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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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VWA regulates S5a binding to ubiquitin and Ubl. (A) S5a kd cells transfected with the indicated S5a constructs were Velcade treated and evaluated for polyubiquitin, PSMA1, and S5a content (input). S5a IP from the indicated transfections reveals the higher binding capacity of S5a ΔVWA for polyubiquitin and its inability to bind proteasomes (IP-S5a). Proteasome purifications (PSMA1 IP) show the lack of S5aΔVWA binding and reduced polyubiquitin content upon S5aΔVWA expression. (B) Tibialis anterior muscles were electroporated with the indicated expression vectors ,and 4 d later mice were deprived of food for 2 d as indicated. In fed mice, muscles were dissected 6 d after electroporation. Polyubiquitin and S5a contents of muscles were revealed by immunoblots. (C) Recombinant full-length or ΔVWA-domain S5a were bacterially expressed and purified (left; Coomassie). Affinities and thermodynamic parameters of interactions with ubiquitin were determined by ITC (right). (D) Left , S5a kd cells were transfected with Ha-ATF4 and the indicated S5a constructs were evaluated for their ATF4 content (input). S5a IP from the indicated transfections reveals the enhanced purification of polyubiquitinated ATF4 only upon S5a ΔVWA transfection (IP-S5a). Right, polyubiquitinated ATF4 was incubated with proteasomes (purified from S5a kd cells) and increasing amounts of the indicated recombinant S5a. Proteasomal and polyubiquitinated ATF4 contents were evaluated by the indicated immunoblots (PSMA1 and HA, respectively).
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Figure 5: VWA regulates S5a binding to ubiquitin and Ubl. (A) S5a kd cells transfected with the indicated S5a constructs were Velcade treated and evaluated for polyubiquitin, PSMA1, and S5a content (input). S5a IP from the indicated transfections reveals the higher binding capacity of S5a ΔVWA for polyubiquitin and its inability to bind proteasomes (IP-S5a). Proteasome purifications (PSMA1 IP) show the lack of S5aΔVWA binding and reduced polyubiquitin content upon S5aΔVWA expression. (B) Tibialis anterior muscles were electroporated with the indicated expression vectors ,and 4 d later mice were deprived of food for 2 d as indicated. In fed mice, muscles were dissected 6 d after electroporation. Polyubiquitin and S5a contents of muscles were revealed by immunoblots. (C) Recombinant full-length or ΔVWA-domain S5a were bacterially expressed and purified (left; Coomassie). Affinities and thermodynamic parameters of interactions with ubiquitin were determined by ITC (right). (D) Left , S5a kd cells were transfected with Ha-ATF4 and the indicated S5a constructs were evaluated for their ATF4 content (input). S5a IP from the indicated transfections reveals the enhanced purification of polyubiquitinated ATF4 only upon S5a ΔVWA transfection (IP-S5a). Right, polyubiquitinated ATF4 was incubated with proteasomes (purified from S5a kd cells) and increasing amounts of the indicated recombinant S5a. Proteasomal and polyubiquitinated ATF4 contents were evaluated by the indicated immunoblots (PSMA1 and HA, respectively).

Mentions: How might S5a prefer binding to Ubl and ubiquitin only in its proteasome-bound state? Several catalytic activities of the proteasome are restricted to occur only in the intact assembled proteasome (Yao and Cohen, 2002; Yao et al., 2006; Verma et al., 2004; Murata et al., 2009; Lee et al., 2010). Several of these restriction mechanisms include an intramolecular fold that upon proteasomal binding enables catalytic activity (Hamazaki et al., 2006; Yao et al., 2006). To evaluate whether S5a binding to ubiquitin is restricted to the proteasome, we deleted the N-terminal VWA domain from S5a, thus excluding the protein's distribution from the proteasome (Fu et al., 1998). As shown in Figure 5A, expression of wild-type (WT) but not a UIM mutant S5a (S5a A219/290Q) in S5a kd cells reduced polyubiquitin levels. In contrast, S5aΔVWA expression induced polyubiquitin levels. On proteasomal purification (PSMA1 IP), we observe elevated levels of S5a in the proteasome (WT and UIM mutant) but not S5aΔVWA, as expected. Furthermore, the expression of S5aΔVWA seemed to reduce the amount of polyubiquitin bound to proteasomes in spite of their higher level upon S5aΔVWA expression (Figure 5A; compare input to PSMA1 IP). In contrast, upon S5a purification, we noted an increase in polyubiquitin association in WT but not the UIM S5a mutant. S5aΔVWA expression increased polyubiquitin binding even further and did not show any proteasomal association (Figure 5A; S5a IP). These results indicate that S5aΔVWA expression competes with the proteasome for binding to polyubiquitinated proteins. Therefore, in addition to its role as a proteasome-binding domain, the VWA domain has an inhibitory role toward S5a binding to ubiquitin.


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)

VWA regulates S5a binding to ubiquitin and Ubl. (A) S5a kd cells transfected with the indicated S5a constructs were Velcade treated and evaluated for polyubiquitin, PSMA1, and S5a content (input). S5a IP from the indicated transfections reveals the higher binding capacity of S5a ΔVWA for polyubiquitin and its inability to bind proteasomes (IP-S5a). Proteasome purifications (PSMA1 IP) show the lack of S5aΔVWA binding and reduced polyubiquitin content upon S5aΔVWA expression. (B) Tibialis anterior muscles were electroporated with the indicated expression vectors ,and 4 d later mice were deprived of food for 2 d as indicated. In fed mice, muscles were dissected 6 d after electroporation. Polyubiquitin and S5a contents of muscles were revealed by immunoblots. (C) Recombinant full-length or ΔVWA-domain S5a were bacterially expressed and purified (left; Coomassie). Affinities and thermodynamic parameters of interactions with ubiquitin were determined by ITC (right). (D) Left , S5a kd cells were transfected with Ha-ATF4 and the indicated S5a constructs were evaluated for their ATF4 content (input). S5a IP from the indicated transfections reveals the enhanced purification of polyubiquitinated ATF4 only upon S5a ΔVWA transfection (IP-S5a). Right, polyubiquitinated ATF4 was incubated with proteasomes (purified from S5a kd cells) and increasing amounts of the indicated recombinant S5a. Proteasomal and polyubiquitinated ATF4 contents were evaluated by the indicated immunoblots (PSMA1 and HA, respectively).
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Figure 5: VWA regulates S5a binding to ubiquitin and Ubl. (A) S5a kd cells transfected with the indicated S5a constructs were Velcade treated and evaluated for polyubiquitin, PSMA1, and S5a content (input). S5a IP from the indicated transfections reveals the higher binding capacity of S5a ΔVWA for polyubiquitin and its inability to bind proteasomes (IP-S5a). Proteasome purifications (PSMA1 IP) show the lack of S5aΔVWA binding and reduced polyubiquitin content upon S5aΔVWA expression. (B) Tibialis anterior muscles were electroporated with the indicated expression vectors ,and 4 d later mice were deprived of food for 2 d as indicated. In fed mice, muscles were dissected 6 d after electroporation. Polyubiquitin and S5a contents of muscles were revealed by immunoblots. (C) Recombinant full-length or ΔVWA-domain S5a were bacterially expressed and purified (left; Coomassie). Affinities and thermodynamic parameters of interactions with ubiquitin were determined by ITC (right). (D) Left , S5a kd cells were transfected with Ha-ATF4 and the indicated S5a constructs were evaluated for their ATF4 content (input). S5a IP from the indicated transfections reveals the enhanced purification of polyubiquitinated ATF4 only upon S5a ΔVWA transfection (IP-S5a). Right, polyubiquitinated ATF4 was incubated with proteasomes (purified from S5a kd cells) and increasing amounts of the indicated recombinant S5a. Proteasomal and polyubiquitinated ATF4 contents were evaluated by the indicated immunoblots (PSMA1 and HA, respectively).
Mentions: How might S5a prefer binding to Ubl and ubiquitin only in its proteasome-bound state? Several catalytic activities of the proteasome are restricted to occur only in the intact assembled proteasome (Yao and Cohen, 2002; Yao et al., 2006; Verma et al., 2004; Murata et al., 2009; Lee et al., 2010). Several of these restriction mechanisms include an intramolecular fold that upon proteasomal binding enables catalytic activity (Hamazaki et al., 2006; Yao et al., 2006). To evaluate whether S5a binding to ubiquitin is restricted to the proteasome, we deleted the N-terminal VWA domain from S5a, thus excluding the protein's distribution from the proteasome (Fu et al., 1998). As shown in Figure 5A, expression of wild-type (WT) but not a UIM mutant S5a (S5a A219/290Q) in S5a kd cells reduced polyubiquitin levels. In contrast, S5aΔVWA expression induced polyubiquitin levels. On proteasomal purification (PSMA1 IP), we observe elevated levels of S5a in the proteasome (WT and UIM mutant) but not S5aΔVWA, as expected. Furthermore, the expression of S5aΔVWA seemed to reduce the amount of polyubiquitin bound to proteasomes in spite of their higher level upon S5aΔVWA expression (Figure 5A; compare input to PSMA1 IP). In contrast, upon S5a purification, we noted an increase in polyubiquitin association in WT but not the UIM S5a mutant. S5aΔVWA expression increased polyubiquitin binding even further and did not show any proteasomal association (Figure 5A; S5a IP). These results indicate that S5aΔVWA expression competes with the proteasome for binding to polyubiquitinated proteins. Therefore, in addition to its role as a proteasome-binding domain, the VWA domain has an inhibitory role toward S5a binding to ubiquitin.

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