Figure 9.

The dramatic accumulation of PtdIns(4)P in sac1^ts/sjl2Δ/sjl3Δ mutant cells alters Golgi-dependent glycosylation pathways. (A) The indicated strains 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 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. The star indicates the migration position of underglycosylated mature CPY. (B) WT, sac1^ts, and sac1^ts/sjl2Δ/sjl3Δ mutant 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. Cellular transport was stopped by the addition of NaN₃ and NaF after the pulse-chase, and the cells were converted to spheroplasts. Internal and external fractions were separated by centrifugation and analyzed for the presence of invertase by immunoprecipitation. (C) sac1^ts and sac1^ts/sjl3Δ mutant 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. Cells were then treated as in B. All data are representative of multiple experiments.