Mutation of the predicted phosphorylation sites in Sbh1p. (A) The indicated sites were mutated to alanine, and mutants expressed in a Δsbh1Δsbh2 strain. SBH1Δsbh2 yeast were used as a control. Strains were grown on YPD plates at 30°C and 37°C for 2d. (B) Upper: Membranes from the indicated strains were labelled with gamma γ-[32P]ATP and Sbh1 proteins analyzed as above. Lower: The indicated amount of microsomes was dissolved in SDS sample buffer, proteins separated by gel electrophoresis and Sbh1p and Pdi1p (as a loading control) detected by immunoblotting. (C) As in (A). (D) Sbh1 mutant proteins from strains shown in (C) were either labelled with g-[32P]ATP in microsomes as above (left panel), or labelled in intact cells with [32P]phosphate; proteins were immunoprecipitated, separated on 15% SDS gels, and detected by autoradiography. Sec63p was used as a control phosphoprotein. Immunoprecipitated proteins were also detected by immunoblotting (lower panels).