Figure 3—figure supplement 2C) or just ribosomal proteins (Figure 3E). To address whether the strong effects on ribosomal protein ubiquitination seen in tom1∆ cells were due specifically to loss of Tom1's E3 activity, the GlyGly SILAC analysis was repeated with WT and tom1CA cells. Quantitative analysis of the data confirmed a disproportionate loss of ribosomal protein ubiquitination (Figure 3—figure supplement 2D). Figure 3 with 3 supplements see all Download asset Open asset Diminished ubiquitination and accumulation of insoluble ribosomal proteins in tom1 cells. (A) Diminished ubiquitination of ribosomal proteins in tom1∆. Scatter plot of the SILAC ratios (tom1∆/WT) for GlyGly-modified peptides identified in biological replicate 1 versus 2. Sites with the largest decrease in ubiquitination are annotated. The other pairwise comparisons are in Figure 3—figure supplement 2A. n = 3 biological replicates. (B) Column scatter plot representing the distribution of ratios (tom1∆/WT) for proteins of the large (circles) and small (square) ribosome subunits. A red bar indicates the mean. (C) Violin plot of gene ontology analysis of ubiquitinated proteins that had one or more ubiquitination site that decreased by ≥ 2-fold. The most strongly affected categories are shown. The number in parentheses refers to the disproportionate enrichment for the category in the top 10% of identifications and is the Benjamini and Hochberg corrected p-value from a Fisher Exact test. (D) Violin plot representing the distribution of ubiquitin site occupancy ratios (tom1∆/WT) for the large (blue) and small (green) ribosome subunits, and non-ribosomal proteins (red). (E) The 25 ribosomal ubiquitination sites with the largest decrease in ubiquitin occupancy in tom1∆. +++p<0.001; ++p<0.01; +p<0.05. Each site was observed in at least two of the three biological replicates. The error bars represent 95% confidence intervals. Note that ubiquitination at K37 and K69 in Rpl26b was decreased by 2.4-fold and 1.6-fold in tom1∆, respectively (Supplementary file 3B). (F, G) Insoluble ribosomal proteins accumulate in tom1∆. (F) Detergent-insoluble pellet fractions isolated from lysate (Total) of indicated cells were analyzed by SDS-PAGE and staining with Coomassie blue or immunoblotting with the indicated antibodies. The pellet fraction is overloaded 20-fold compared to the total and supernatant fractions. n = 2 biological replicates. (G) Scatter plot representing ∆iBAQ of biological replicate B vs. A for insoluble proteins in tom1∆ mutants. Ribosomal proteins with the largest increase in the pellet fraction upon TOM1 deletion, and Rpl1 and Rpl3 are annotated. Pearson's r-value is indicated on top of the plot. The other pairwise comparisons are in Figure 3—figure supplement 3B. n = 3 biological replicates. (H) Gene ontology analysis of proteins exhibiting increased insolubility in tom1∆. Analysis is the same as for panel C. https://doi.org/10.7554/eLife.19105.008 Endogenous ribosomal proteins aggregate in tom1∆ mutants If Tom1 mediates degradation of unassembled ribosomal proteins in unperturbed cells, there should not only be a decrease in ribosomal ubiquitin conjugates in tom1 mutants, but a commensurate increase in unassembled ribosomal proteins that fail to be degraded. Since preliminary sucrose gradient fractionations did not reveal high levels of unassembled ribosomal proteins in tom1∆ cells (data not shown), we reasoned that over time, undegraded excess ribosomal proteins might aggregate and collect in insoluble deposits. To investigate this matter, we prepared detergent-insoluble fractions from WT cells treated with or without bortezomib (btz) and tom1∆ cells, and evaluated them for their content of ribosomal proteins. Detergent-insoluble proteins, including Rpl1 and Rpl3, were greatly increased in tom1∆ cells compared to WT cells (Figure 3F, Pellet). High accumulation of insoluble proteins in tom1∆ cells was evident regardless of the method or buffer employed for cell lysis (Figure 3—figure supplement 3A). This observation was confirmed and extended by mass spectrometry coupled with label-free absolute quantification using iBAQ (intensity-Based Absolute Quantification) (Geiger et al., 2012). The insoluble proteins that exhibited the largest increase in tom1∆ cells were ribosomal proteins including Rpl1 and Rpl3 (Figure 3G, Figure 3—figure supplement 3B, and Supplementary file 3C). Gene ontology analysis (Figure 3H) and a plot of the top 20 detergent-insoluble proteins (Figure 3—figure supplement 3C) indicated that ribosomal proteins, including those of both the 60S and 40S subunits (Figure 3—figure supplement 3D) comprise the major class of aggregating proteins in tom1∆ cells. Tom1 works through residues that are normally inaccessible in the structure of the mature ribosome GlyGly profiling and analysis of insoluble proteins in tom1 mutants both pointed to a broad role of Tom1 in ubiquitinating and degrading excess, unassembled ribosomal proteins. This raised a critical question that is common to all PQC pathways yet is poorly understood: how does Tom1 ubiquitinate so many different ribosomal proteins, yet manage to maintain some level of specificity for unassembled forms? To begin to address this question, we constructed a mutant Rpl26a that did not bind to rRNA. Two positively-charged clusters in Rpl26 – RRKARK (amino acids 12–17) and a patch formed by R27, R28, R51, and R52 – mediate binding to 5.8S rRNA and assembly into ribosomes (Babiano et al., 2012) (Figure 4A). We mutated various combinations of these residues to glutamate and observed that some mutants exhibited even less accumulation than overexpressed WT Rpl26aFLAG (Figure 4B). Figure 4 with 1 supplement see all Download asset Open asset A short stretch of positively-charged residues in Rpl26a that mediates rRNA binding promotes association with Tom1. (A) Top: The first 54 amino acids of Rpl26a. Sequences adjacent to rRNA are boxed. Arginine residues targeted for mutation are in red. Bottom: Relative positions of arginines and rRNA based on the atomic model of the yeast 80S ribosome (PDB files 3U5D and 3U5E). Orange and pink ribbons correspond to 25S and 5.8S rRNA, respectively. Blue ribbon corresponds to Rpl26. Predicted regions (#1 and #2) for rRNA binding are highlighted in yellow and boxed. (B) Differential accumulation of WT and mutant Rpl26aFLAG upon galactose induction. n = 2 biological replicates. (C) Top: Ribosome assembly of WT Rpl26aFLAG or Rpl26a-4EFLAG induced in rpl26a∆rpl26b∆ cells. Bottom: Same as above except that MG132 was added 30 min after addition of galactose. T indicates total extract. n = 2 biological replicates. (D) Polyubiquitination of Rpl26a-4EFLAG. Assay was performed as described for Figure 1D. Samples in '+' lanes were treated with deubiquitinating enzyme Usp2 prior to processing for SDS-PAGE, to demonstrate that high MW species were modified with ubiquitin. n = 2 biological replicates. (E) The Rpl26-4E mutation disrupts binding to Tom1. from cells of the indicated genotypes were subjected to pull-down with by SDS-PAGE and immunoblotting for the indicated proteins. n = 2 biological replicates. (F) Protein level of Rpl26aFLAG mutants upon galactose induction in WT and tom1∆ cells. n = 2 biological replicates. (G) Fluorescence of induced in WT cells. Nop56-RFP marks nucleoli. nuclear as by n = 2 biological replicates. WT did not accumulate or assemble into ribosomes in rpl26a∆rpl26b∆ cells (Figure top However, upon inhibition of the proteasome with MG132 or Rpl26a-4EFLAG accumulated (Figure supplement and was detected in the low MW fractions of a sucrose gradient (Figure it was to a than unassembled WT Rpl26aFLAG (Figure Rpl26a-4EFLAG exhibited association with 3xHATom1 (Figure and only accumulation in tom1∆ (Figure suggesting that it is not primarily a Tom1 but is to another pathway. Consistent with this Rpl26a-4EFLAG accumulated in mutants (Figure supplement The of Rpl26a-4EFLAG to be targeted by Tom1 was not due to a defect in its nuclear localization (Figure and Figure supplement We suggest that upon its into the a for that is to the nuclear and 2006). Taken together, our suggest that residues of Rpl26a that mediate with rRNA and are into the ribosome are also required for ubiquitination by Tom1 Rpl26a fails to To this observation and its we on because assembly of has in some et al., Upon its binds the The then the for into the Importantly, a structure is for and and the binding site of on the has et al., and In our GlyGly profiling were for three ubiquitination sites on and and 1.6-fold decrease in tom1∆, (Supplementary file 3B). The structure of the ribosome indicates that and exhibited the on Tom1, rRNA and are not for modification in the mature ribosome et al., 2011). the structure of the revealed that and a model of to indicates that it and Upon of into the is through the of (Kressler et al., and To test if this the of to Tom1 be the if assembly of was following nuclear and of we performed an in vitro ubiquitination with purified 3xHATom1 immunoprecipitates ubiquitinated in but not in the that at least eight lysines of should in the Figure of within the required its extended because it was the was at (Figure These with those on Rpl26a, suggest that Tom1 recognizes and ubiquitinates ribosomal proteins via residues that are only in the unassembled Notably, of yeast cells revealed that which the that binds with Tom1 Figure of et al., However, it is if loss of Tom1 the was due to degradation or of molecules into ribosomes. Figure Download asset Open asset Tom1 acts through residues that are normally inaccessible in the structure of the mature ribosome. (A) of within the mature ribosome (PDB residues are with and red. in binding and are indicated. The of the binding site are not The which is in the but is not is in red. (B) of by 3xHATom1. Anti-HA immunoprecipitates from untagged (NT), 3xHATOM1 (WT), and (CA) cells were supplemented or not with E1/E2/ubiquitin/ATP (Ubi) and purified and proteins. Samples were analyzed by SDS-PAGE and staining with Coomassie blue or immunoblotting with the indicated antibodies. WT, and refer to and respectively. See detailed methods in Material and methods. n = 3 biological replicates. (C) Tom1 targets lysines that are inaccessible in mature ribosomes. residues shown are those from large subunit ribosomal proteins in Figure that are in the model for the structure of the yeast ribosome The structure of a HECT ubiquitin that a gap of 25 be present for Tom1 to a lysine for Two of the sites and are in the 60S large subunit but become inaccessible upon of the 80S ribosome. To more if Tom1 targets lysines that are inaccessible in the mature ribosome we examined the of the major Tom1-dependent ubiquitination sites on large subunit proteins reported in Figure this we used the structure of the HECT domain of conjugated to both a ubiquitin and a We whether the amino of a lysine within the mature large subunit make with the site of Of the lysines that be observed in the in the 60S and in the assembled 80S were not to the (Figure Taken together, our data suggest strongly that Tom1-dependent ubiquitination occur on ribosomal proteins prior to their assembly into the on residues that normally are in the in or are from Tom1 is required for maintaining cellular homeostasis of ribosomal proteins We next our to the effects of Tom1 If a to degrade excess ribosomal proteins to the defect of tom1 mutants et al., (Figure 1—figure supplement and we reasoned that that imbalances in the production of ribosome should this To test we performed three different tom1CA cells not WT were sensitive to overexpression of (Figure but not (Figure supplement from the GAL10 promoter. we a in which ribosomal proteins as a are made in excess of rRNA via the and of proteins in rRNA synthesis including for RNA largest and of by RNA