Figure 7.
Vacuolar protein-sorting defect of did mutants. (A) Aberrant secretion of a vacuolar enzyme in the did mutants. Sorting of the vacuolar hydrolase CPY was determined by pulse-chase analysis. Cells spheroplasts were labeled for 10 min with 35S-TransLabel, and after a 30-min chase period, CPY from intracellular (I) and extracellular (E) fractions was precipitated with anti-CPY antibodies and analyzed by SDS-PAGE. The intermediate secreted from did1 cells had a slightly slower electrophoretic mobility than did the p2 form observed in wild-type cells and the other strains. This might be due to altered glycosylation of CPY in the did1 mutant, although this has not been tested. (B) FM 4-64 staining of did1–did4 mutants. Cells were labeled with FM 4-64 for 10 min and chased for 1 h at 30°C. The cells were then viewed with epifluorescence optics.