Figure 2.

Sti1 dependency caused by K399C is relieved by increasing Hsp70 interaction. (A) A portion of a Coomassie-stained gel of a representative in vitro pull-down experiment of purified His₆-tagged wild-type and mutant Hsp82 with Ssa2 is shown above a graph displaying quantification of three replicate experiments (see also Figure S3). SdN mutants reduce interaction with Ssa2 to 50% of wild type. Values are averages relative to wild type and normalized to amount of Hsp82. Error bars are the SD. (B) Relative abundance of cochaperones copurifying in early and late complexes with His6-Hsp82^K399C were derived from quantitative mass-spec. Left, early complexes; right, late complexes enriched by addition of AMP-PNP. (C) Cultures of sti1∆ strain 1670 expressing the indicated Hsp82 mutants as the sole source of Hsp90 were diluted and plated as in Figure 1D. Removal of negative charge at position 402 (E402A, F or S) partially rescues K399C Sti1 dependence, while positive charge (E402K or R) restored wild-type growth. (D) Western analysis of indicated proteins from lysates and His6-Hsp82 complexes (Pull-down) isolated from hsc82∆,hsp82∆ strain MR318 expressing the indicated Hsp82 mutants as the sole source of Hsp90. Aha1 and Sba1 samples were treated with AMP-PNP. E402R, alone or in combination with K399C (C + R), increased the amount of Hsp70 recovered. (E) Quantification of the amounts of Hsp70 (Ssa2) recovered by in vitro pull down with indicated Hsp82 mutants. E402R, alone or with K399C (C + R), increased the amount of Hsp70 recovered. Values are averages of three replicate experiments and error bars are the SD. (F) Intrinsic (−Aha1) or Aha1-stimulated (+Aha1) ATPase activity of Hsp82. Neither were affected by K399C or E402R, alone or in combination (C + R). Values are averages of three replicates and error bars are the SD. nt, nontagged wild-type Hsp82; WT, wild type.