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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 and STUbL may have common cellular substrates.Wss1 counteracts ubiquitylation of SUMO conjugates by STUbL. HA-Wss1 constructs or pYEPGAP-URA3 vector alone (V, see Supplementary file 4 for constructs description) was transformed into EJY251-11b cells that express HIS6-FLAG-tagged (HF)-SUMO as the only SUMO source (Supplementary file 3). Cells were grown to early exponential phase in galactose and proline-containing medium and treated or not with 75 μM MG-132 for 3 hr. Whole cell lysates (WCLs) were prepared under denaturing conditions and SUMO conjugates were isolated on Ni-NTA agarose. The fractions were analyzed by western blotting with specific antibodies (α-SUMO and α-Ub). Bottom western blot shows HA-Wss1 constructs in WCL.DOI:http://dx.doi.org/10.7554/eLife.06763.012
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fig4s1: Wss1 and STUbL may have common cellular substrates.Wss1 counteracts ubiquitylation of SUMO conjugates by STUbL. HA-Wss1 constructs or pYEPGAP-URA3 vector alone (V, see Supplementary file 4 for constructs description) was transformed into EJY251-11b cells that express HIS6-FLAG-tagged (HF)-SUMO as the only SUMO source (Supplementary file 3). Cells were grown to early exponential phase in galactose and proline-containing medium and treated or not with 75 μM MG-132 for 3 hr. Whole cell lysates (WCLs) were prepared under denaturing conditions and SUMO conjugates were isolated on Ni-NTA agarose. The fractions were analyzed by western blotting with specific antibodies (α-SUMO and α-Ub). Bottom western blot shows HA-Wss1 constructs in WCL.DOI:http://dx.doi.org/10.7554/eLife.06763.012

Mentions: Wss1 has been reported to trim Ub from STUbL substrates (Mullen et al., 2010) so we asked if expression of Wss1 reduced the levels of ubiquitylated SUMO conjugates. Indeed, expression of Wss1 reduced the amount of ubiquitylated SUMO species in the cell but this reduction was independent of its metalloprotease active site (Figure 4—figure supplement 1). This suggests that the effect of Wss1 on levels of ubiquitylated SUMO conjugates could be due to competition with STUbL for binding of SUMOylated proteins.


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 and STUbL may have common cellular substrates.Wss1 counteracts ubiquitylation of SUMO conjugates by STUbL. HA-Wss1 constructs or pYEPGAP-URA3 vector alone (V, see Supplementary file 4 for constructs description) was transformed into EJY251-11b cells that express HIS6-FLAG-tagged (HF)-SUMO as the only SUMO source (Supplementary file 3). Cells were grown to early exponential phase in galactose and proline-containing medium and treated or not with 75 μM MG-132 for 3 hr. Whole cell lysates (WCLs) were prepared under denaturing conditions and SUMO conjugates were isolated on Ni-NTA agarose. The fractions were analyzed by western blotting with specific antibodies (α-SUMO and α-Ub). Bottom western blot shows HA-Wss1 constructs in WCL.DOI:http://dx.doi.org/10.7554/eLife.06763.012
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559962&req=5

fig4s1: Wss1 and STUbL may have common cellular substrates.Wss1 counteracts ubiquitylation of SUMO conjugates by STUbL. HA-Wss1 constructs or pYEPGAP-URA3 vector alone (V, see Supplementary file 4 for constructs description) was transformed into EJY251-11b cells that express HIS6-FLAG-tagged (HF)-SUMO as the only SUMO source (Supplementary file 3). Cells were grown to early exponential phase in galactose and proline-containing medium and treated or not with 75 μM MG-132 for 3 hr. Whole cell lysates (WCLs) were prepared under denaturing conditions and SUMO conjugates were isolated on Ni-NTA agarose. The fractions were analyzed by western blotting with specific antibodies (α-SUMO and α-Ub). Bottom western blot shows HA-Wss1 constructs in WCL.DOI:http://dx.doi.org/10.7554/eLife.06763.012
Mentions: Wss1 has been reported to trim Ub from STUbL substrates (Mullen et al., 2010) so we asked if expression of Wss1 reduced the levels of ubiquitylated SUMO conjugates. Indeed, expression of Wss1 reduced the amount of ubiquitylated SUMO species in the cell but this reduction was independent of its metalloprotease active site (Figure 4—figure supplement 1). This suggests that the effect of Wss1 on levels of ubiquitylated SUMO conjugates could be due to competition with STUbL for binding of SUMOylated proteins.

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