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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 promotes sumoylation of Cdc48 and other cellular proteins.(A) Wss1-induced sumoylation of cellular proteins does not depend on SUMO E3-ligases. Control cells (BY4742) and cells knocked out for known and putative SUMO E3-ligase genes (YKO clones, Open Biosystems) were transformed with wild-type HA-Wss1 (WT) or pYEPGAP-URA3 -vector alone (V) and sumoylation profile was examined by WCL western blotting with SUMO -specific antibodies (80 μg of protein per lane for each sample). The asterisk indicates HMW-SUMO conjugates, and the black arrowheads show sumoylated Cdc48. (B) In vitro sumoylation with His6-Cdc48, HF-Doa1, and HA-Wss1 proteins. The proteins were expressed in bacteria and affinity purified. In vitro sumoylation reaction was performed with recombinant SUMO and SUMO E1–E2 enzymes (see ‘Materials and methods’). The reaction was analyzed by western blotting with specific antibodies. (C) Effect of Wss1 mutations on in vitro sumoylation of Cdc48. Recombinant HA-Wss1 proteins were used in in vitro sumoylation reaction with His6-Cdc48 protein. The reaction was analyzed by western blotting with Cdc48-specific antibodies.DOI:http://dx.doi.org/10.7554/eLife.06763.021
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fig6s1: Wss1 promotes sumoylation of Cdc48 and other cellular proteins.(A) Wss1-induced sumoylation of cellular proteins does not depend on SUMO E3-ligases. Control cells (BY4742) and cells knocked out for known and putative SUMO E3-ligase genes (YKO clones, Open Biosystems) were transformed with wild-type HA-Wss1 (WT) or pYEPGAP-URA3 -vector alone (V) and sumoylation profile was examined by WCL western blotting with SUMO -specific antibodies (80 μg of protein per lane for each sample). The asterisk indicates HMW-SUMO conjugates, and the black arrowheads show sumoylated Cdc48. (B) In vitro sumoylation with His6-Cdc48, HF-Doa1, and HA-Wss1 proteins. The proteins were expressed in bacteria and affinity purified. In vitro sumoylation reaction was performed with recombinant SUMO and SUMO E1–E2 enzymes (see ‘Materials and methods’). The reaction was analyzed by western blotting with specific antibodies. (C) Effect of Wss1 mutations on in vitro sumoylation of Cdc48. Recombinant HA-Wss1 proteins were used in in vitro sumoylation reaction with His6-Cdc48 protein. The reaction was analyzed by western blotting with Cdc48-specific antibodies.DOI:http://dx.doi.org/10.7554/eLife.06763.021

Mentions: We next examined the role of Wss1 in the regulation of SUMO metabolism by perturbing a variety of structural elements implicated in activity and binding. Wss1 proteins were expressed from a pYepGAP vector, and sumoylation of cellular proteins was analyzed by western blotting. Contrary to the accepted role of Wss1 as a protease, but consistent with its role as a SUMO ligase, Wss1 expression results in a marked buildup of high molecular weight SUMO conjugates at the top of the gel and two major species at 120 kD and 140 kD (Figure 6A). A similar effect was observed with WLM*, but not with the ΔSIM2 mutant, demonstrating that SUMO-binding and not protease activity was involved. None of the known SUMO ligases was required for stimulation of sumoylation by Wss1 (Figure 6—figure supplement 1) suggesting that the observed activity was due to Wss1 directly. Most strikingly, Siz1 appears to be responsible for most sumoylation under basal conditions, and Wss1 expression partially rescued the decreased SUMO conjugation seen in siz1Δ cells.10.7554/eLife.06763.020Figure 6.Wss1 sumoylates cellular proteins and promotes their binding to Cdc48.


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 promotes sumoylation of Cdc48 and other cellular proteins.(A) Wss1-induced sumoylation of cellular proteins does not depend on SUMO E3-ligases. Control cells (BY4742) and cells knocked out for known and putative SUMO E3-ligase genes (YKO clones, Open Biosystems) were transformed with wild-type HA-Wss1 (WT) or pYEPGAP-URA3 -vector alone (V) and sumoylation profile was examined by WCL western blotting with SUMO -specific antibodies (80 μg of protein per lane for each sample). The asterisk indicates HMW-SUMO conjugates, and the black arrowheads show sumoylated Cdc48. (B) In vitro sumoylation with His6-Cdc48, HF-Doa1, and HA-Wss1 proteins. The proteins were expressed in bacteria and affinity purified. In vitro sumoylation reaction was performed with recombinant SUMO and SUMO E1–E2 enzymes (see ‘Materials and methods’). The reaction was analyzed by western blotting with specific antibodies. (C) Effect of Wss1 mutations on in vitro sumoylation of Cdc48. Recombinant HA-Wss1 proteins were used in in vitro sumoylation reaction with His6-Cdc48 protein. The reaction was analyzed by western blotting with Cdc48-specific antibodies.DOI:http://dx.doi.org/10.7554/eLife.06763.021
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fig6s1: Wss1 promotes sumoylation of Cdc48 and other cellular proteins.(A) Wss1-induced sumoylation of cellular proteins does not depend on SUMO E3-ligases. Control cells (BY4742) and cells knocked out for known and putative SUMO E3-ligase genes (YKO clones, Open Biosystems) were transformed with wild-type HA-Wss1 (WT) or pYEPGAP-URA3 -vector alone (V) and sumoylation profile was examined by WCL western blotting with SUMO -specific antibodies (80 μg of protein per lane for each sample). The asterisk indicates HMW-SUMO conjugates, and the black arrowheads show sumoylated Cdc48. (B) In vitro sumoylation with His6-Cdc48, HF-Doa1, and HA-Wss1 proteins. The proteins were expressed in bacteria and affinity purified. In vitro sumoylation reaction was performed with recombinant SUMO and SUMO E1–E2 enzymes (see ‘Materials and methods’). The reaction was analyzed by western blotting with specific antibodies. (C) Effect of Wss1 mutations on in vitro sumoylation of Cdc48. Recombinant HA-Wss1 proteins were used in in vitro sumoylation reaction with His6-Cdc48 protein. The reaction was analyzed by western blotting with Cdc48-specific antibodies.DOI:http://dx.doi.org/10.7554/eLife.06763.021
Mentions: We next examined the role of Wss1 in the regulation of SUMO metabolism by perturbing a variety of structural elements implicated in activity and binding. Wss1 proteins were expressed from a pYepGAP vector, and sumoylation of cellular proteins was analyzed by western blotting. Contrary to the accepted role of Wss1 as a protease, but consistent with its role as a SUMO ligase, Wss1 expression results in a marked buildup of high molecular weight SUMO conjugates at the top of the gel and two major species at 120 kD and 140 kD (Figure 6A). A similar effect was observed with WLM*, but not with the ΔSIM2 mutant, demonstrating that SUMO-binding and not protease activity was involved. None of the known SUMO ligases was required for stimulation of sumoylation by Wss1 (Figure 6—figure supplement 1) suggesting that the observed activity was due to Wss1 directly. Most strikingly, Siz1 appears to be responsible for most sumoylation under basal conditions, and Wss1 expression partially rescued the decreased SUMO conjugation seen in siz1Δ cells.10.7554/eLife.06763.020Figure 6.Wss1 sumoylates cellular proteins and promotes their binding to Cdc48.

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