Mutants that affect vacuolar domain formation or dissipation. (A) The Rim101 pathway. (B) WT or rim21Δ cells expressing Vph1-GFP were grown to Stat-phase, and the percentage of cells with vacuolar domains was determined. Portions of the cultures were pelleted, and the media were replaced with 100 mM Tris, pH 9.3. (C) The indicated strains were grown as in B, and the percentage of cells with vacuolar domains was determined. (D) Percentages of vacuolar domain types in cells expressing Vph1-GFP and with the indicated genotypes were determined in Stat-phase. (E) Midlogarithmic growth–phase cultures were untreated (SC) or treated with 10 µg/ml CHX or 60 mM NaOAc, pH 5.2, for 3 h, and the percentages of cells with vacuolar domains were determined. (F) WT or sec18-1 cells were grown at 24°C to midlogarithmic growth phase, and a portion of the cultures was shifted to 37°C for 3 h. The percentage of cells with vacuolar domains was determined. Graphs show means of 100–300 cells ± SD from two independent experiments. CHX, cycloheximide; MVB, multivesicular body.