Mutation of the known phosphate acceptor site in SBH1. (A) The indicated sites were mutated to alanine, and mutant Sbh1 proteins expressed in a Δsbh1Δsbh2 strain. The SBH1Δsbh2 strain was used as a control. Strains were grown on YPD plates at 30°C and 37°C for 2d. (B) Sbh1 mutant proteins from strains shown in (A) were labelled in intact cells with [32P]phosphate; cells were lysed and proteins immunoprecipitated, separated on 15% SDS gels, and detected by autoradiography. Immunoprecipitated proteins were also detected by immunoblotting (lower panels). (C) SBH1Δsbh2 and sbh1-T5AΔsbh2 strains were grown to early exponential phase, cycloheximide added to prevent further protein synthesis, samples taken at the indicated times, cells lysed, proteins resolved on SDS gels and Sbh1 proteins detected by immunoblotting. (D) Alignment of Sbh1p and Sbh2p. The identified phosphorylation site in Sbh1p is indicated in orange, transmembrane domains are shown in blue. The bracket denotes the peptide of Sbh1p against which our polyclonal antibody was raised.