Predicted regions (#1 and #2) for rRNA binding are highlighted in yellow and boxed
Predicted regions (#1 and #2) for rRNA binding are highlighted in yellow and boxed. in mutants.DOI: http://dx.doi.org/10.7554/eLife.19105.024 elife-19105-supp3.xlsx (907K) DOI:?10.7554/eLife.19105.024 Abstract Overproduced candida 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. cells display reduced ubiquitination of multiple RPs, excellent build up of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a serious perturbation Verbascoside to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in adult ribosomes. Collectively, these data point to an important part for Tom1 in normal physiology and quick us to refer to this pathway as ERISQ, for excessive ribosomal protein quality control. A similar pathway, mediated from the Tom1 homolog Huwe1, restricts build up of overexpressed hRpl26 in human being cells. We propose that ERISQ is definitely a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001 promoter. Build up of Rpl26aFLAG in most mutants was much like crazy type (WT) and well below the level recognized in (Number 1figure product 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 and cells (Number 1A and Number 1figure product 1A and B). Open in a separate window Number 1. Ubc4/5 and Tom1 are the E2 and IL7 E3 enzymes responsible for ERISQ.(A) Rpl26aFLAG accumulates in and cells was evaluated by SDS-PAGE and Verbascoside 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 website resin. Input and bound proteins were evaluated as with (A). n = 3 biological replicates. (C) Rpl26aFLAG accumulates in cells. As with (A) except the Tom1 ligase-dead ((WT), or (CA) cells, as indicated. Observe detailed methods in Material and methods. n = 3 biological replicates. DOI: http://dx.doi.org/10.7554/eLife.19105.003 Figure 1figure product 1. Open in a separate window Recognition of ERISQ defect in and promoter were used. Rpl26aFLAG induced in cells was used like a positive control. n = 1 biological replicate. (B) Quantification of data in (A). DOI: http://dx.doi.org/10.7554/eLife.19105.004 Number 1figure product 2. Open in a separate windowpane Characterization of tagged and ligase-dead Tom1.(A) Protein level of 3HATom1 in cells expressing Tom1 (NT), 3HATom1 (WT) and 3HATom1CA (CA). n = 2 biological replicates. (B) Protein level of overexpressed Rpl26aFLAG induced in cells expressing Tom1 (NT), Tom1HA, 3HATom1 and mutants. To test whether Ubc4/Ubc5 advertised ubiquitination of unassembled ribosomal proteins, we examined ubiquitin conjugates of overexpressed Rpl26aFLAG that accumulated in proteasome-deficient cells (Sung et al., 2016). Ubiquitinated Rpl26aFLAG was recognized in but not in cells (Number 1B), indicating that Ubc4/Ubc5 promote ubiquitination of excessive Rpl26a. Tom1 is an E3 ubiquitin ligase of the HECT (homologous to E6AP C terminus) family. To investigate Tom1 function, we constructed strains in which the endogenous locus was mutated such that the catalytic cysteine3235 was changed to alanine (and cells, like cells treated with the proteasome inhibitor bortezomib (Sung et al., 2016), accumulated unassembled Rpl26aFLAG that co-fractionated with 3HATom1CA (Number 2A; note that 3HATom1 and 3HATom1CA fractionated similarly). Co-immunoprecipitation of 3HATom1 or 3HATom1CA with Rpl26aFLAG was only recognized Verbascoside in these low MW fractions (Number 2B). Moreover, ubiquitinated Rpl26aFLAG recognized in low MW fractions from bortezomib-treated cells was almost entirely lost from cells (Number 2B). Consistent with the reported localization of Tom1 (Huh et al., 2003), Rpl26aFLAG or Rpl26aGFP that accumulated upon their transient overexpression in cells were found in the nucleus and nucleolus (Number 2C). Taken collectively, 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. Open in a separate window Number 2. Tom1 functions in non-ribosomal fractions.(A) Sucrose gradient fractionation behavior of 3xHATom1 and Rpl26aFLAG upon galactose induction of Rpl26aFLAG in or cells. T shows total draw out. n = 2 biological replicates. (B) Tom1 is required for ubiquitination of unassembled Rpl26aFLAG. Remaining: experimental plan. Right: cells were treated with bortezomib for 30 min after induction of.