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Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Balakirev MY, Mullally JE, Favier A, Assard N, Sulpice E, Lindsey DF, Rulina AV, Gidrol X, Wilkinson KD - Elife (2015)

Bottom Line: Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins.In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent.Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches en technologies et sciences pour le vivant-Biologie à Grande Echelle, Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA), Grenoble, France.

ABSTRACT
Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

No MeSH data available.


Related in: MedlinePlus

Wss1 protects cells from genotoxic threats.(A) Wss1 overexpression (Wss1oe, pYEPGAP-URA3-WT construct) suppresses accumulation of HMW-SUMO conjugates in smt3-331 cells (SBY331 and MBY13 strains, Supplementary file 3). Whole cell lysate (WCL) and chromatin fraction were analyzed by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Asterisk indicates HMW–SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple)–SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 expression suppresses temperature sensitivity of smt3-331 cells. Smt3-331 WSS1, smt3-331 wss1Δ, and smt3-331 wss1Δ cells overexpressing Wss1 protein (Wss1oe, pYEPGAP-URA3-WT construct) were spotted on SD-ura plates, and incubated at 30°C or 37°C. (B) Wss1 protects cells from Top1-associated cytotoxicity. The wss1Δ tdp1Δ mutant sickness depends on Top1 expression. The model stains expressing MBP-tagged Top1 protein under control of GAL promoter were constructed on BY4742 and MBY24 (WSS1-GFP) genetic backgrounds (MBY42-44,49 and MBY46,47 correspondingly, Supplementary file 3). The cells were spotted on selective media and incubated at 30°C. The bottom western blots for each panel show the corresponding expression of MBP-Top1 protein detected with MBP-specific antibody. As expected, no MBP-Top1 protein was detected in cells grown on glucose medium. (C) Wss1 prevents accumulation of HMW-SUMO conjugates in UV-irradiated cells (BY4742 and MBY15 strains, Supplementary file 3). Single UV dose (254 nm, 200 J/m2) induces increase in sumoylation of cellular proteins as shown by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Both whole cell lysate (WCL) and chromatin fraction were analyzed. Asterisk indicates HMW SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple) SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 protects cells from UV-induced DNA damage. WSS1 (BY4742) and wss1Δ (MBY15) cells were spotted on YPD plates, exposed (UV) or not (C) to a single UV dose (254 nm, 100 J/m2), and incubated for 2 days at 30°C.DOI:http://dx.doi.org/10.7554/eLife.06763.028
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fig9s1: Wss1 protects cells from genotoxic threats.(A) Wss1 overexpression (Wss1oe, pYEPGAP-URA3-WT construct) suppresses accumulation of HMW-SUMO conjugates in smt3-331 cells (SBY331 and MBY13 strains, Supplementary file 3). Whole cell lysate (WCL) and chromatin fraction were analyzed by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Asterisk indicates HMW–SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple)–SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 expression suppresses temperature sensitivity of smt3-331 cells. Smt3-331 WSS1, smt3-331 wss1Δ, and smt3-331 wss1Δ cells overexpressing Wss1 protein (Wss1oe, pYEPGAP-URA3-WT construct) were spotted on SD-ura plates, and incubated at 30°C or 37°C. (B) Wss1 protects cells from Top1-associated cytotoxicity. The wss1Δ tdp1Δ mutant sickness depends on Top1 expression. The model stains expressing MBP-tagged Top1 protein under control of GAL promoter were constructed on BY4742 and MBY24 (WSS1-GFP) genetic backgrounds (MBY42-44,49 and MBY46,47 correspondingly, Supplementary file 3). The cells were spotted on selective media and incubated at 30°C. The bottom western blots for each panel show the corresponding expression of MBP-Top1 protein detected with MBP-specific antibody. As expected, no MBP-Top1 protein was detected in cells grown on glucose medium. (C) Wss1 prevents accumulation of HMW-SUMO conjugates in UV-irradiated cells (BY4742 and MBY15 strains, Supplementary file 3). Single UV dose (254 nm, 200 J/m2) induces increase in sumoylation of cellular proteins as shown by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Both whole cell lysate (WCL) and chromatin fraction were analyzed. Asterisk indicates HMW SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple) SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 protects cells from UV-induced DNA damage. WSS1 (BY4742) and wss1Δ (MBY15) cells were spotted on YPD plates, exposed (UV) or not (C) to a single UV dose (254 nm, 100 J/m2), and incubated for 2 days at 30°C.DOI:http://dx.doi.org/10.7554/eLife.06763.028

Mentions: It has been reported that Wss1 suppresses the temperature sensitivity (ts) of the SUMO-mutant allele smt3-331 (Biggins et al., 2001). To ask if this suppression required the SUMO ligase or protease activity of Wss1, we probed the role of Wss1 in smt3-331 cells. The ts-suppression required both correct SUMO binding via SIM2 and an intact protease domain, but not binding to Cdc48 mediated by R218, R219, and F152 of Wss1. The expression of a C-terminal Wss1-GFP fusion (at much lower levels) showed nearly the same suppression (Figure 7A). Looking at sumoylation, we found that smt3-331 has negligible monomeric SUMO and an elevated level of HMW-SUMO conjugates, pointing to a defect in SUMO metabolism (Figure 7A). Wss1 induced a significant decrease in SUMO conjugates in smt3-331 cells (Figure 7A and Figure 9—figure supplement 1A) but not in the wild-type SMT3 strain (Figure 6). The effect of Wss1 mutants on sumoylation was similar to their effect on ts-suppression, suggesting a correlation between accumulation of HMW-SUMO and cell survival (Figure 7A and Figure 9—figure supplement 1A).10.7554/eLife.06763.022Figure 7.Wss1 regulates HMW-SUMO in smt3-331 cells.


Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Balakirev MY, Mullally JE, Favier A, Assard N, Sulpice E, Lindsey DF, Rulina AV, Gidrol X, Wilkinson KD - Elife (2015)

Wss1 protects cells from genotoxic threats.(A) Wss1 overexpression (Wss1oe, pYEPGAP-URA3-WT construct) suppresses accumulation of HMW-SUMO conjugates in smt3-331 cells (SBY331 and MBY13 strains, Supplementary file 3). Whole cell lysate (WCL) and chromatin fraction were analyzed by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Asterisk indicates HMW–SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple)–SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 expression suppresses temperature sensitivity of smt3-331 cells. Smt3-331 WSS1, smt3-331 wss1Δ, and smt3-331 wss1Δ cells overexpressing Wss1 protein (Wss1oe, pYEPGAP-URA3-WT construct) were spotted on SD-ura plates, and incubated at 30°C or 37°C. (B) Wss1 protects cells from Top1-associated cytotoxicity. The wss1Δ tdp1Δ mutant sickness depends on Top1 expression. The model stains expressing MBP-tagged Top1 protein under control of GAL promoter were constructed on BY4742 and MBY24 (WSS1-GFP) genetic backgrounds (MBY42-44,49 and MBY46,47 correspondingly, Supplementary file 3). The cells were spotted on selective media and incubated at 30°C. The bottom western blots for each panel show the corresponding expression of MBP-Top1 protein detected with MBP-specific antibody. As expected, no MBP-Top1 protein was detected in cells grown on glucose medium. (C) Wss1 prevents accumulation of HMW-SUMO conjugates in UV-irradiated cells (BY4742 and MBY15 strains, Supplementary file 3). Single UV dose (254 nm, 200 J/m2) induces increase in sumoylation of cellular proteins as shown by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Both whole cell lysate (WCL) and chromatin fraction were analyzed. Asterisk indicates HMW SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple) SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 protects cells from UV-induced DNA damage. WSS1 (BY4742) and wss1Δ (MBY15) cells were spotted on YPD plates, exposed (UV) or not (C) to a single UV dose (254 nm, 100 J/m2), and incubated for 2 days at 30°C.DOI:http://dx.doi.org/10.7554/eLife.06763.028
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fig9s1: Wss1 protects cells from genotoxic threats.(A) Wss1 overexpression (Wss1oe, pYEPGAP-URA3-WT construct) suppresses accumulation of HMW-SUMO conjugates in smt3-331 cells (SBY331 and MBY13 strains, Supplementary file 3). Whole cell lysate (WCL) and chromatin fraction were analyzed by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Asterisk indicates HMW–SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple)–SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 expression suppresses temperature sensitivity of smt3-331 cells. Smt3-331 WSS1, smt3-331 wss1Δ, and smt3-331 wss1Δ cells overexpressing Wss1 protein (Wss1oe, pYEPGAP-URA3-WT construct) were spotted on SD-ura plates, and incubated at 30°C or 37°C. (B) Wss1 protects cells from Top1-associated cytotoxicity. The wss1Δ tdp1Δ mutant sickness depends on Top1 expression. The model stains expressing MBP-tagged Top1 protein under control of GAL promoter were constructed on BY4742 and MBY24 (WSS1-GFP) genetic backgrounds (MBY42-44,49 and MBY46,47 correspondingly, Supplementary file 3). The cells were spotted on selective media and incubated at 30°C. The bottom western blots for each panel show the corresponding expression of MBP-Top1 protein detected with MBP-specific antibody. As expected, no MBP-Top1 protein was detected in cells grown on glucose medium. (C) Wss1 prevents accumulation of HMW-SUMO conjugates in UV-irradiated cells (BY4742 and MBY15 strains, Supplementary file 3). Single UV dose (254 nm, 200 J/m2) induces increase in sumoylation of cellular proteins as shown by western blotting with SUMO-specific antibodies (80 μg of protein per lane for each sample). Both whole cell lysate (WCL) and chromatin fraction were analyzed. Asterisk indicates HMW SUMO conjugates. Histograms represents ImageJ quantification of total (blue) or HMW (purple) SUMO conjugates, normalized to the maximum value. Bottom panel: Wss1 protects cells from UV-induced DNA damage. WSS1 (BY4742) and wss1Δ (MBY15) cells were spotted on YPD plates, exposed (UV) or not (C) to a single UV dose (254 nm, 100 J/m2), and incubated for 2 days at 30°C.DOI:http://dx.doi.org/10.7554/eLife.06763.028
Mentions: It has been reported that Wss1 suppresses the temperature sensitivity (ts) of the SUMO-mutant allele smt3-331 (Biggins et al., 2001). To ask if this suppression required the SUMO ligase or protease activity of Wss1, we probed the role of Wss1 in smt3-331 cells. The ts-suppression required both correct SUMO binding via SIM2 and an intact protease domain, but not binding to Cdc48 mediated by R218, R219, and F152 of Wss1. The expression of a C-terminal Wss1-GFP fusion (at much lower levels) showed nearly the same suppression (Figure 7A). Looking at sumoylation, we found that smt3-331 has negligible monomeric SUMO and an elevated level of HMW-SUMO conjugates, pointing to a defect in SUMO metabolism (Figure 7A). Wss1 induced a significant decrease in SUMO conjugates in smt3-331 cells (Figure 7A and Figure 9—figure supplement 1A) but not in the wild-type SMT3 strain (Figure 6). The effect of Wss1 mutants on sumoylation was similar to their effect on ts-suppression, suggesting a correlation between accumulation of HMW-SUMO and cell survival (Figure 7A and Figure 9—figure supplement 1A).10.7554/eLife.06763.022Figure 7.Wss1 regulates HMW-SUMO in smt3-331 cells.

Bottom Line: Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins.In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent.Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

View Article: PubMed Central - PubMed

Affiliation: Institut de recherches en technologies et sciences pour le vivant-Biologie à Grande Echelle, Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA), Grenoble, France.

ABSTRACT
Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

No MeSH data available.


Related in: MedlinePlus