Fig. 6.

High levels of Sts1 rescue certain lid mutant mislocalization and growth defects. (A) Yeast was grown in SD–Trp and imaged by fluorescence microscopy; three replicates of at least 100 cells were counted. Two-ANOVA was used to determine statistical significance of differences in localization (****P<0.0001; ns, not significant). Scale bar: 5 μm. The indicated mutant yeast expressed Rpn5-GFP and was transformed with either p414GPD (empty vector) or p414GPD-STS1. Strains used: MHY6964, MHY9583 and MHY9174. (B) Serial dilution growth assays. Matched wild-type (WT) and mutant transformants were grown as follows, from left to right: SD –Trp –Arg+1 µM canavanine, 34°C (5 days); SD–Trp, 30°C (5 days); and SD–Trp, 34°C (2 days). Strains used: MHY500, MHY5748, MHY9509 and MHY9134. (C) Recombinant GST-Srp1–Sts1-6His complex (1 μM) was immobilized on glutathione (GSH) resin and incubated with 1 μM purified CP, RP, 26S proteasome or 26S proteasomes reconstituted from RP and CP (‘RP+CP’) to detect interactions. All input complexes were isolated from yeast using anti-Flag affinity purifications. Proteins from the GST pulldowns (PD) were examined by anti-Flag immunoblotting.