Figure 1.

Hrr25p phosphorylates Crz1p in vitro and in vivo. (A) Hrr25p phosphorylates Crz1p in vitro. Recombinant ZZ-Crz1p was incubated with recombinant wild-type or catalytically inactive Hrr25p and γ³²P-ATP. Phosphorylation of ZZ-Crz1 was analyzed by SDS-PAGE and autoradiography. The bottom panel shows ZZ-Crz1p stained by Coomassie. (B) Crz1p phosphorylated by Hrr25p is a target of calcineurin. ZZ-Crz1p was phosphorylated by Hrr25p as in A and incubated with buffer alone or calcineurin, calmodulin, and Ca²⁺ or EGTA. Samples were analyzed by SDS-PAGE and autoradiography. The bottom panel shows ZZ-Crz1p stained by Coomassie. (C) Hrr25p phosphorylates and binds to Crz1p in vivo. IgG sepharose was used to isolate ZZ-CRZ1p from extracts of cells (KKY271) expressing vector (pRS316), overexpressing wild-type (pAM4-1), or catalytically inactive HA-HRR25-K38A(pAM8-2). Samples were analyzed by SDS-PAGE and Western blotting. ZZ-Crz1p was visualized using an α-Crz1 antibody; HA-Hrr25p was visualized using an α-HAantibody.