Fig. 4. Turnover of Pma1-10 requires the epsin UIM domain. (A) FM 4-64 endocytosis assay. ENT1⁺ [ent1 ent2 ede1 cells carrying pEDE1 and pENT1 (LHY3185)] and ent1^UIM [ent1 ent2 ede1 cells carrying pent1^uim (LHY3156)] cells were grown in YPD, labeled for 15 minutes at 30°C with 40 µM FM 4-64. Cells were then washed and resuspended in fresh YPD for 45 minutes at 30°C before visualization. (B) Pulse-chase analysis. ENT1⁺ (L3852) and ent1^UIM cells growing exponentially in minimal medium were induced to express HA-Pma1-10 (pKK7) for 1 hour at 25°C. Cells were then shifted to 37°C for 5 minutes before pulse-labeling with Expre³⁵S³⁵S followed by chase for various times. Immunoprecipitations from lysate with anti-HA antibody were normalized to acid-precipitable cpm, and analyzed by SDS-PAGE and fluorography. (C) Indirect immunofluorescence localization of Pma1-10 in ent1^UIM cells. Cells growing exponentially were induced to express HA-Pma1-10 (pKK42) for 1 hour at 37°C and then `chased' in the presence of 2 mM methionine for 2 hours at 37°C. Cells were fixed before induction (0), after induction (on), and after shut-off (off), and stained with anti-HA antibody followed by CY3-conjugated secondary antibody. Images were captured at the same exposure time and adjusted with Adobe Photoshop software using the same settings.