Figure 4.
HSP90 mutations affect the levels of Ste11 and Ste7, and Ste7 hyperphosphorylation. (A) Ste11 protein levels are reduced in Hsp90 mutant strains. Left panel, HA-epitope–tagged Ste11 was overexpressed in Hsp90 mutant strains as indicated; equal amounts of “rapid protein extracts” of two colonies each (1 and 2) were immunoblotted with anti-HA antibodies. Right panel, endogenous wild-type Ste11 was concentrated by immunoprecipitation and immunoblotted with a Ste11-specific antiserum; lane Δ, extracts from a Δste11 control strain. (B) Phosphorylation of Ste7 is reduced by HSP90 mutations. Strains expressing Ste7 containing the myc epitope were exposed for 2 h to α-factor where indicated. Extracts were analyzed by an immunoblot assay using a rabbit polyclonal antibody (a gift from S. Lindquist), which only recognizes the yeast Hsp82 (top panel), and the mouse monoclonal antibody 9E10 against the myc epitope (bottom panel). Two different exposures of the latter immunoblot are presented. The position of the hyperphosphorylated Ste7 (Ste7-P) is indicated. The ratios of hyperphosphorylated Ste7 (Ste7-P) over unphosphorylated Ste7 of all samples were standardized on the ratio obtained in the presence of α-factor with the strain expressing wild-type Hsp82.