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

To better understand the role of Rpn13 during proteasomal degradation, we used full-length mouse Rpn13 (Rpn131-407), as well as its N-terminal pleckstrin-like receptor for ubiquitin (Pru) domain (amino acid residues 1–150; hereafter referred to as Rpn131-150) and distinct C-terminal region (amino acid residues 150–407; hereafter referred to as Rpn13150-407) (Fig. 1A), as baits in yeast two-hybrid screens with a thymus cDNA library prey. This approach identified SGTA as a potential interacting partner of Rpn13150-407 (data not shown). To validate the yeast two-hybrid data, purified GST-tagged Rpn13, Rpn131-150 and Rpn13150-407, were used as baits in pull-down experiments primed with lysate from HeLa cells overexpressing FLAG-tagged SGTA. This showed a specific physical interaction of full-length Rpn131-407 and the Rpn13150-407 fragment with exogenous FLAG-SGTA (Fig. 2A, lanes 3–5). To map the Rpn13-binding site on SGTA, a variety of SGTA deletion mutants were purified as recombinant GST fusion proteins and their interaction with exogenous FLAG-tagged Rpn13 present in HeLa cell lysate was examined. This approach identified the central region of SGTA, comprising residues 85-210, as necessary and sufficient for Rpn13 binding (Fig. 2B, cf. lanes 3–7). This corresponds to the central tetratricopeptide repeat (TPR) domain of SGTA (Fig. 1B), a region previously implicated in binding both molecular chaperones and viral proteins (Dutta and Tan, 2008; Fielding et al., 2006; Liou and Wang, 2005; Walczak et al., 2014). In contrast to its interaction with Rpn13, FLAG-SGTA did not bind to purified human Rpn10 or its fragments (Fig. 2C, lanes 6–8), confirming the specificity of its interaction with the Rpn13 ubiquitin receptor (Fig. 2C, cf. lanes 3–5). Since HeLa lysate is most likely to contain a number of endogenous SGTA and/or Rpn13-binding partners, we further tested the nature of the interaction by using recombinant Rpn13 and SGTA. The SGTA–Rpn13 interaction could be recapitulated using purified proteins (Fig. 2D and E), and we concluded that the two components bind directly to each other.