Figure 3. The vacuole is required for cell-cycle progression from early G1.

(A) The vac17Δ pep12-60^tsf double mutant shows synthetic growth defects at the restrictive temperature, 37°C. Wild-type, vac17Δ, pep12-60^tsf and vac17Δ pep12-60^tsf strains were cultured in liquid media and serial dilutions were spotted onto YPD plates. The plates were incubated at 24°C, 30°C and 37°C for 2 days. (B) The vac17Δ pep12-60^tsf double mutant arrests in G1 phase at the restrictive temperature 37°C. Percent cells in G1 phase (solid lines). Yeast strains tested; wild-type, vac17Δ, pep12-60^tsf, and vac17Δ pep12-60^tsf. Cultures were incubated at 24°C overnight, and then sifted to 37°C for 0, 2, 4, 8, 12, or 24 hr. The percentage of G1 cells (1N DNA) was measured using propidium iodide (PI) staining and assessed by flow cytometry. The same cultures were analyzed for lethality (percent dead cells) (dashed lines). After incubation at 37°C, the number of yeast cells were assessed with a hemocytometer, and their ability to form colonies at 24°C on YPD plates was tested. Lethality was inferred from the number of cells that survived the treatment. Error bars; SD calculated from four independent experiments. *** (p-value < 1 × 10⁻³). (C) The vac17Δ pep12-60^tsf double mutant arrests in early G1 phase at the restrictive temperature 37°C. Cells were scored for the presence of Whi5-3xGFP in the nucleus. Wild-type, vac17Δ, pep12-60^tsf, and vac17Δ pep12-60^tsf cells, which express Whi5-3xGFP from its endogenous locus, were incubated at 24°C overnight, and then sifted to 37°C for 0 or 4 hr. Error bars; SD calculated from three independent experiments with at least 100 cells counted in each strain/experiment. *** (p-value < 1 × 10⁻³). (D) Arrested cells that have 1N DNA content are unbudded. Wild-type and vac17Δ pep12-60^tsf cells were incubated at 24°C overnight, and then sifted to 37°C for 24 hr. After fixation, yeast were stained with PI, and cells with 1N DNA were sorted by flow cytometry. The sorted cells were observed by microscopy. For both wild-type and the vac17Δ pep12-60^tsf double mutant 99% of the cells with 1N DNA were unbudded. Sorted cells from three individual experiments were counted. At least 400 cells were counted for each experiment.

DOI: http://dx.doi.org/10.7554/eLife.08160.007

Figure 3—figure supplement 1. The vacuole is required for cell-cycle progression from early G1 phase.

(A) The vac17Δ pep12-60^tsf double mutant is arrested in G1 phase at the restrictive temperature, 37°C. Percent cells in G1 (1N DNA) and G2 (2N DNA) phase after incubation at 37°C for 0, 2, 4, 8, 12, or 24 hr. Percent cells arrested in G1 are also shown in Figure 3B. Error bars; SD calculated from four independent experiments. (B) vac17Δ pep12Δ and vac17Δ vps45Δ double mutants exhibit an accumulation of cells arrested in G1 phase. Flow cytometry analysis of PI staining of yeast strains; wild-type, vac17Δ, pep12Δ, vps45Δ, vac17Δ pep12Δ, and vac17Δ vps45Δ. (C) Quantification of percent cells in G1 and G2 phase. Error bars; SD calculated from four independent experiments. (D) The vac17Δ pep12-60^tsf double mutant arrests in early G1 phase at the restrictive temperature, 37°C. Wild-type, vac17Δ, pep12-60^tsf, and vac17Δ pep12-60^tsf cells which express Whi5-3xGFP from its endogenous locus, were incubated at 24°C overnight, and then sifted to 37°C for 0 or 4 hr. Images of Whi5-3xGFP localization after cells were incubated at 37°C for 4 hr. Quantification is shown in Figure 3C.

DOI: http://dx.doi.org/10.7554/eLife.08160.008