Termination of vacuole transport initiates at the bud cortex. (A, C, and F) Fluorescence microscopy of a vac17Δ strain transformed with plasmids encoding GFP-tagged VAC17 or vac17 point mutants with or without mCherry- or Venus-tagged Myo2. (A) vac17-S222A-GFP and vac17-T240A-GFP resulted in mislocalization of the vacuole (FM4-64) to the bud tip (arrowheads) or mother-bud neck (arrows). (B) Quantification of >30 large-budded cells per strain per n. (C) Vacuoles colocalize with Myo2-Venus at the bud tip (arrowheads) or mother-bud neck (arrows) in cells expressing vac17-S222A or vac17-T240A. (D) Quantification of >40 large-budded cells per strain per n for the colocalization of Myo2 and the vacuole. (E) Quantification of >40 large-budded cells per strain per n for the localization of Myo2. (F) vac17-S222A-GFP and vac17-T240A-GFP colocalize with mCherry-Myo2 at the bud tip (arrowheads) or mother-bud neck (arrows). DIC, differential interference contrast. (G) Quantification of >58 large-budded cells per strain per n for the colocalization of Myo2 and Vac17. (H) Quantification of >58 large-budded cells per strain per experiment for the localization of Myo2. Error bars indicate SEM. n = 3. *, P < 0.05; **, P < 0.01; two-tailed Student’s t test.