VWA domain of S5a restricts the ability to bind ubiquitin and Ubl to the 26S proteasome.
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.
Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.Show MeSH
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.
Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.