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VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome.

Piterman R, Braunstein I, Isakov E, Ziv T, Navon A, Cohen S, Stanhill A - Mol. Biol. Cell (2014)

Bottom Line: We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association.Multiubiquitination events within the VWA domain can further regulate S5a association.Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.

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S5a multiubiquitination. (A) S5a IP was performed from LMW and HMW cellular fractions and immunoblotted for proteasomal (PSMA1) and S5a content. Note the appearance of a high-mobility S5a form in the LMW fractions (arrow). (B) Exogenous GFP-S5a was expressed in cells and distribution evaluated as in Figure 1A using a GFP immunoblot. Fractions along the gradient were presented as is or treated with the deubiquitinating enzyme USP2. Note the disappearance of the higher-mobility S5a-reactive bands upon USP2 addition. (C) Coomassie gel of GFP (control) and GFP-S5a, indicating the ubiquitinated bands excised for MS identification. MS spectra of the three identified sites and their aligned localization within the S5a VWA domain. (D) Cyclohexamide experiments revealing the long half-life of S5a and PSMA1 as opposed to the short half-life of an ERAD substrate (γV-CH1). (E) Cells transfected with HA-ATF4 and the indicated S5a were evaluated for their ATF4, S5a, and hPlic content (input). S5a IP from the indicated transfections was evaluated for copurification of hPlic and ATF4, revealing the partial ability of S5aKtoR mutant to enhance Ubl-dependent interaction with hPlic (IP-S5a).
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Figure 6: S5a multiubiquitination. (A) S5a IP was performed from LMW and HMW cellular fractions and immunoblotted for proteasomal (PSMA1) and S5a content. Note the appearance of a high-mobility S5a form in the LMW fractions (arrow). (B) Exogenous GFP-S5a was expressed in cells and distribution evaluated as in Figure 1A using a GFP immunoblot. Fractions along the gradient were presented as is or treated with the deubiquitinating enzyme USP2. Note the disappearance of the higher-mobility S5a-reactive bands upon USP2 addition. (C) Coomassie gel of GFP (control) and GFP-S5a, indicating the ubiquitinated bands excised for MS identification. MS spectra of the three identified sites and their aligned localization within the S5a VWA domain. (D) Cyclohexamide experiments revealing the long half-life of S5a and PSMA1 as opposed to the short half-life of an ERAD substrate (γV-CH1). (E) Cells transfected with HA-ATF4 and the indicated S5a were evaluated for their ATF4, S5a, and hPlic content (input). S5a IP from the indicated transfections was evaluated for copurification of hPlic and ATF4, revealing the partial ability of S5aKtoR mutant to enhance Ubl-dependent interaction with hPlic (IP-S5a).

Mentions: Monoubiquitination events on Rpn10 have been described in yeast and Drosophila (Isasa et al., 2010; Lipinszki et al., 2012); however the multiubiquitination pattern in the two species was quite different. Recent reports also indicate ubiquitination of proteasomal ubiquitin receptors S5a and Rpn13 as a regulatory step in proteasomal substrate regulation (Besche et al., 2014; Jacobson et al., 2014). To evaluate the ubiquitination status of S5a in mammals, we immunoprecipitated S5a from proteasome-free and -bound fractions. As seen in Figure 6A, lower-mobility migration of S5a could be detected mainly in the proteasome-free fractions. The specific distribution of this event on S5a is readily seen when ectopic expression of green fluorescent protein (GFP)-S5a is performed and the reactivity of GFP-S5a is observed along the various fractions. As noted in Figure 6B (top), the lower-mobility GFP-S5a was restricted mainly to the LMW fractions, and this altered mobility could be abolished by incubation with a deubiquitinating enzyme (Figure 6B, bottom), indicating that ubiquitin is the posttranslational modification responsible for the abnormal migration of S5a in the LMW fractions. Using the robustness of the ubiquitination events on ectopically expressed S5a enabled us to excise lower-mobility, Coomassie-stained bands from affinity-purified GFP-S5a and identify three in vivo monoubiquitination events on S5a, all occurring within the VWA domain (Figure 6C). These results are the first to characterize mammalian S5a ubiquitination sites and differ from those obtained by in vitro ubiquitination of Rpn10 (Isasa et al., 2010) and the reported C-terminal end of Drosophila S5a/P54 (Lipinszki et al., 2009). All three sites in S5a (K74, K122, and K126) are absent in the yeast Rpn10, and K122 is absent in Drosophila P54 (see Figure 6C for sequence alignment). Multiubiquitination of S5a does not seem to regulate protein stability, as S5a did not show a reduced half-life compared with other, long-lived proteasomal proteins (Figure 6D), in line with previous reports in Drosophila showing that ubiquitination events on P54 do not alter its own stability (Lipinszki et al., 2009). We were able to confirm the previously reported reduced ubiquitin binding, but not proteasomal binding, of S5a-Ubi (a C-terminal fusion with ubiquitin reported to mimic Rpn10 ubiquitination and reduce ubiquitin binding; Isasa et al., 2010; Supplemental Figure S3). Using a S5a mutant that is unable to undergo ubiquitination (KtoR mutant; unpublished data) did not reveal any changes in the ability to bind 26S proteasomes (Supplemental Figure S3). Use of this mutant can provide a tool to address the question of whether the enhanced capacity to bind polyubiquitinated ATF4 by S5aΔVWA is due to the lack of multiubiquitination or is part of an intrinsic property of VWA to regulate S5a ubiquitin binding. As seen in Figure 6E, S5a KtoR did not increase S5a binding to polyubiquitinated ATF4 as observed with S5aΔVWA. Because our previous results demonstrated S5a dependence for hPlic recruitment to the proteasome (Figure 3), we also addressed the ability of S5a KtoR and S5aΔVWA mutants to bind hPlic. Both mutations exhibited enhancement of hPlic binding, confirming the difference between Ubl and ubiquitin binding by S5a (our data; Walters et al., 2002), as indicated by the ability of the KtoR mutant to enhance only Ubl binding. Thus we conclude that the multiubiquitination events found in the LMW fractions on S5a (Figure 3A) can contribute to the ability of the VWA domain to restrict S5a ability to bind Ubl only upon proteasomal integration.


VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome.

Piterman R, Braunstein I, Isakov E, Ziv T, Navon A, Cohen S, Stanhill A - Mol. Biol. Cell (2014)

S5a multiubiquitination. (A) S5a IP was performed from LMW and HMW cellular fractions and immunoblotted for proteasomal (PSMA1) and S5a content. Note the appearance of a high-mobility S5a form in the LMW fractions (arrow). (B) Exogenous GFP-S5a was expressed in cells and distribution evaluated as in Figure 1A using a GFP immunoblot. Fractions along the gradient were presented as is or treated with the deubiquitinating enzyme USP2. Note the disappearance of the higher-mobility S5a-reactive bands upon USP2 addition. (C) Coomassie gel of GFP (control) and GFP-S5a, indicating the ubiquitinated bands excised for MS identification. MS spectra of the three identified sites and their aligned localization within the S5a VWA domain. (D) Cyclohexamide experiments revealing the long half-life of S5a and PSMA1 as opposed to the short half-life of an ERAD substrate (γV-CH1). (E) Cells transfected with HA-ATF4 and the indicated S5a were evaluated for their ATF4, S5a, and hPlic content (input). S5a IP from the indicated transfections was evaluated for copurification of hPlic and ATF4, revealing the partial ability of S5aKtoR mutant to enhance Ubl-dependent interaction with hPlic (IP-S5a).
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Figure 6: S5a multiubiquitination. (A) S5a IP was performed from LMW and HMW cellular fractions and immunoblotted for proteasomal (PSMA1) and S5a content. Note the appearance of a high-mobility S5a form in the LMW fractions (arrow). (B) Exogenous GFP-S5a was expressed in cells and distribution evaluated as in Figure 1A using a GFP immunoblot. Fractions along the gradient were presented as is or treated with the deubiquitinating enzyme USP2. Note the disappearance of the higher-mobility S5a-reactive bands upon USP2 addition. (C) Coomassie gel of GFP (control) and GFP-S5a, indicating the ubiquitinated bands excised for MS identification. MS spectra of the three identified sites and their aligned localization within the S5a VWA domain. (D) Cyclohexamide experiments revealing the long half-life of S5a and PSMA1 as opposed to the short half-life of an ERAD substrate (γV-CH1). (E) Cells transfected with HA-ATF4 and the indicated S5a were evaluated for their ATF4, S5a, and hPlic content (input). S5a IP from the indicated transfections was evaluated for copurification of hPlic and ATF4, revealing the partial ability of S5aKtoR mutant to enhance Ubl-dependent interaction with hPlic (IP-S5a).
Mentions: Monoubiquitination events on Rpn10 have been described in yeast and Drosophila (Isasa et al., 2010; Lipinszki et al., 2012); however the multiubiquitination pattern in the two species was quite different. Recent reports also indicate ubiquitination of proteasomal ubiquitin receptors S5a and Rpn13 as a regulatory step in proteasomal substrate regulation (Besche et al., 2014; Jacobson et al., 2014). To evaluate the ubiquitination status of S5a in mammals, we immunoprecipitated S5a from proteasome-free and -bound fractions. As seen in Figure 6A, lower-mobility migration of S5a could be detected mainly in the proteasome-free fractions. The specific distribution of this event on S5a is readily seen when ectopic expression of green fluorescent protein (GFP)-S5a is performed and the reactivity of GFP-S5a is observed along the various fractions. As noted in Figure 6B (top), the lower-mobility GFP-S5a was restricted mainly to the LMW fractions, and this altered mobility could be abolished by incubation with a deubiquitinating enzyme (Figure 6B, bottom), indicating that ubiquitin is the posttranslational modification responsible for the abnormal migration of S5a in the LMW fractions. Using the robustness of the ubiquitination events on ectopically expressed S5a enabled us to excise lower-mobility, Coomassie-stained bands from affinity-purified GFP-S5a and identify three in vivo monoubiquitination events on S5a, all occurring within the VWA domain (Figure 6C). These results are the first to characterize mammalian S5a ubiquitination sites and differ from those obtained by in vitro ubiquitination of Rpn10 (Isasa et al., 2010) and the reported C-terminal end of Drosophila S5a/P54 (Lipinszki et al., 2009). All three sites in S5a (K74, K122, and K126) are absent in the yeast Rpn10, and K122 is absent in Drosophila P54 (see Figure 6C for sequence alignment). Multiubiquitination of S5a does not seem to regulate protein stability, as S5a did not show a reduced half-life compared with other, long-lived proteasomal proteins (Figure 6D), in line with previous reports in Drosophila showing that ubiquitination events on P54 do not alter its own stability (Lipinszki et al., 2009). We were able to confirm the previously reported reduced ubiquitin binding, but not proteasomal binding, of S5a-Ubi (a C-terminal fusion with ubiquitin reported to mimic Rpn10 ubiquitination and reduce ubiquitin binding; Isasa et al., 2010; Supplemental Figure S3). Using a S5a mutant that is unable to undergo ubiquitination (KtoR mutant; unpublished data) did not reveal any changes in the ability to bind 26S proteasomes (Supplemental Figure S3). Use of this mutant can provide a tool to address the question of whether the enhanced capacity to bind polyubiquitinated ATF4 by S5aΔVWA is due to the lack of multiubiquitination or is part of an intrinsic property of VWA to regulate S5a ubiquitin binding. As seen in Figure 6E, S5a KtoR did not increase S5a binding to polyubiquitinated ATF4 as observed with S5aΔVWA. Because our previous results demonstrated S5a dependence for hPlic recruitment to the proteasome (Figure 3), we also addressed the ability of S5a KtoR and S5aΔVWA mutants to bind hPlic. Both mutations exhibited enhancement of hPlic binding, confirming the difference between Ubl and ubiquitin binding by S5a (our data; Walters et al., 2002), as indicated by the ability of the KtoR mutant to enhance only Ubl binding. Thus we conclude that the multiubiquitination events found in the LMW fractions on S5a (Figure 3A) can contribute to the ability of the VWA domain to restrict S5a ability to bind Ubl only upon proteasomal integration.

Bottom Line: We identify the VWA domain of S5a as a domain that limits ubiquitin and Ubl binding to occur only upon proteasomal association.Multiubiquitination events within the VWA domain can further regulate S5a association.Our results provide a molecular explanation to how ubiquitin and Ubl binding to S5a is restricted to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.

Show MeSH