Limits...
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 localization upon DNA damage.(A) Intracellular localization of Wss1. Wss1-GFP fusion protein was expressed from pUG35 plasmid under control of the inducible MET25 promoter. Live cells (BY4742) were stained with DAPI and analyzed by fluorescence microscopy. Low (SD-Ura medium) and high (SD-Ura-Met) level of expression conditions are shown. Right column shows cells pre-incubated with 0.2 µg/ml 4-NQO. Histogram shows percentage of cells containing Wss1 foci in control (n = 628) and 4-NQO-treated (n = 498) cells. Scale bar is 2 µm. (B) Cellular stress induces vacuolar accumulation of Wss1. Wss1-GFP fusion protein was expressed in BY4742 and smt3-331cells from pUG35 plasmid. BY4742 cells were treated or not with 0.2 µg/ml 4-NQO for 3 hr. The cells were then stained with FM4-64 and analyzed by fluorescence microscopy. Histogram shows percentage of cells with vacuolar GFP signal in control (n = 638) and 4-NQO-treated (n = 513) BY4742 cells as well as in smt3-331 (n = 201, SBY331) cells and parental SMT3 strain (n = 316, SBY21). Scale bar is 2 µm. (C) Top1 overexpression induces accumulation of Wss1 in vacuole. The images show accumulation of Wss1-GFP in vacuole in pGAL-MBP-TOP1 WSS1-GFP tdp1Δ cells (MBY47 strain) after induction of MBP-Top1 protein in galactose medium. Live cells were stained with vacuolar marker FM4-64 and analyzed by fluorescence microscopy. All selected images show bright fluorescent spots within the cytoplasm and on the vacuolar membrane suggesting the activation of specific autophagic pathway. Scale bar is 2 µm. See also corresponding source files: Figure 10—source data 2.xlsx (for supplement A and B) and Figure 10—source data 1.xlsx (for supplement C).DOI:http://dx.doi.org/10.7554/eLife.06763.032
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4559962&req=5

fig10s1: Wss1 localization upon DNA damage.(A) Intracellular localization of Wss1. Wss1-GFP fusion protein was expressed from pUG35 plasmid under control of the inducible MET25 promoter. Live cells (BY4742) were stained with DAPI and analyzed by fluorescence microscopy. Low (SD-Ura medium) and high (SD-Ura-Met) level of expression conditions are shown. Right column shows cells pre-incubated with 0.2 µg/ml 4-NQO. Histogram shows percentage of cells containing Wss1 foci in control (n = 628) and 4-NQO-treated (n = 498) cells. Scale bar is 2 µm. (B) Cellular stress induces vacuolar accumulation of Wss1. Wss1-GFP fusion protein was expressed in BY4742 and smt3-331cells from pUG35 plasmid. BY4742 cells were treated or not with 0.2 µg/ml 4-NQO for 3 hr. The cells were then stained with FM4-64 and analyzed by fluorescence microscopy. Histogram shows percentage of cells with vacuolar GFP signal in control (n = 638) and 4-NQO-treated (n = 513) BY4742 cells as well as in smt3-331 (n = 201, SBY331) cells and parental SMT3 strain (n = 316, SBY21). Scale bar is 2 µm. (C) Top1 overexpression induces accumulation of Wss1 in vacuole. The images show accumulation of Wss1-GFP in vacuole in pGAL-MBP-TOP1 WSS1-GFP tdp1Δ cells (MBY47 strain) after induction of MBP-Top1 protein in galactose medium. Live cells were stained with vacuolar marker FM4-64 and analyzed by fluorescence microscopy. All selected images show bright fluorescent spots within the cytoplasm and on the vacuolar membrane suggesting the activation of specific autophagic pathway. Scale bar is 2 µm. See also corresponding source files: Figure 10—source data 2.xlsx (for supplement A and B) and Figure 10—source data 1.xlsx (for supplement C).DOI:http://dx.doi.org/10.7554/eLife.06763.032

Mentions: To examine the site(s) of Wss1 action, we analyzed cellular localization of this protein. GFP-Wss1 showed nuclear staining with an occasional single spot per cell nucleus as previously reported (Figure 10A and Figure 10—figure supplement 1A) (van Heusden and Steensma, 2008). Notably, when examined in tdp1Δ cells, GFP-Wss1 showed multiple foci, reflecting, probably, multiple sites of DNA damage (Figure 10A). Consistent with this conclusion, treatment of TDP1 cells with the UV mimetic 4-NQO also induced multiple Wss1 foci (Figure 10—figure supplement 1A). The foci were not observed with the GFP-Wss1-ΔSIM2 construct, suggesting SUMO-dependent targeting of Wss1 to these foci (Figure 10A).10.7554/eLife.06763.029Figure 10.Wss1 localization upon 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 localization upon DNA damage.(A) Intracellular localization of Wss1. Wss1-GFP fusion protein was expressed from pUG35 plasmid under control of the inducible MET25 promoter. Live cells (BY4742) were stained with DAPI and analyzed by fluorescence microscopy. Low (SD-Ura medium) and high (SD-Ura-Met) level of expression conditions are shown. Right column shows cells pre-incubated with 0.2 µg/ml 4-NQO. Histogram shows percentage of cells containing Wss1 foci in control (n = 628) and 4-NQO-treated (n = 498) cells. Scale bar is 2 µm. (B) Cellular stress induces vacuolar accumulation of Wss1. Wss1-GFP fusion protein was expressed in BY4742 and smt3-331cells from pUG35 plasmid. BY4742 cells were treated or not with 0.2 µg/ml 4-NQO for 3 hr. The cells were then stained with FM4-64 and analyzed by fluorescence microscopy. Histogram shows percentage of cells with vacuolar GFP signal in control (n = 638) and 4-NQO-treated (n = 513) BY4742 cells as well as in smt3-331 (n = 201, SBY331) cells and parental SMT3 strain (n = 316, SBY21). Scale bar is 2 µm. (C) Top1 overexpression induces accumulation of Wss1 in vacuole. The images show accumulation of Wss1-GFP in vacuole in pGAL-MBP-TOP1 WSS1-GFP tdp1Δ cells (MBY47 strain) after induction of MBP-Top1 protein in galactose medium. Live cells were stained with vacuolar marker FM4-64 and analyzed by fluorescence microscopy. All selected images show bright fluorescent spots within the cytoplasm and on the vacuolar membrane suggesting the activation of specific autophagic pathway. Scale bar is 2 µm. See also corresponding source files: Figure 10—source data 2.xlsx (for supplement A and B) and Figure 10—source data 1.xlsx (for supplement C).DOI:http://dx.doi.org/10.7554/eLife.06763.032
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559962&req=5

fig10s1: Wss1 localization upon DNA damage.(A) Intracellular localization of Wss1. Wss1-GFP fusion protein was expressed from pUG35 plasmid under control of the inducible MET25 promoter. Live cells (BY4742) were stained with DAPI and analyzed by fluorescence microscopy. Low (SD-Ura medium) and high (SD-Ura-Met) level of expression conditions are shown. Right column shows cells pre-incubated with 0.2 µg/ml 4-NQO. Histogram shows percentage of cells containing Wss1 foci in control (n = 628) and 4-NQO-treated (n = 498) cells. Scale bar is 2 µm. (B) Cellular stress induces vacuolar accumulation of Wss1. Wss1-GFP fusion protein was expressed in BY4742 and smt3-331cells from pUG35 plasmid. BY4742 cells were treated or not with 0.2 µg/ml 4-NQO for 3 hr. The cells were then stained with FM4-64 and analyzed by fluorescence microscopy. Histogram shows percentage of cells with vacuolar GFP signal in control (n = 638) and 4-NQO-treated (n = 513) BY4742 cells as well as in smt3-331 (n = 201, SBY331) cells and parental SMT3 strain (n = 316, SBY21). Scale bar is 2 µm. (C) Top1 overexpression induces accumulation of Wss1 in vacuole. The images show accumulation of Wss1-GFP in vacuole in pGAL-MBP-TOP1 WSS1-GFP tdp1Δ cells (MBY47 strain) after induction of MBP-Top1 protein in galactose medium. Live cells were stained with vacuolar marker FM4-64 and analyzed by fluorescence microscopy. All selected images show bright fluorescent spots within the cytoplasm and on the vacuolar membrane suggesting the activation of specific autophagic pathway. Scale bar is 2 µm. See also corresponding source files: Figure 10—source data 2.xlsx (for supplement A and B) and Figure 10—source data 1.xlsx (for supplement C).DOI:http://dx.doi.org/10.7554/eLife.06763.032
Mentions: To examine the site(s) of Wss1 action, we analyzed cellular localization of this protein. GFP-Wss1 showed nuclear staining with an occasional single spot per cell nucleus as previously reported (Figure 10A and Figure 10—figure supplement 1A) (van Heusden and Steensma, 2008). Notably, when examined in tdp1Δ cells, GFP-Wss1 showed multiple foci, reflecting, probably, multiple sites of DNA damage (Figure 10A). Consistent with this conclusion, treatment of TDP1 cells with the UV mimetic 4-NQO also induced multiple Wss1 foci (Figure 10—figure supplement 1A). The foci were not observed with the GFP-Wss1-ΔSIM2 construct, suggesting SUMO-dependent targeting of Wss1 to these foci (Figure 10A).10.7554/eLife.06763.029Figure 10.Wss1 localization upon 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