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

The effect of expression of isolated proteasomal ubiquitin receptors was addressed by co-transfecting HeLa cells grown in 12-well cell culture dishes with a combination of 0.5 µg substrate DNA, 0.2 µg pcDNA5-SGTA-V5 or control vector and 0.3 µg plasmid encoding the Rpn13 variant, Rpn10 or appropriate empty vector controls using Lipofectamine 2000 (Invitrogen) according to manufacturer's instruction. Cells were processed 22 h post-transfection as previously described (Leznicki and High, 2012) and results visualised by quantitative western blotting (LiCor Biosciences). The effects of overexpressing SGTA-V5 and the K160E/R164E mutant version were monitored by using a TRex Flp-In HeLa cell line stably expressing OP91 under the control of a tetracycline-inducible promoter. After transfection with plasmids expressing V5-tagged SGTA variants, or a Pex19 control, samples were grown for 24 h. Cells were then induced by treatment with 1 µg/ml tetracycline, grown for further 24 h, harvested directly into sample buffer and analysed by quantitative western blotting (cf. Wunderley et al., 2014). For the quantification of substrate levels, the relevant experiments were independently repeated at least three times, the amount of substrate quantified with Odyssey 2.1 software and plotted relative to the matched control using GraphPad Prism 4.0 software.