Ypt1 regulates Hrr25 kinase activity on vesicles. (A) Hrr25 kinase activity is ts in the ypt1-3 mutant. WT (SFNY 2443) and mutant (SFNY 2445) cells were grown at 23°C or shifted to 37°C for 2 h. Hrr25-HA was precipitated from lysates onto Protein A–conjugated agarose beads and kinase activity was assayed using MBP as a substrate as described in the Materials and methods. (B) Quantitation of kinase activity from WT and the ypt1-3 mutant from three separate experiments. The data were normalized to the amount of Hrr25-HA in the precipitate. Error bars represent SD; n = 3; *, P < 0.05, Student’s t test. (C and D) Hrr25-HA was precipitated from permissively grown ypt1-3 cells and preincubated with increasing concentrations of Ypt1 Q67L (C) or Ypt1 (D) for 15 min at 25°C and then assayed as described in the Materials and methods. The ratio of phosphorylated MBP to Hrr25-HA is calculated at the bottom of each lane. Note that the kinase activity of the mutant at 0 ng was set at 1.0. The assays in C and D were performed multiple times. The data that are shown are representative. (E) Lst1 is hypophosphorylated in the ypt1-3 and sec12-4 (NY738) mutants. WT and mutant cells were shifted to 37°C for 1 h, lysates were prepared and Lst1 was immunoprecipitated (lanes 1–12). Samples in lanes 5, 6, 9, and 10 were treated with CIP, while samples in lanes 7, 8, 11, and 12 were treated with CIP plus EDTA.