Fig. 6. yck^ts cells show wild-type processing of the soluble vacuolar protein CPY and wild-type localization of V-ATPase. (A) Processing of the soluble vacuolar protein CPY. Wild-type (WT), apm3, yck^ts and yck^ts apm3- cells were grown to exponential growth phase at 24°C in glucose medium, then incubated at 37°C for 30 minutes. Cells were pulse-labeled for 5 minutes with [³⁵S]-translabel, chased for the times indicated (min) and subjected to immunoprecipitation with CPY antiserum. The immunoprecipitates were analyzed by electrophoresis in 10% polyacrylamide-SDS gels, which were then subjected to fluorography. p1, ER form; p2, Golgi form; m, mature vacuolar form. (B) Immunolocalization of the vacuolar H⁺ATPase subunit Vat2p. Cells were grown to exponential growth phase at 24°C in galactose medium, then incubated at 37°C for 90 minutes with cycloheximide as described in Fig. 2. Cells were prepared for immunofluorescence with the anti-Vma2p/Vat2p antibody. Cells were visualized by Normarski optics or with a FITC filter set.