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 evaluate the cellular role of S5a and to address the cellular consequences of S5a impairments, we established a cell line in which we used RNA interference (RNAi) to obtain >90% reduction in S5a expression (Figure 1A). On continuous culturing of the established cell line (S5a kd), we noted that the cells adapted to the knockdown by overexpressing S5a, thus compensating for the reduced levels of S5a (Figure 1A). This finding reflects the fact that no compensation for the reduction of S5a was established in the S5a kd cells at early time points; thus changes observed in these cells are a direct consequence of S5a reduction and not part of a compensatory response to the reduction. This can also be observed by the elevated basal levels of p27Kip1 in nonsynchronized S5a kd cells, an elevation that is reduced upon S5a reappearance (Figure 1A). The high basal levels of p27Kip1 in the S5a kd cells is part of a cell cycle impairment observed in these cells (an attenuated transition into the G1 phase during normal cell cycle conditions and an increased sensitivity of these cells toward a sublethal dosage of the protein misfolding agent arsenite; Supplemental Figure S1). Subsequently all experiments in the S5a kd cells were performed on freshly thawed cells after S5a expression evaluation. Furthermore, proteasome complexes purified from control and S5a kd cells indicated complex integrity (Coomassie staining and complex sedimentation) and functionality (LLVY peptide hydrolysis); therefore S5a kd cells maintain an intact and functional proteasome. When challenging S5a kd cells with various protein misfolding agents (arsenite, tunicamycin, thapsigargin, or the proteasome inhibitor Velcade) we did not observe changes in polyubiquitin accumulation between S5a kd and control cells. Instead, the main observed difference was at the basal level of polyubiquitin in the nonstressed cells. Figure 1B indicates a threefold increase in the polyubiquitin signal in S5a kd cells under conditions without stress, consistent with a recent report showing higher basal levels of polyubiquitin upon S5a reduction (Sparks et al., 2014). Thus S5a seems to play an important role in maintaining efficient processing of polyubiquitinated substrates by the proteasome. In yeast the Rpn10 proteasomal subunit was found in free and proteasome-bound fractions (van Nocker et al., 1996; Matiuhin et al., 2008). The S5a kd cells were further characterized with respect to the distribution of S5a in glycerol velocity gradients. On evaluating S5a distribution in the S5a kd cells, we did not observe S5a presence in proteasome-free fractions in spite of the excess material loaded on the S5a kd cell lysate gradient (Figure 1C). These results imply that proteasome-bound S5a is a stable component of the proteasome complex that does not exchange with the proteasome-“free” isoform, consistent with its stoichiometric presence in the proteasome (Berko et al., 2014).
Affiliation: Department of Biochemistry, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, Haifa 31096, Israel.