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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

Characterization of Wss1-Cdc48-Doa1 interaction.(A, B) Interaction of Wss1 with Cdc48 and adaptor proteins reveals Wss1 specificity for Doa1. All proteins were expressed in bacteria and purified to homogeneity. The proteins were pulled down with amylose beads in the presence or absence of recombinant MBP-Wss1 protein. Panel A: the ‘input’ (I) and ‘bound’ fractions (B) were analyzed by PAGE with Coomassie staining (volume of the ‘bound’ fraction was adjusted to have the same dilution as in ‘input’ fraction). Because HF-Doa1 migrates at the same position as MBP-Wss1 no additional band was seen in MBP-Wss1 bound fraction (lane 7). To ascertain that HF-Doa1 binds to MBP-Wss1 all bound fractions were additionally analyzed by western blotting with α-His6 antibodies (panel B). (C) Interaction of Wss1 with preformed Cdc48 complexes confirms ternary 1:1:1 Wss1/Doa1/Cdc48 complex. The Cdc48 complexes were preformed from purified GST-Cdc48 with a double molar excess of the indicated protein adaptors. Next, MBP-Wss1 was added in stoichiometric amount and Cdc48 complexes were separated on glutathione beads. The bound complexes were analyzed by PAGE with Coomassie staining, and quantified by ImageJ. The bottom picture shows the models of Cdc48 complexes resulted from ImageJ measurements. (D) Mass spectrometry analysis of Doa1-interacting proteins identifies Wss1. Purified HF-Doa1 protein was mixed with whole cell lysate and retrieved, along with the partner proteins, on Ni-NTA resin (see ‘Materials and methods’). In the control experiment, the lysate was passed through the resin without HF-Doa1. The proteins specifically retrieved with HF-Doa1 were identified by mass spectrometry. Top seven interactors are shown in the table (see also Supplementary file 2 for complete list). (E) Wss1-dependent binding of Cdc48 and Doa1 to SUMO beads. Purified of His6-Cdc48 and his6-Flag (HF)-Doa1 proteins were pulled down with SUMO beads with (right part of the blots) or without (left part) HA-Wss1 protein. The input (I) and bound (B) fractions were analyzed by western blotting. Input: 20%.DOI:http://dx.doi.org/10.7554/eLife.06763.018
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fig5s2: Characterization of Wss1-Cdc48-Doa1 interaction.(A, B) Interaction of Wss1 with Cdc48 and adaptor proteins reveals Wss1 specificity for Doa1. All proteins were expressed in bacteria and purified to homogeneity. The proteins were pulled down with amylose beads in the presence or absence of recombinant MBP-Wss1 protein. Panel A: the ‘input’ (I) and ‘bound’ fractions (B) were analyzed by PAGE with Coomassie staining (volume of the ‘bound’ fraction was adjusted to have the same dilution as in ‘input’ fraction). Because HF-Doa1 migrates at the same position as MBP-Wss1 no additional band was seen in MBP-Wss1 bound fraction (lane 7). To ascertain that HF-Doa1 binds to MBP-Wss1 all bound fractions were additionally analyzed by western blotting with α-His6 antibodies (panel B). (C) Interaction of Wss1 with preformed Cdc48 complexes confirms ternary 1:1:1 Wss1/Doa1/Cdc48 complex. The Cdc48 complexes were preformed from purified GST-Cdc48 with a double molar excess of the indicated protein adaptors. Next, MBP-Wss1 was added in stoichiometric amount and Cdc48 complexes were separated on glutathione beads. The bound complexes were analyzed by PAGE with Coomassie staining, and quantified by ImageJ. The bottom picture shows the models of Cdc48 complexes resulted from ImageJ measurements. (D) Mass spectrometry analysis of Doa1-interacting proteins identifies Wss1. Purified HF-Doa1 protein was mixed with whole cell lysate and retrieved, along with the partner proteins, on Ni-NTA resin (see ‘Materials and methods’). In the control experiment, the lysate was passed through the resin without HF-Doa1. The proteins specifically retrieved with HF-Doa1 were identified by mass spectrometry. Top seven interactors are shown in the table (see also Supplementary file 2 for complete list). (E) Wss1-dependent binding of Cdc48 and Doa1 to SUMO beads. Purified of His6-Cdc48 and his6-Flag (HF)-Doa1 proteins were pulled down with SUMO beads with (right part of the blots) or without (left part) HA-Wss1 protein. The input (I) and bound (B) fractions were analyzed by western blotting. Input: 20%.DOI:http://dx.doi.org/10.7554/eLife.06763.018

Mentions: The two most abundant Wss1 interactors were Doa1 and Cdc48. These interactions were further confirmed by immunoprecipitation of HA-Wss1 from DOA1-FLAG cell lysates (Figure 5B). Doa1 is a Cdc48 cofactor that binds the C-terminus of the chaperone through its PUL domain (Mullally et al., 2006). Because both Cdc48 binding motifs of Wss1, SHP, and VIM, should interact with the N-terminal domain of Cdc48 (Yeung et al., 2008; Hanzelmann and Schindelin, 2011; Stapf et al., 2011), we asked whether Wss1, Doa1, and Cdc48 form a ternary complex. Using purified recombinant proteins, we found that (i) Wss1 can bind Cdc48 and Doa1 separately (Figure 5C and Figure 5—figure supplement 2A,B), (ii) the proteins form a ternary 1:1:1 Wss1/Doa1/Cdc48 complex (Figure 5C and Figure 5—figure supplement 2C), and (iii) binding of Wss1 to Doa1 is specific, with few other Cdc48 cofactors observed in the pull-downs (Figure 5—figure supplement 2A–C).


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)

Characterization of Wss1-Cdc48-Doa1 interaction.(A, B) Interaction of Wss1 with Cdc48 and adaptor proteins reveals Wss1 specificity for Doa1. All proteins were expressed in bacteria and purified to homogeneity. The proteins were pulled down with amylose beads in the presence or absence of recombinant MBP-Wss1 protein. Panel A: the ‘input’ (I) and ‘bound’ fractions (B) were analyzed by PAGE with Coomassie staining (volume of the ‘bound’ fraction was adjusted to have the same dilution as in ‘input’ fraction). Because HF-Doa1 migrates at the same position as MBP-Wss1 no additional band was seen in MBP-Wss1 bound fraction (lane 7). To ascertain that HF-Doa1 binds to MBP-Wss1 all bound fractions were additionally analyzed by western blotting with α-His6 antibodies (panel B). (C) Interaction of Wss1 with preformed Cdc48 complexes confirms ternary 1:1:1 Wss1/Doa1/Cdc48 complex. The Cdc48 complexes were preformed from purified GST-Cdc48 with a double molar excess of the indicated protein adaptors. Next, MBP-Wss1 was added in stoichiometric amount and Cdc48 complexes were separated on glutathione beads. The bound complexes were analyzed by PAGE with Coomassie staining, and quantified by ImageJ. The bottom picture shows the models of Cdc48 complexes resulted from ImageJ measurements. (D) Mass spectrometry analysis of Doa1-interacting proteins identifies Wss1. Purified HF-Doa1 protein was mixed with whole cell lysate and retrieved, along with the partner proteins, on Ni-NTA resin (see ‘Materials and methods’). In the control experiment, the lysate was passed through the resin without HF-Doa1. The proteins specifically retrieved with HF-Doa1 were identified by mass spectrometry. Top seven interactors are shown in the table (see also Supplementary file 2 for complete list). (E) Wss1-dependent binding of Cdc48 and Doa1 to SUMO beads. Purified of His6-Cdc48 and his6-Flag (HF)-Doa1 proteins were pulled down with SUMO beads with (right part of the blots) or without (left part) HA-Wss1 protein. The input (I) and bound (B) fractions were analyzed by western blotting. Input: 20%.DOI:http://dx.doi.org/10.7554/eLife.06763.018
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fig5s2: Characterization of Wss1-Cdc48-Doa1 interaction.(A, B) Interaction of Wss1 with Cdc48 and adaptor proteins reveals Wss1 specificity for Doa1. All proteins were expressed in bacteria and purified to homogeneity. The proteins were pulled down with amylose beads in the presence or absence of recombinant MBP-Wss1 protein. Panel A: the ‘input’ (I) and ‘bound’ fractions (B) were analyzed by PAGE with Coomassie staining (volume of the ‘bound’ fraction was adjusted to have the same dilution as in ‘input’ fraction). Because HF-Doa1 migrates at the same position as MBP-Wss1 no additional band was seen in MBP-Wss1 bound fraction (lane 7). To ascertain that HF-Doa1 binds to MBP-Wss1 all bound fractions were additionally analyzed by western blotting with α-His6 antibodies (panel B). (C) Interaction of Wss1 with preformed Cdc48 complexes confirms ternary 1:1:1 Wss1/Doa1/Cdc48 complex. The Cdc48 complexes were preformed from purified GST-Cdc48 with a double molar excess of the indicated protein adaptors. Next, MBP-Wss1 was added in stoichiometric amount and Cdc48 complexes were separated on glutathione beads. The bound complexes were analyzed by PAGE with Coomassie staining, and quantified by ImageJ. The bottom picture shows the models of Cdc48 complexes resulted from ImageJ measurements. (D) Mass spectrometry analysis of Doa1-interacting proteins identifies Wss1. Purified HF-Doa1 protein was mixed with whole cell lysate and retrieved, along with the partner proteins, on Ni-NTA resin (see ‘Materials and methods’). In the control experiment, the lysate was passed through the resin without HF-Doa1. The proteins specifically retrieved with HF-Doa1 were identified by mass spectrometry. Top seven interactors are shown in the table (see also Supplementary file 2 for complete list). (E) Wss1-dependent binding of Cdc48 and Doa1 to SUMO beads. Purified of His6-Cdc48 and his6-Flag (HF)-Doa1 proteins were pulled down with SUMO beads with (right part of the blots) or without (left part) HA-Wss1 protein. The input (I) and bound (B) fractions were analyzed by western blotting. Input: 20%.DOI:http://dx.doi.org/10.7554/eLife.06763.018
Mentions: The two most abundant Wss1 interactors were Doa1 and Cdc48. These interactions were further confirmed by immunoprecipitation of HA-Wss1 from DOA1-FLAG cell lysates (Figure 5B). Doa1 is a Cdc48 cofactor that binds the C-terminus of the chaperone through its PUL domain (Mullally et al., 2006). Because both Cdc48 binding motifs of Wss1, SHP, and VIM, should interact with the N-terminal domain of Cdc48 (Yeung et al., 2008; Hanzelmann and Schindelin, 2011; Stapf et al., 2011), we asked whether Wss1, Doa1, and Cdc48 form a ternary complex. Using purified recombinant proteins, we found that (i) Wss1 can bind Cdc48 and Doa1 separately (Figure 5C and Figure 5—figure supplement 2A,B), (ii) the proteins form a ternary 1:1:1 Wss1/Doa1/Cdc48 complex (Figure 5C and Figure 5—figure supplement 2C), and (iii) binding of Wss1 to Doa1 is specific, with few other Cdc48 cofactors observed in the pull-downs (Figure 5—figure supplement 2A–C).

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