The dramatic accumulation of PtdIns(4)P in
sac1ts/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,
sac1ts, and
sac1ts/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) sac1ts
and sac1ts/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.