(A) PMA1 was overexpressed in newborn daughter cells expressing Vph1-mCherry using a β-estradiol inducible system where a GAL4-Estrogen binding domain-VP16 (GEV) fusion protein drives GAL1 promoter expression of an extra copy of PMA1. (n ≥ 30 cells per condition). (B) Wild-type and pma1-105 cells expressing Vph1-mCherry were aged and quinacrine stained (n ≥ 30 cells per timepoint). White arrowheads indicate mother cell vacuoles with reduced acidity. Orange arrowheads indicate acidic mother-cell vacuoles. (C) Replicative lifespan of wild-type, pma1-105, vma2, and vma2 pma1-105 cells by micromanipulation. Median lifespan is indicated. For the difference between wild-type and pma1-105, p < 0.0001, one-tailed logrank test. (n = 114 cells for PMA1, n = 119 for pma1-105, n = 36 for vma2, and n = 39 for vma2 pma1-105). (D) PMA1-mCherry was overexpressed in cells undergoing their first division that expressed endogenous Pma1-mCherry and that were treated with β-estradiol and then with β-estradiol plus nocodazole (Noc). (E) As in D, cells that expressed Vph1-mCherry were induced to overexpress PMA1 and were quinacrine stained. (n ≥ 30 cells per condition). Arrowheads indicate the vacuoles of interest.