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

Our data show that SGTA binds to a C-terminal region of Rpn13, Rpn13150-407, that is distinct from the N-terminal Pru domain, which interacts with the proteasome and ubiquitin (Chen et al., 2010; Husnjak et al., 2008; Schreiner et al., 2008). On this basis, we speculated that overexpression of SGTA increases its occupancy of Rpn13, thereby antagonising the proteasomal degradation of MLPs and resulting in an increase of their steady-state expression level (Fig. 3A; see also Wunderley et al., 2014). To test this hypothesis, we investigated the effect of Rpn13 overexpression on the association of exogenous SGTA with proteasomes (cf. Fig. 3; supplementary material Figs S1 and S2). Although exogenous V5-tagged SGTA was expressed under all conditions tested (Fig. 4Ai and Bi, see SGTA panel), it was only detected in the proteasome-associated fraction recovered from cells expressing HTBH-tagged Rpn11 after treatment with MG132 [Fig. 4Aii and Bii, SGTA panel, product labelled ex(V5).]. Notably, the amounts of exogenous SGTA and its endogenous counterpart that were recovered with the proteasome are both reduced upon Rpn13150-407 co-expression (Fig. 4Bii, SGTA panel, lanes 2 and 8). By contrast, although present at higher levels (Fig. 4Ai and Bi, FLAG-Rpn13 panel; see also Fig. 5), overexpression of full-length Rpn13, or of its N-terminal fragment, have far less of an effect on the proteasomal association of SGTA (Fig. 4Bii, SGTA panel, lanes 2, 4, 6 and 8). On this basis, we conclude that – when overexpressed – the C-terminal Rpn13150-407 fragment can compete for binding to available SGTA, thereby reducing its association with the proteasome.