Figure 4.

Inactivation of Sac1p does not rescue transport defects associated with pik1^ts mutant cells. (A) Hsp150p secretion in WT, sac1^ts, pik1^ts, and pik1^ts/sac1^ts cells. Cells were preincubated at 38°C for 10 min, metabolically labeled with an ³⁵S-protein–labeling mixture during a 10-min pulse, and then chased in the presence of excess unlabeled methionine and cysteine for 30 min. Labeled cells were subjected to centrifugation and Hsp150p was immunoprecipitated from both the media (external) fraction and the cellular (internal) fraction and resolved by SDS-PAGE. (B) CPY processing in sac1^ts, pik1^ts, and pik1^ts/sac1^ts cells. Cells were preincubated at 38°C for 10 min, metabolically labeled with a ³⁵S-protein–labeling mixture during a 10-min pulse, and then chased in the presence of excess of unlabeled methionine and cysteine for the indicated times. CPY was then immunoprecipitated and resolved by SDS-PAGE. The migration positions of precursor and mature forms of CPY are indicated on the left. All data are representative of multiple experiments.