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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 regulates HMW-SUMO in smt3-331 cells.(A) Suppression of Smt3-331 phenotype by Wss1. Wss1 constructs were expressed in smt3-331 wss1Δ cells (MBY13, Supplementary file 3) as HA-fusions using pYEPGAP-URA3 vector (V, etc) or as GFP fusion using pUG35 vector (Wss1-GFP, Supplementary file 4). Left panel: ts-suppression assay on SD-ura plates. Right panel: western blot analysis of SMT3 (first lane labeled SMT3, MBY14 strain) and smt3-331 cell lysates with α-SUMO. Asterisk shows HMW-SUMO. Histogram: ImageJ quantification of total (blue) or HMW-SUMO (red) conjugates, normalized to the value obtained with vector alone (V). (B) Wss1 controls HMW-SUMO. Cellular protein synthesis was inhibited in exponentially growing smt3-331 WSS1 (SBY331, Supplementary file 3) and smt3-331 wss1Δ cells (MBY13) by 50 μg/ml cycloheximide (CHX). WCL was analyzed by western blotting with α-SUMO. SUMO signal was measured by ImageJ in three zones I–III and normalized to the initial value. The plots show the kinetics of SUMO change (error bars represent standard deviations). The percentage on the right indicates total amount of SUMO signal remained after 2 hr.DOI:http://dx.doi.org/10.7554/eLife.06763.02210.7554/eLife.06763.023Figure 7—source data 1.Western blot quantification for Figures 7A and 7B.Figure 7—source data 1DOI:http://dx.doi.org/10.7554/eLife.06763.023
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fig7: Wss1 regulates HMW-SUMO in smt3-331 cells.(A) Suppression of Smt3-331 phenotype by Wss1. Wss1 constructs were expressed in smt3-331 wss1Δ cells (MBY13, Supplementary file 3) as HA-fusions using pYEPGAP-URA3 vector (V, etc) or as GFP fusion using pUG35 vector (Wss1-GFP, Supplementary file 4). Left panel: ts-suppression assay on SD-ura plates. Right panel: western blot analysis of SMT3 (first lane labeled SMT3, MBY14 strain) and smt3-331 cell lysates with α-SUMO. Asterisk shows HMW-SUMO. Histogram: ImageJ quantification of total (blue) or HMW-SUMO (red) conjugates, normalized to the value obtained with vector alone (V). (B) Wss1 controls HMW-SUMO. Cellular protein synthesis was inhibited in exponentially growing smt3-331 WSS1 (SBY331, Supplementary file 3) and smt3-331 wss1Δ cells (MBY13) by 50 μg/ml cycloheximide (CHX). WCL was analyzed by western blotting with α-SUMO. SUMO signal was measured by ImageJ in three zones I–III and normalized to the initial value. The plots show the kinetics of SUMO change (error bars represent standard deviations). The percentage on the right indicates total amount of SUMO signal remained after 2 hr.DOI:http://dx.doi.org/10.7554/eLife.06763.02210.7554/eLife.06763.023Figure 7—source data 1.Western blot quantification for Figures 7A and 7B.Figure 7—source data 1DOI:http://dx.doi.org/10.7554/eLife.06763.023

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 regulates HMW-SUMO in smt3-331 cells.(A) Suppression of Smt3-331 phenotype by Wss1. Wss1 constructs were expressed in smt3-331 wss1Δ cells (MBY13, Supplementary file 3) as HA-fusions using pYEPGAP-URA3 vector (V, etc) or as GFP fusion using pUG35 vector (Wss1-GFP, Supplementary file 4). Left panel: ts-suppression assay on SD-ura plates. Right panel: western blot analysis of SMT3 (first lane labeled SMT3, MBY14 strain) and smt3-331 cell lysates with α-SUMO. Asterisk shows HMW-SUMO. Histogram: ImageJ quantification of total (blue) or HMW-SUMO (red) conjugates, normalized to the value obtained with vector alone (V). (B) Wss1 controls HMW-SUMO. Cellular protein synthesis was inhibited in exponentially growing smt3-331 WSS1 (SBY331, Supplementary file 3) and smt3-331 wss1Δ cells (MBY13) by 50 μg/ml cycloheximide (CHX). WCL was analyzed by western blotting with α-SUMO. SUMO signal was measured by ImageJ in three zones I–III and normalized to the initial value. The plots show the kinetics of SUMO change (error bars represent standard deviations). The percentage on the right indicates total amount of SUMO signal remained after 2 hr.DOI:http://dx.doi.org/10.7554/eLife.06763.02210.7554/eLife.06763.023Figure 7—source data 1.Western blot quantification for Figures 7A and 7B.Figure 7—source data 1DOI:http://dx.doi.org/10.7554/eLife.06763.023
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fig7: Wss1 regulates HMW-SUMO in smt3-331 cells.(A) Suppression of Smt3-331 phenotype by Wss1. Wss1 constructs were expressed in smt3-331 wss1Δ cells (MBY13, Supplementary file 3) as HA-fusions using pYEPGAP-URA3 vector (V, etc) or as GFP fusion using pUG35 vector (Wss1-GFP, Supplementary file 4). Left panel: ts-suppression assay on SD-ura plates. Right panel: western blot analysis of SMT3 (first lane labeled SMT3, MBY14 strain) and smt3-331 cell lysates with α-SUMO. Asterisk shows HMW-SUMO. Histogram: ImageJ quantification of total (blue) or HMW-SUMO (red) conjugates, normalized to the value obtained with vector alone (V). (B) Wss1 controls HMW-SUMO. Cellular protein synthesis was inhibited in exponentially growing smt3-331 WSS1 (SBY331, Supplementary file 3) and smt3-331 wss1Δ cells (MBY13) by 50 μg/ml cycloheximide (CHX). WCL was analyzed by western blotting with α-SUMO. SUMO signal was measured by ImageJ in three zones I–III and normalized to the initial value. The plots show the kinetics of SUMO change (error bars represent standard deviations). The percentage on the right indicates total amount of SUMO signal remained after 2 hr.DOI:http://dx.doi.org/10.7554/eLife.06763.02210.7554/eLife.06763.023Figure 7—source data 1.Western blot quantification for Figures 7A and 7B.Figure 7—source data 1DOI:http://dx.doi.org/10.7554/eLife.06763.023
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