FIGURE 1:

Ark1 and Prk1 are required for nutrient modulation of TORC2 signaling. (A) Cells of the indicated genotypes were grown to log phase in YPD medium at 30°C, and levels of Ypk-pT662 and Ypk1 protein were assayed by Western blot. An asterisk indicates a background band that overlaps with the Ypk1 signal. Quantification of Ypk-pT662 and total Ypk1 protein are shown. Error bars represent the SD of the mean of three biological replicates. *** indicates a p value smaller than 0.005 compared with the wild type. (B) Cells of the indicated phenotype were grown in YPG/E medium until early log phase, and 2% galactose was added to overexpress Ark1. Cells were collected at the indicated time intervals and levels of Ark1, Ypk-pT662 and Ypk1 protein were assayed by Western blot. An asterisk indicates a background band. (C) Wild-type and ark1∆ prk1∆ cells were grown in YPD medium to early log phase and were then rapidly washed into YPG/E medium at 30°C. Cells were collected at the indicated time intervals, and Western blotting was used to assay levels of Ypk-pT662 and Ypk1 protein. (D) A series of 10-fold dilutions of the indicated strains were grown at 30°C for 2 d on rich (YPD) or poor (YPG/E) nutrient conditions. A small number of colonies that have suppressor mutations appear when ark1∆ prk1∆ cells are grown on YPG/E medium.