Figure 3: Characterization of sec22Δ vacuoles.

Morphology of vacuoles and pH sensitivity were analysed in sec22Δ and wild type. (a) Cellular (filled bars) and vacuolar (squares) volumes. In case of several vacuolar lobes stained per cell, the volumes of the lobes were added. Cell volumes are 35.2±1.2 fl (wild type) and 35.8±1.7 fl (sec22Δ); and vacuolar volumes are 8.8±0.9 fl and 9.0±1.2 fl. FM4-64 staining was used to label vacuoles in living yeast cells. Fifty cells from three independent cultivations were evaluated. Data are presented as means±s.d. (b) Percent ratio of vacuolar to cellular volumes at different external Cs⁺ concentrations (n=30 cells). Data are presented as means±s.d. (c) Cells with fragmented vacuoles at three different concentrations of external CsCl, as well as at 100 mM external KCl. Cells showing two to five smaller sized lobes were defined as fragmented in contrast to a regular anatomy with a single, large vacuole. Data are presented as means±s.d. (n=3 with 100 cells in each independent replicate). All analyses in (a,b,c) were performed in YPD-rich medium after addition of cytostatic 5-FU. Both the absolute and relative vacuolar volumes, as well as vacuolar morphology, were similar in wild type and sec22Δ. However, at experimental conditions (50 μM Cs⁺) sec22Δ has a significantly higher percentage of fragmented vacuoles (18.3±4.5%) than wild type (6.0±2.1%) (P≤0.01), which has also been confirmed at 100 μM Cs⁺. (d) Wild-type and mutant cells grown in the presence of 50 μM CsCl. Normal and fragmented (white arrows) vacuoles are visualized by FM4-64 fluorescence. Scale bar: 10 μm. (e) pH sensitivity of yeast strains. Wild type, sec22Δ, vma22Δ, vma2Δ and sec22Δ::Sec22 (sec22Δ complemented with wild-type Sec22) were tested on YPD plates buffered at different pH (dilution series 1/1, 1/5, 1/25, 1/125 and 1/325). Colony sizes, 7–8 mm.