Figure 3.

Endocytic transport to the vacuole in vps18^tsf mutant cells. (A) Wild-type (WT, SEY6210) and vps18^tsf (SRY18T-1) cells were stained with the vital fluorescent endocytic marker FM4–64 at 26°C or 38°C. After a chase in YPD at 26°C or 38°C, the stained cells were visualized by Nomarski (left) and fluorescence (right) microscopy. (B) The stability of the a-factor pheromone transporter Ste6p was determined by pulse–chase analysis. Wild-type (WT, SEY6210.1, MATa) and vps18^tsf (SRY18T-4, MATa) cells harboring pDB192 (2μSTE6) were grown at 26°C and shifted to 38°C for 5 min prior to labeling for 15 min at 38°C. After addition of chase (time 0′), the culture was further incubated at 38°C and samples were harvested at the time points indicated (Chase). The samples were subjected to immunoprecipitation with Ste6p-specific antiserum and analyzed by SDS-PAGE.