Fig. 2. The phosphorylatable threonines in position 652 and 654 of Sec63p are required for binding to the N-terminal domain of Sec63p. (A) Immunoprecipitation of F-Fpr1-63_C14 (WT) and the mutants carrying single alanine exchanges in position 652 (T652A), 654 (T654A), 661 (S661A) or the exchange at position 652 and 654 (T652; 654A). The precipitates were either mock (–) or phosphatase (+) treated and subjected to anti-Flag detection after 12.5% SDS-PAGE. (B) Extracts of cells expressing F-Fpr1-63_C14 (WT) or its mutants were anti-Flag immunoprecipitated and the precipitates were subsequently incubated with extracts containing Sec62_C125-Dha. Bound fractions were probed with anti-Flag (upper panel) or anti-Ha antibodies (lower panel) after 12.5% SDS-PAGE. An extract of cells expressing Sec62_C125-Dha is shown in lane a. (C) Sepharose-coupled F-Fpr1-63_C47 was treated without or with phosphatase (– or +) and incubated with extracts containing Sec62_C125-Dha. The bound (lanes a-d) and unbound fractions (lanes e, f) were probed with anti-Flag (a, b) and anti-Ha antibodies (lanes c-f). (D) Same as C but with F-Fpr1-63_C47 (WT), and its T/A mutants in position 652 and 654. LC and HC indicate the light and heavy chains of the antibody, respectively.