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
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Mentions: To further evaluate the role of S5a as a proteasomal Ubl recruitment factor, we affinity purified 26S proteasomes from control and S5a kd cells and evaluated them for the Ubl-dependent shuttling factor hPlic-2 (UBQLN2; the mammalian homologue of yeast Dsk2; Kleijnen et al., 2000). As noted in Figure 3A, most hPlic is not associated with higher–molecular weight (HMW) proteasomal fractions, consistent with its role as a proteasome shuttle factor (Kleijnen et al., 2003). To evaluate the dependence of hPlic on S5a proteasomal recruitment, proteasomes purified from control and S5a kd cells were evaluated for their hPlic content. A significant reduction in hPlic content was observed in the S5a kd cells (Figure 3B), implying a central role for S5a in Ubl proteasomal recruitment. To extend this evaluation to additional Ubl's, we used the ability of fusion proteins with a Ubl domain to enable proteasomal degradation (Kraut and Matouschek, 2011). We fused a Ubl domain from RAD23 to Sic1 and monitored the ability of the Ubl domain to enable proteasomal binding and degradation. On incubation of the Ubl-Sic1 substrate with proteasomes purified from control and S5a kd cells, we noted reduced binding of the substrate to the S5a kd proteasomes (Figure 3C). The outcome of the reduced proteasomal binding of Ubl in the S5a kd cells is reflected in an in vitro degradation assay in which the attenuated degradation rates of the Ubl substrate are observed in the S5a kd cells (Figure 3D).
Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.