Fig. 4. Defective vacuole and/or endocytic acidification causes a cell-wall defect. (A) vma1, vma3, btn1and btn1vma1 cells are sensitive to zymolyase. Enzyme sensitivity over time (minutes) of the indicated cells grown at 25°C. Cell lysis was monitored by measuring OD600 nm. (B) Acidic medium rescues zymolyase sensitivity and vacuole pH of btn1 cells. (i) Zymolyase sensitivity of wild-type cells or btn1 cells grown at 25°C in medium at pH 6 or pH 4. (ii) Vacuole pH as monitored by CDCFDA fluorescence. btn1 cells grown at 25°C in a medium of pH 6 are marked by post-staining with DAPI, those grown at pH 4 are unmarked. (C) Growth in acidic medium acidifies the vacuoles but differentially affects the zymolyase sensitivity of btn1vma1 cells and those deleted for vma1 or vma3. (i) Zymolyase sensitivity after 90 minutes and (ii) zymolyase sensitivity over time of indicated cells grown in medium of pH 4 or pH 6 at 25°C. Unpaired Student's t-tests were performed (n=3, ^*P<0.05, ^**P<0.01, ^***P<0.001). (D) Vacuole pH as monitored by CDCFDA fluorescence of the indicated cells grown in a medium of pH 4 or pH 6 at 25°C. Typical results are presented for A, Bi and Cii. Scale bars: 10 µm.