A. Phosphorylation of Vac17 is decreased in cdc28-4^ts. cdc28-4^ts and wild type (W303) strains grown at the permissive temperature (24°C) in log phase, then shifted to the restrictive temperature (36°C). Cells were harvested before and 90 minutes after the shift. Cell extracts were treated with alkaline phosphatase and analyzed by immunoblot. B. Inhibition of Cdc28 kinase activity results in a decrease in Vac17 phosphorylation. 1NM-PP1 (10 mM in DMSO) or DMSO alone were added to a final concentration of 10 nM to log phase cdc28-as1 cells. Samples were collected after 30 min and analyzed by immunoblot. C. Activation of Cdc28 via constitutive overexpression of Clb2 or Clb5 results in hyper-phosphorylation of Vac17. Cells expressing either Gal-CLB2-TAP or Gal-CLB5-TAP were grown in YEP + 2% glycerol + 2% ethanol media in log phase. Galactose was added to a final concentration of 2% to induce expression of Clb2-TAP or Clb5-TAP. Cells were harvested before and 3 hours after induction. Cell extracts were subjected to immunoblot analysis. D. Vac17 contains four Cdk1 consensus sites. Full consensus sites (red), minimum consensus sites (blue). Myo2 BD: Myo2-binding domain; PEST: PEST sequence; Vac8 BD: Vac8-binding domain. E. Direct phosphorylation of Vac17 by Clb2-Cdc28 or Clb5-Cdc28 in vitro. 1 µg of histone H1 or 2 µg recombinant Vac17 fragments purified from E. coli were incubated with [γ³²P]-ATP in the presence or absence of Clb2-Cdc28 or Clb5- Cdc28. Phosphorylation was analyzed by autoradiography following SDS-PAGE. F. Phosphorylation sites of the Vac17 peptides determined by mass spectrometry. Phosphorylated peptides with phosphorylation sites underlined.

A. Mutation of the four Cdk1 consensus sites of Vac17 results in a partial defect in vacuole inheritance. Vacuole inheritance of wild type and vac17-4A cells were scored at each time point as described in Figure 1D. Two independent cell cycles shown. B. Mutation of four Cdk1 consensus sites in Vac17 does not affect the cell cycle. Cell cycle progression was estimated from the bud-mother index (BMI, ratio of bud to mother cell diameter). >80 cells scored for each time point. Two independent experiments shown. C. Vacuole inheritance is further impaired in the myo2-3A vac17-4A double mutant. >200 cells scored for each sample. Averages and standard deviations from three independent experiments. D. Substitution of the Cdk1 sites of Vac17 decrease its binding to Myo2. Myo2-GFP was immunoprecipitated with an anti-GFP antibody. Co-precipitation of Vac17-HA or vac17-4A-HA assayed by immunoblot. C. Substitution of the Cdk1 sites of Vac17 does not affect its ability to bind Vac8. Vac17-GFP or vac17-4A-GFP was immunoprecipitated with an anti-GFP antibody. Co-precipitation of Vac8 assayed by immunoblot. Asterisks: non-specific bands.

A. Vac17 is phosphorylated. 15 µl of water, alkaline phosphatase (AP), and/or sodium orthovanadate were added to cell extracts (1 ml) and incubated for at 30°C 1 hour. Immunoblot analysis was performed using anti-Vac17 and anti-Pgk1 antibodies. B–C. Phosphorylation of Vac17 is cell cycle-dependent. B. Wild type cells were arrested in G1 phase with α-factor (α), S phase with hydroxyurea (HU), or M phase with nocodazole (Noc). As: untreated asynchronous culture. Cell extracts analyzed by immnunoblot. C. Wild type cells were synchronized by α-factor arrest and release. Samples were collected at indicated times after release to monitor phosphorylation of Vac17 during the cell cycle. D. Phosphorylation of Vac17 parallels vacuole inheritance. Wild type cells labeled with FM4-64, 1 hour, arrested with α-factor for 3 hours, then released. Percentage of buds with inherited vacuoles (blue) at the indicated times after release. 80–200 cells scored for each time point. Phosphorylated Vac17 levels (red) were quantified using Labworks 4.0 software. Data from two independent experiments.

A. Vac17 phosphorylation is impaired in vac8Δ cells. Cell extracts from wild type, vac8Δ, myo2-N1304S and vac17Δ cells grown to log phase, were treated with alkaline phosphatase (AP). Because Vac17 levels in vac8Δ and myo2 are 10-fold higher than wild-type, extracts from the mutants were diluted 10-fold using cell extracts from the vac17Δ mutant. Aliquots (15 µl) analyzed by immunoblot. B. Localization of Vac17-GFP in wild type, vac8 and myo2 mutant cells. Cells carrying the VAC17-GFP plasmid were labeled with FM4–64 for 1.5 hours, and chased for one doubling time (~3 hours). Overlay images indicate vacuoles (red) and Vac17-GFP (green). >250 cells scored for the mutant cells. 9.7% vac8Δ and 20.6% myo2-N1304S cells had detectable Vac17-GFP. Localization of Vac17-GFP in cells where fluorescence was detectable was the same as the examples shown. C. Vac17 and Myo2 co-localize in vac8Δ cells. Wild type VAC17-GFP was visualized in a vac8Δ MYO2-mRFP strain. Scale bars, 5 µm.