A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins
- California Institute of Technology, United States
- Howard Hughes Medical Institute, California Institute of Technology, United States
Figures
Figure 1 with 3 supplements
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 …
Figure 1—figure supplement 1
Identification of ERISQ defect in tom1∆ and ubc4∆.
(A) One hundred fifteen different knockout mutant strains each containing a plasmid that expressed Rpl26aFLAG from the GAL10 promoter were used. Rpl26aFLAG induced in rpl26a∆rpl26b∆ cells was used …
Figure 1—figure supplement 2
Characterization of tagged and ligase-dead Tom1.
(A) Protein level of 3×HATom1 in cells expressing Tom1 (NT), 3×HATom1 (WT) and 3×HATom1CA (CA). n = 2 biological replicates. (B) Protein level of overexpressed Rpl26aFLAG induced in cells expressing …
Figure 1—figure supplement 3
Tom1 mediates ubiquitination of overexpressed Rpl26a.
(A) Polyubiquitination of Rpl26aFLAG. 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 …
Figure 2
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. …
Figure 3 with 3 supplements
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 …
Figure 3—figure supplement 1
Tom1 targets a broad range of overexpressed and endogenous ribosomal proteins.
(A) Relative levels of the transiently overexpressed, indicated ribosomal proteins (all tagged with a His6-HA-protein A ZZ domain epitope) in WT and tom1∆ mutants. n = 2 biological replicates. …
Figure 3—figure supplement 2
Quantitative GlyGly proteomic analyses of tom1 mutants.
(A) Scatter plots of the SILAC ratios (tom1∆/WT) of biological replicate 3 vs. 2 (left) and biological replicate 3 vs. 1 (right) for GlyGly-modified peptides in tom1∆ and WT cells. Sites with the …
Figure 3—figure supplement 3
Endogenous ribosomal proteins accumulate as insoluble species in tom1∆ mutants.
(A) Accumulation of insoluble proteins in tom1 mutant cells is independent of lysis method or buffer. Cells of the indicated genotypes were lysed with glass beads in the presence of 3 different …
Figure 4 with 1 supplement
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 …
Figure 4—figure supplement 1
Rpl26a-4E mutant is unstable and degraded by Doa10 in the nucleus/nucleolus.
(A) Similar accumulation of Rpl26aFLAG and Rpl26a-4EFLAG upon galactose induction in the presence of bortezomib. Total cell lysates were evaluated by SDS-PAGE and immunoblotting with the indicated …
Figure 5
Tom1 acts through residues that are normally inaccessible in the structure of the mature ribosome.
(A) Structure of Rpl4 within the mature ribosome (PDB ID 4V88). Lysine residues are colored blue, with K55, K308, and K338 colored red. Areas involved in binding Acl4 and ctKap104 are indicated. The …
Figure 6 with 1 supplement
Defective ribosome assembly homeostasis and proteostatic collapse in tom1 mutant cells.
(A–C) Hypersensitivity of tom1CA cells to imbalances in ribosome components. (A) Cells of the indicated genotypes were spotted in serial 10-fold dilutions on glucose or galactose medium and …
Figure 6—figure supplement 1
Tom1 is required for maintaining proteostasis.
(A) Cells of the indicated genotypes were spotted in serial 10-fold dilutions on glucose or galactose medium and incubated at 30°C for 2 days. ev refers to empty vector. n = 2 biological replicates. …
Figure 7
ERISQ is conserved in human cells.
(A) Proteasome inhibition enables overexpression of human Rpl26. Left: transiently expressed hRpl26FLAG in T-REx-293 cells treated with 10 µM MG132 or 1 µM bortezomib (btz) for 3 hr. Right: …
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Figure 7—source data 1
Quantification of hRpl26-FLAG and GAPDH levels from three biological replicates.
- https://doi.org/10.7554/eLife.19105.018
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Figure 7—source data 2
Quantification of hRpl26-FLAG and GAPDH levels from one biological replicate.
- https://doi.org/10.7554/eLife.19105.019
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Figure 7—source data 3
Quantification of hRpl26-FLAG and GAPDH levels from three biological replicates.
- https://doi.org/10.7554/eLife.19105.020
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Figure 7—source data 4
Quantification of hRpl26-FLAG and GAPDH levels from one biological replicate.
- https://doi.org/10.7554/eLife.19105.021
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.19105.022
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Supplementary file 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.19105.023
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Supplementary file 3
(A) Tom1 interactors identified by MS. (B) Dataset from SILAC-GlyGly analysis. (C) Insoluble proteins identified in tom1 mutants.
- https://doi.org/10.7554/eLife.19105.024
