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

Standard molecular biology techniques were used to clone the variants of mouse Rpn13, human Rpn10 and human SGTA into pGEX-4T1 vector for bacterial production of GST-tagged recombinant proteins. The His Trx-SGTA fusion protein is as previously described (Leznicki et al., 2011). Plasmid pGEX-4T2- TetraUb was a kind gift of Caixia Guo and Errol Friedberg (University of Texas, Dallas, TX). For expression in mammalian cells, full-length Rpn13 and SGTA were cloned into NpFLAG-CMV2 plasmid, whereas full-length Rpn10 was in pcDNA3.1-myc. Where used, SGTA-V5, Bag6-V5 and OP91 were in pcDNA5 and OPG-TASK85 in pcDNA3.1 are as previously described (Leznicki and High, 2012; Wunderley et al., 2014). The K160E/R164E mutant of SGTA is defective for Hsp70 binding (Walczak et al., 2014; Xu et al., 2012), and was generated by site-directed mutagenesis and validated by DNA sequencing prior to use. The Ub-R-GFP cDNA was obtained from Addgene (plasmid number 11939) deposited by Nico Dantuma (Karolinska Institute, Sweden) (Dantuma et al., 2000), and re-cloned into pcDNA5 vector using a TOPO cloning kit (Invitrogen).