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Dss1 is a 26S proteasome ubiquitin receptor.

Paraskevopoulos K, Kriegenburg F, Tatham MH, Rösner HI, Medina B, Larsen IB, Brandstrup R, Hardwick KG, Hay RT, Kragelund BB, Hartmann-Petersen R, Gordon C - Mol. Cell (2014)

Bottom Line: Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48.The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions.Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK.

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UBSs in Dss1 Are Required for Proteasome Function(A) K48-linked (left panel) and K63-linked (right panel) ubiquitin chains (input) (3 μg per assay) were coprecipitated with GST-Dss1, GST-Dss1 UBS-I mutant (L40A/W41A/W45A), GST-Dss1 UBS-II mutant (F18A/F21A/W26A), and GST-Dss1 UBS-I and UBS-II mutant (F18A/F21A/W26A/L40A/W41A/W45A). GST and GST-Rhp23 proteins were included as negative and positive controls, respectively. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to ubiquitin. Equal loading was checked by staining with Ponceau S.(B) The dss1Δ strains transformed with the indicated expression constructs were analyzed for growth on solid media at 30°C and 37°C. The pictures were taken after 72 hr.(C) The dss1Δ strains transformed with the indicated expression constructs were analyzed for the presence of ubiquitin-protein conjugates by blotting. Expression of the various Dss1 proteins was confirmed by blotting for the GFP tag. Tubulin served as a loading control. wt, wild-type.(D) A dss1Δ strain was transformed with the indicated expression vectors for Dss1-GFP fusion proteins and used for immunoprecipitations with antibodies to GFP. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to the proteasome subunit Mts4/Rpn1 and GFP on Dss1. Dss1 expression was not visible in whole cell lysates but was clearly enriched in the precipitated material. FL, full-length.(E) Plating assay of the dss1Δrhp23Δrpn10Δ strain with the indicated expression constructs. The dss1Δrpn10Δ strain transformed with the indicated constructs was crossed to dss1Δrhp23Δ cells to generate a triple deletion. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vector.(F) Plating assays of the dss1Δrhp23Δrpn10Δ and dss1Δrhp23Δrpn10ΔUIM strains with the indicated Rpn10 and Dss1 expression constructs, as shown in Figure 4E were quantified. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vectors. Viable spores were counted and normalized to the controls (Rpn10 FL and Dss1 wild-type). Data are presented as mean ± SEM (n = 6).See also Figure S4.
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fig4: UBSs in Dss1 Are Required for Proteasome Function(A) K48-linked (left panel) and K63-linked (right panel) ubiquitin chains (input) (3 μg per assay) were coprecipitated with GST-Dss1, GST-Dss1 UBS-I mutant (L40A/W41A/W45A), GST-Dss1 UBS-II mutant (F18A/F21A/W26A), and GST-Dss1 UBS-I and UBS-II mutant (F18A/F21A/W26A/L40A/W41A/W45A). GST and GST-Rhp23 proteins were included as negative and positive controls, respectively. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to ubiquitin. Equal loading was checked by staining with Ponceau S.(B) The dss1Δ strains transformed with the indicated expression constructs were analyzed for growth on solid media at 30°C and 37°C. The pictures were taken after 72 hr.(C) The dss1Δ strains transformed with the indicated expression constructs were analyzed for the presence of ubiquitin-protein conjugates by blotting. Expression of the various Dss1 proteins was confirmed by blotting for the GFP tag. Tubulin served as a loading control. wt, wild-type.(D) A dss1Δ strain was transformed with the indicated expression vectors for Dss1-GFP fusion proteins and used for immunoprecipitations with antibodies to GFP. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to the proteasome subunit Mts4/Rpn1 and GFP on Dss1. Dss1 expression was not visible in whole cell lysates but was clearly enriched in the precipitated material. FL, full-length.(E) Plating assay of the dss1Δrhp23Δrpn10Δ strain with the indicated expression constructs. The dss1Δrpn10Δ strain transformed with the indicated constructs was crossed to dss1Δrhp23Δ cells to generate a triple deletion. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vector.(F) Plating assays of the dss1Δrhp23Δrpn10Δ and dss1Δrhp23Δrpn10ΔUIM strains with the indicated Rpn10 and Dss1 expression constructs, as shown in Figure 4E were quantified. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vectors. Viable spores were counted and normalized to the controls (Rpn10 FL and Dss1 wild-type). Data are presented as mean ± SEM (n = 6).See also Figure S4.

Mentions: As expected from the NMR data, mutation of either UBS-I (L40A, W41A, W45A) or UBS-II (F18A, F21A, W26A) clearly reduced binding to ubiquitin, and no ubiquitin binding was observed for Dss1 mutated at both sites (Figure 4A). Consistent with UBS-I being the stronger of the two binding sites, mutation of this site also had a greater effect on ubiquitin binding (Figure 4A).


Dss1 is a 26S proteasome ubiquitin receptor.

Paraskevopoulos K, Kriegenburg F, Tatham MH, Rösner HI, Medina B, Larsen IB, Brandstrup R, Hardwick KG, Hay RT, Kragelund BB, Hartmann-Petersen R, Gordon C - Mol. Cell (2014)

UBSs in Dss1 Are Required for Proteasome Function(A) K48-linked (left panel) and K63-linked (right panel) ubiquitin chains (input) (3 μg per assay) were coprecipitated with GST-Dss1, GST-Dss1 UBS-I mutant (L40A/W41A/W45A), GST-Dss1 UBS-II mutant (F18A/F21A/W26A), and GST-Dss1 UBS-I and UBS-II mutant (F18A/F21A/W26A/L40A/W41A/W45A). GST and GST-Rhp23 proteins were included as negative and positive controls, respectively. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to ubiquitin. Equal loading was checked by staining with Ponceau S.(B) The dss1Δ strains transformed with the indicated expression constructs were analyzed for growth on solid media at 30°C and 37°C. The pictures were taken after 72 hr.(C) The dss1Δ strains transformed with the indicated expression constructs were analyzed for the presence of ubiquitin-protein conjugates by blotting. Expression of the various Dss1 proteins was confirmed by blotting for the GFP tag. Tubulin served as a loading control. wt, wild-type.(D) A dss1Δ strain was transformed with the indicated expression vectors for Dss1-GFP fusion proteins and used for immunoprecipitations with antibodies to GFP. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to the proteasome subunit Mts4/Rpn1 and GFP on Dss1. Dss1 expression was not visible in whole cell lysates but was clearly enriched in the precipitated material. FL, full-length.(E) Plating assay of the dss1Δrhp23Δrpn10Δ strain with the indicated expression constructs. The dss1Δrpn10Δ strain transformed with the indicated constructs was crossed to dss1Δrhp23Δ cells to generate a triple deletion. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vector.(F) Plating assays of the dss1Δrhp23Δrpn10Δ and dss1Δrhp23Δrpn10ΔUIM strains with the indicated Rpn10 and Dss1 expression constructs, as shown in Figure 4E were quantified. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vectors. Viable spores were counted and normalized to the controls (Rpn10 FL and Dss1 wild-type). Data are presented as mean ± SEM (n = 6).See also Figure S4.
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fig4: UBSs in Dss1 Are Required for Proteasome Function(A) K48-linked (left panel) and K63-linked (right panel) ubiquitin chains (input) (3 μg per assay) were coprecipitated with GST-Dss1, GST-Dss1 UBS-I mutant (L40A/W41A/W45A), GST-Dss1 UBS-II mutant (F18A/F21A/W26A), and GST-Dss1 UBS-I and UBS-II mutant (F18A/F21A/W26A/L40A/W41A/W45A). GST and GST-Rhp23 proteins were included as negative and positive controls, respectively. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to ubiquitin. Equal loading was checked by staining with Ponceau S.(B) The dss1Δ strains transformed with the indicated expression constructs were analyzed for growth on solid media at 30°C and 37°C. The pictures were taken after 72 hr.(C) The dss1Δ strains transformed with the indicated expression constructs were analyzed for the presence of ubiquitin-protein conjugates by blotting. Expression of the various Dss1 proteins was confirmed by blotting for the GFP tag. Tubulin served as a loading control. wt, wild-type.(D) A dss1Δ strain was transformed with the indicated expression vectors for Dss1-GFP fusion proteins and used for immunoprecipitations with antibodies to GFP. The precipitated material was analyzed by SDS-PAGE and western blotting using antibodies to the proteasome subunit Mts4/Rpn1 and GFP on Dss1. Dss1 expression was not visible in whole cell lysates but was clearly enriched in the precipitated material. FL, full-length.(E) Plating assay of the dss1Δrhp23Δrpn10Δ strain with the indicated expression constructs. The dss1Δrpn10Δ strain transformed with the indicated constructs was crossed to dss1Δrhp23Δ cells to generate a triple deletion. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vector.(F) Plating assays of the dss1Δrhp23Δrpn10Δ and dss1Δrhp23Δrpn10ΔUIM strains with the indicated Rpn10 and Dss1 expression constructs, as shown in Figure 4E were quantified. Following crossing, 10,000 spores were plated under selection for the deleted genes and the expression vectors. Viable spores were counted and normalized to the controls (Rpn10 FL and Dss1 wild-type). Data are presented as mean ± SEM (n = 6).See also Figure S4.
Mentions: As expected from the NMR data, mutation of either UBS-I (L40A, W41A, W45A) or UBS-II (F18A, F21A, W26A) clearly reduced binding to ubiquitin, and no ubiquitin binding was observed for Dss1 mutated at both sites (Figure 4A). Consistent with UBS-I being the stronger of the two binding sites, mutation of this site also had a greater effect on ubiquitin binding (Figure 4A).

Bottom Line: Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48.The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions.Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK.

Show MeSH
Related in: MedlinePlus