Figure 2. Hch1 reduces client accumulation and activity.

(a) HCH1 (MR318) or hch1 (CLY7) cells expressing Hsp82 WT or W585T (MR923-MR926) were transformed with a plasmid encoding the mammalian steroid hormone receptor GR and the β-galactosidase reporter pUCΔSS-26X. Resultant colonies were grown to mid-log phase followed by the addition of synthetic hormone. Following a five-hour incubation, β-galactosidase activity was quantified (each sample was run in triplicate and error bars represent s.d. from the mean). These same strains were lysed and analysed using SDS–PAGE and the indicated antibodies. (b) The same strains used in a were transformed with the GAL-inducible Ste11ΔN construct as well as the β-galactosidase reporter PRE-lacZ (ref. ⁵⁵). Transformants were grown to mid-log phase in selective media supplemented with raffinose followed by a 6-h galactose induction. Cells were then measured for β-galactosidase activity as in a. These strains were also lysed following galactose induction and run on a 4–20% SDS gel and probed with the indicated antibodies. (c) HCH1 or hch1 cells expressing either WT or W585T Hsp82 as indicated were transformed with pRS316GAL-v-SRC. Transformants were grown overnight in minimal media and then diluted 10-fold and grown for four days at 30° on minimal media supplemented with glucose (URA D.O.) or galactose (URA-GAL). (d) Cells from c were grown overnight in URA-RAF and switched to URA-GAL media overnight. Cells were lysed and analyzed as in b.