Binding of SGTA to Rpn13 selectively modulates protein quality control

Leznicki, Pawel; Korac-Prlic, Jelena; Kliza, Katarzyna; Husnjak, Koraljka; Nyathi, Yvonne; Dikic, Ivan; High, Stephen

Given that the central TPR domain of SGTA (Fig. 1B) is responsible for binding Rpn13 (see Fig. 2B), we speculated that this interaction is sensitive to point mutations that perturb the binding of other components to this region (Walczak et al., 2014). Hence, a previously defined K160E/R164E mutant version of SGTA that is defective in binding to Hsc70 was created (Walczak et al., 2014; Xu et al., 2012) and its ability to bind Rpn13 tested. Whereas a recombinant fusion protein containing wild-type SGTA (Leznicki and High, 2012) bound to both full-length Rpn13 and its C-terminal domain (Fig. 6A, His Trx-SGTA panel), no interaction with the K160E/R164E variant was detected (Fig. 6B, His Trx-SGTA panel). Likewise, when equivalent versions of these SGTA variants were expressed in parental HEK293T or HEK293Rpn11-HTBH cells (Fig. 6Ci and Di), the amount of the SGTA K160E/R164E-V5 mutant recovered with the proteasome was substantially reduced [Fig. 6Dii, SGTA panels, lanes 3–6, see product labelled ex(V5).]. On this basis, we conclude that the SGTA K160E/R164E mutant is defective in its Rpn13-mediated association with the proteasome. Finally, we used the SGTA K160E/R164E mutant as an alternative tool to test the contribution of the proteasomal binding of SGTA to its role in MLP quality control. Once again (Figs 3 and 5; see also Wunderley et al., 2014), overexpression of SGTA-V5 led to a substantial increase in OP91 (Fig. 6E, OP91 panel, cf. lanes 1 and 2), with a four-fold increase in its steady-state level (Fig. 6F). Strikingly, although both versions of SGTA-V5 were expressed at the same level (Fig. 6E, V5 panel, lanes 2 and 3), the K160E/R164E mutant was far less effective at enhancing the steady-state level of OP91 (Fig. 6E, OP91 panel, lanes 2 and 3; Fig. 6F). We, therefore, conclude that the binding of SGTA to the Rpn13 subunit of the proteasome makes an important contribution to the role of SGTA during the quality control of MLPs.