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Fig. 1. The interaction between the C-terminus of Sec63p and the N-terminus of Sec62p is phosphorylation dependent.

(A) Representation of Sec63p and its C-terminal-derived fusion constructs F-Fpr1-63_C47 and F-Fpr1-63_C14. The sequence of the last 14 residues of Sec63p is given in the one-letter code. (B) Extracts of yeast cells expressing F-Fpr1-63_C14 together with Sec62_C-125-Dha (lanes a, b, c) or alone (lanes d, e, f) were incubated with anti-Ha or anti-Flag antibodies to precipitate Sec62_C-125-Dha (lanes b, c) or F-Fpr1-63_C14 (lanes e, f), respectively. Extracts (Ex), supernatant (Sn) and precipitates (P) were probed with anti-Flag antibody directly (lanes a-f) or after the treatment of the precipitate with calf intestinal phosphatase (CIP) (lane e). (C) In vivo phosphorylation of F-Fpr1-63_C14. Yeast cells expressing F-Fpr1-63_C14 (lanes a, b) or F-Fpr1-63_{C14T652; 654A} (lane c) were labeled with [³²P]O₄. Extracts were incubated either with anti-Flag antibody (lanes b, c) or not (lane a) followed by the precipitation of the antibody, SDS-PAGE of the bound fraction, and autoradiography of the dried gel. Anti-Flag western blot of the immunoprecipitates of the unlabeled cells is shown in lanes d and e. (D) Sepharose-coupled F-Fpr1-63_C14 was incubated without (–) or with (+) phosphatase (lanes a, b) followed by incubation with Sec62_C125-Dha (lane c). Precipitates and supernatants were subjected to 12.5% SDS-PAGE and immunoblot detection with anti-Flag (lanes a, b) or anti-Ha antibody (lanes c-g). LC indicates the light chain of the antibody.

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