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

Previous studies have shown that all cellular Rpn13 is incorporated into the proteasome at steady state (Hamazaki et al., 2006; Qiu et al., 2006), and we speculated that, in a cellular context, the newly defined Rpn13–SGTA interaction mediates the proteasomal recruitment of SGTA. To test this hypothesis, we first purified proteasomes by using the HEK293Rpn11-HTBH cell line that constitutively expresses a tagged form of the Rpn11 subunit in addition to the endogenous protein (Wang et al., 2007). This cell line provides a convenient approach to isolate native proteasomes and has been used in several studies (Chen et al., 2010; Tsimokha et al., 2014; Wang et al., 2010). We found that a small fraction of endogenous SGTA was recovered with intact proteasomes following their isolation by using a streptavidin pull down (supplementary material Fig. S1, lanes 1–4). The association of endogenous SGTA with the proteasome was most apparent when cells had been pre-treated with the proteasome inhibitor MG132 prior to purification (supplementary material Fig. S1, cf. lanes 3 and 4). On the basis of these data, we concluded that Rpn13 provides a binding site for SGTA at the proteasome, and we next explored the functional consequences of this interaction. Whereas the increase in proteasomal SGTA observed upon treatment with MG132 was consistent with the stabilisation of a direct interaction with Rpn13 (cf. Fig. 2), we cannot rule out the alternative possibility that SGTA also binds to Rpn13 through ubiquitylated substrates that accumulate on the proteasome in the presence of the inhibitor (Isakov and Stanhill, 2011).