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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 proteolysis.(A) Cellular stress destabilizes Wss1 protein. Kinetics of Wss1 degradation was studied on WSS1 and wss1Δ background in smt3-331 and SMT3 (BY4742) cells. The cells were transformed with HA-Wss1 constructs (pYEPGAP-URA3-WT, WLM*, and ΔSIM2) and grown at 30°C. Cellular protein synthesis was inhibited in mid-exponential phase by 50 μg/ml cycloheximide (CHX), and Wss1 protein was analyzed in WCL by western blotting with α-HA. When indicated 0.2 μg/ml 4-NQO was added 3 hr before CHX treatment. The plots show α−HA signal quantified by ImageJ and normalized to initial level for WT (blue diamonds), WLM*(red squares), and ΔSIM2 (green triangles) proteins. Error bars represent standard deviations from triplicate observations. (B) Wss1 does not cleave His6-Ub-SUMO-HA substrate. Affinity isolated activated HA-Wss1protein (see also Figure 8E) undergoes self-processing and degrades Cdc48 protein but not His6-Ub-SUMO-HA substrate. (C) Mapping of Wss1 fragments with recombinant truncated constructs purified from bacteria. See also corresponding source file: Figure 8—source data 1.xlsx.DOI:http://dx.doi.org/10.7554/eLife.06763.026
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fig8s1: Wss1 proteolysis.(A) Cellular stress destabilizes Wss1 protein. Kinetics of Wss1 degradation was studied on WSS1 and wss1Δ background in smt3-331 and SMT3 (BY4742) cells. The cells were transformed with HA-Wss1 constructs (pYEPGAP-URA3-WT, WLM*, and ΔSIM2) and grown at 30°C. Cellular protein synthesis was inhibited in mid-exponential phase by 50 μg/ml cycloheximide (CHX), and Wss1 protein was analyzed in WCL by western blotting with α-HA. When indicated 0.2 μg/ml 4-NQO was added 3 hr before CHX treatment. The plots show α−HA signal quantified by ImageJ and normalized to initial level for WT (blue diamonds), WLM*(red squares), and ΔSIM2 (green triangles) proteins. Error bars represent standard deviations from triplicate observations. (B) Wss1 does not cleave His6-Ub-SUMO-HA substrate. Affinity isolated activated HA-Wss1protein (see also Figure 8E) undergoes self-processing and degrades Cdc48 protein but not His6-Ub-SUMO-HA substrate. (C) Mapping of Wss1 fragments with recombinant truncated constructs purified from bacteria. See also corresponding source file: Figure 8—source data 1.xlsx.DOI:http://dx.doi.org/10.7554/eLife.06763.026

Mentions: Thus, Wss1 acts as a HMW-SUMO-processing protease in smt3-331 but not in the SMT3 strain. Several observations suggest that the proteolytic activity of Wss1 might be specifically activated in smt3-331 cells. Thus, we found that in smt3-331 strain the level of exogenously expressed HA-Wss1 and, particularly, HA-WLM* protein was greatly decreased in WSS1 compared to wss1Δ cells pointing to a mechanism involving Wss1-mediated degradation (Figure 8A). Assuming similar rates of transcription, the massively higher level of ΔSIM2-Wss1 observed in the steady state suggests that this degradation was SUMO dependent. Therefore, consistent with our observations in vitro (Figure 2 and Figure 3), Wss1 appears to undergo autoproteolytic activation and degradation stimulated by interaction with HMW-SUMO. Corroborating this conclusion, Wss1 protein was destabilized in smt3-331 compared to SMT3 cells and its degradation depended on the presence of the native protease and SIM domains (Figure 8—figure supplement 1A). At the same time, we found that wss1Δ increased the level of Cdc48 protein in smt3-331 mutant, but not in SMT3 strain (Figure 8B). In complementation assays, only WT Wss1 was able to reduce the level of Cdc48 protein in wss1Δ cells. These data suggest that Wss1 degrades itself and Cdc48 in smt3-331 cells, and that this activity requires its protease domain, SUMO-interaction and Cdc48 binding. Because Wss1 binding to Cdc48 is not required for ts-suppression (Figure 8A), the degradation of Cdc48 by WT-Wss1 seems to be a sort of collateral damage induced by activation of the Wss1 protease by increased HMW-SUMO in smt3-331 cells. Loss of Cdc48 itself would be detrimental to the cell and the lack of this degradation in F2R-Wss1 could explain why F2R-Wss1 is a better ts-suppressor than WT Wss1 (Figure 8A).10.7554/eLife.06763.024Figure 8.Wss1 protease is activated by DNA damage.


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 proteolysis.(A) Cellular stress destabilizes Wss1 protein. Kinetics of Wss1 degradation was studied on WSS1 and wss1Δ background in smt3-331 and SMT3 (BY4742) cells. The cells were transformed with HA-Wss1 constructs (pYEPGAP-URA3-WT, WLM*, and ΔSIM2) and grown at 30°C. Cellular protein synthesis was inhibited in mid-exponential phase by 50 μg/ml cycloheximide (CHX), and Wss1 protein was analyzed in WCL by western blotting with α-HA. When indicated 0.2 μg/ml 4-NQO was added 3 hr before CHX treatment. The plots show α−HA signal quantified by ImageJ and normalized to initial level for WT (blue diamonds), WLM*(red squares), and ΔSIM2 (green triangles) proteins. Error bars represent standard deviations from triplicate observations. (B) Wss1 does not cleave His6-Ub-SUMO-HA substrate. Affinity isolated activated HA-Wss1protein (see also Figure 8E) undergoes self-processing and degrades Cdc48 protein but not His6-Ub-SUMO-HA substrate. (C) Mapping of Wss1 fragments with recombinant truncated constructs purified from bacteria. See also corresponding source file: Figure 8—source data 1.xlsx.DOI:http://dx.doi.org/10.7554/eLife.06763.026
© Copyright Policy
Related In: Results  -  Collection

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fig8s1: Wss1 proteolysis.(A) Cellular stress destabilizes Wss1 protein. Kinetics of Wss1 degradation was studied on WSS1 and wss1Δ background in smt3-331 and SMT3 (BY4742) cells. The cells were transformed with HA-Wss1 constructs (pYEPGAP-URA3-WT, WLM*, and ΔSIM2) and grown at 30°C. Cellular protein synthesis was inhibited in mid-exponential phase by 50 μg/ml cycloheximide (CHX), and Wss1 protein was analyzed in WCL by western blotting with α-HA. When indicated 0.2 μg/ml 4-NQO was added 3 hr before CHX treatment. The plots show α−HA signal quantified by ImageJ and normalized to initial level for WT (blue diamonds), WLM*(red squares), and ΔSIM2 (green triangles) proteins. Error bars represent standard deviations from triplicate observations. (B) Wss1 does not cleave His6-Ub-SUMO-HA substrate. Affinity isolated activated HA-Wss1protein (see also Figure 8E) undergoes self-processing and degrades Cdc48 protein but not His6-Ub-SUMO-HA substrate. (C) Mapping of Wss1 fragments with recombinant truncated constructs purified from bacteria. See also corresponding source file: Figure 8—source data 1.xlsx.DOI:http://dx.doi.org/10.7554/eLife.06763.026
Mentions: Thus, Wss1 acts as a HMW-SUMO-processing protease in smt3-331 but not in the SMT3 strain. Several observations suggest that the proteolytic activity of Wss1 might be specifically activated in smt3-331 cells. Thus, we found that in smt3-331 strain the level of exogenously expressed HA-Wss1 and, particularly, HA-WLM* protein was greatly decreased in WSS1 compared to wss1Δ cells pointing to a mechanism involving Wss1-mediated degradation (Figure 8A). Assuming similar rates of transcription, the massively higher level of ΔSIM2-Wss1 observed in the steady state suggests that this degradation was SUMO dependent. Therefore, consistent with our observations in vitro (Figure 2 and Figure 3), Wss1 appears to undergo autoproteolytic activation and degradation stimulated by interaction with HMW-SUMO. Corroborating this conclusion, Wss1 protein was destabilized in smt3-331 compared to SMT3 cells and its degradation depended on the presence of the native protease and SIM domains (Figure 8—figure supplement 1A). At the same time, we found that wss1Δ increased the level of Cdc48 protein in smt3-331 mutant, but not in SMT3 strain (Figure 8B). In complementation assays, only WT Wss1 was able to reduce the level of Cdc48 protein in wss1Δ cells. These data suggest that Wss1 degrades itself and Cdc48 in smt3-331 cells, and that this activity requires its protease domain, SUMO-interaction and Cdc48 binding. Because Wss1 binding to Cdc48 is not required for ts-suppression (Figure 8A), the degradation of Cdc48 by WT-Wss1 seems to be a sort of collateral damage induced by activation of the Wss1 protease by increased HMW-SUMO in smt3-331 cells. Loss of Cdc48 itself would be detrimental to the cell and the lack of this degradation in F2R-Wss1 could explain why F2R-Wss1 is a better ts-suppressor than WT Wss1 (Figure 8A).10.7554/eLife.06763.024Figure 8.Wss1 protease is activated by DNA damage.

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