Elevated levels of Vac17 in the vac17ΔH mutant may eventually suppress the vacuole inheritance defect. (A–C) Buds that ultimately received vacuoles were chosen from Fig. 2, A and C and analyzed retrospectively. (A and B) Vacuole inheritance over time following bud emergence (n = 24 cells for wild type; n = 7 for vac17ΔH mutants). Time 0 represents cells where vacuole inheritance occurred concomitantly with bud emergence. After time 0, all cells that achieved vacuole inheritance at any time were included. (C) Analysis of the timing of vacuole inheritance in large buds of vac17ΔH mutant cells that lacked a vacuole at the start of imaging (n = 13). Large buds inherited vacuoles at times ranging from 20 s to 40 min. (D) Western blot analysis of lysates from cells expressing Vac17 from a CEN (low copy) plasmid compared with vector alone (n = 3). Cells were harvested during mid-log phase (OD₆₀₀ 0.4–0.6). The vector control did not contain detectable bands. This confirms that the doublet bands in Vac17-3xEnvy and vac17ΔH-3xEnvy are Vac17 protein. Both bands were included in the quantification. Deletion of the PEST motif, which is essential for Vac17 degradation, results in elevated Vac17 levels. Source data are available for this figure: SourceData FS2.