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Fig. 3. Vacuoles of btn1 cells are more alkaline. (A) Cells deleted for btn1 and prelabelled with Hoechst (blue) to stain nuclei and calcofluor (blue) to stain septa, were mixed with unlabelled wild-type cells, also prelabelled with calcofluor (blue), and incubated with CDCFDA (green, to indicate relative intravacuolar pH) for 10 minutes. A wild-type and a btn1 cell (distinguished by round nuclear staining) are indicated by arrows. Vacuoles in btn1 cell had reduced CDCFDA fluorescence indicating an increased pH. Bar, 10 µm. (B) Correlation between CDCFDA fluorescence and vacuole size for cells deleted for btn1 (open circles) and wild-type cells (filled circles). (C) Time course of hydrolysis of CDCFDA as an indicator of relative intravacuolar pH and measured by quantitative fluorescence in wild-type cells (filled squares), cells deleted for btn1 (open circle) or cells deleted for btn1 and expressing GFP-Btn1 for 18 hours (filled circles). GFP-Btn1 partially rescues the reduced CDCFDA fluorescence of btn1 cells. (D) Wild-type cells (left panel) and btn1 cells (right panel) labelled with LSDND as an indicator of relative vacuole pH. Note that btn1 cells have reduced fluorescence. Bar, 10 µm. (E) Vacuole pH estimation of wild-type and btn1 cells. The vacuoles of btn1 cells are pH 5.1 compared to those in wild-type cells which are 4.14. (F) Mean vacuole diameter of wild-type and btn1 cells exposed to media of different pH. The vacuole diameter of both strains increased as cells were exposed to media of increasing pH.

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