Fig. 6. Sec63p is phosphorylated by protein kinase CK2 in vivo and in vitro. (A) Yeast cells carrying a deletion of CKA1 and the indicated alleles of CKA2 were shifted to 37.5°C and the expression of F-Fpr1-63_C14 was induced. Cell extracts were prepared at the indicated times and probed with anti-Flag antibody after transfer onto nitrocellulose. (B) As A but F-Fpr1-63_C14 of cells grown for 3 hours at 37.5°C was precipitated by anti-Flag antibody and either mock treated (–) or treated with an unspecific phosphatase (+). (C) Autoradiogram (lanes a-e) of CIP-treated F-Fpr1-63_C14 (WT) or F-Fpr1-63_{C14T652; 654A} (T652; 654A) incubated in the presence (+) of [-³²P] GTP with Cka1-ProA attached to IgG-sepharose (CK2) or with mock-treated IgG-beads (–), or with (+) or without (–) heparin. Anti-Flag western blot of the substrates for the kinase assays (lanes f, g). (D) As A but with a CK2 and a ck2ts strain, that contain SEC63-ProA and expressing Sec63p ectopically from the P_MET17-promotor. Cells were grown at 25°C in medium containing 10 mM methionine (+) and shifted to 37.5°C and methionine-free medium (–). Extracts were probed with the Sec62_C125-Dha overlay assay (upper panel) or with anti-Sec63p antibody (lower panel). (Lanes k and l) JD53 cells containing SEC63 (k) or SEC63-ProA (l) were probed with the Sec62_C125-Dha overlay assay (upper panel) or with anti-Sec63p antibody (lower panel).