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

No proteasome-associated OP91 was apparent with the parental cell line (supplementary material Fig. S2B, cf. OP91 panel). Likewise, when intact proteasomes were isolated from HEK293Rpn11-HTBH cells, OP91 was undetectable in the absence of additional factors (Fig. 3B, OP91 panel, lane 4). By contrast, OP91 was readily detectable when the proteasomal fraction was isolated from cells that co-express exogenous SGTA (Fig. 3B, OP91 panel, cf. lanes 4 and 6). Notably, the proteasomal association of this MLP substrate was mirrored by the recruitment of both exogenous and endogenous SGTA to the proteasome [Fig. 3B, SGTA-V5 (exogenous SGTA) and SGTA panels, cf. lanes 3, 4 and 6]. By contrast, although Bag6 co-expression led to a comparable increase in steady-state levels of OP91 (Fig. 3A, OP91 panel, cf. lanes 4–6), the amount of proteasome-associated OP91 was much lower (Fig. 3B, OP91 panel, cf. lanes 4–6). Likewise, the recovery of exogenous Bag6 with the proteasome appeared unaffected by OP91 co-expression (Fig. 3B, Bag6-V5 panel, cf. lanes 2 and 5, red circles). Interestingly, Bag6 co-expression with OP91 did appear to enhance the proteasomal recruitment of endogenous SGTA (Fig. 3B, SGTA panel, cf. lanes 2, 4 and 5, see component labelled ‘end.’). In short, the enhanced steady-state MLP levels observed upon SGTA overexpression correlate with a specific increase in the binding of both OP91 and SGTA to the proteasome. Given that SGTA is known to bind a variety of hydrophobic substrates, including MLPs and tail-anchored membrane proteins (Leznicki et al., 2010, 2011; Liou and Wang, 2005; Wunderley et al., 2014), we conclude that proteasome-associated SGTA might influence MLP stability by regulating the access of such substrates to the catalytic core. To test this hypothesis, we explored the outcome of perturbing the Rpn13-dependent binding of exogenous SGTA to the proteasome.