Sbh1p can be phosphorylated in yeast ER membranes. (A) Left: Wildtype microsomes (5eq) were incubated with g-[32P]ATP (40 μCi) for 30 min. Membranes were lysed in SDS, and Sbh1p and Sss1p immunoprecipitated with polyclonal rabbit antisera in duplicate. Precipitated proteins and 10% of the lysate were resolved on a 15% SDS gel and detected by autoradiography. Upper right: Microsomes (1 eq) of the indicated strains were lysed, proteins resolved on a 15% SDS gel, and Sbh proteins detected with an antiserum directed against the first 18 amino acids of Sbh1p. Lower right: Microsomes from the indicated strains were labelled with γ-[32P]ATP as above and Sbh proteins precipitated in duplicate. Proteins were resolved on a 15% gel and detected by autoradiography. (B) Alignment of S. cerevisiae Sbh1p and Canis familiaris Sec61ß. Transmembrane domains are indicated in blue. Phosphorylation sites predicted by NetPhos are shown in orange, an additional site predicted by Scan Prosite in yellow.