Back to Content
Figure 1.
The N-terminal region of Vac17 is important for vacuole inheritance and binds to Myo2. (A) Schematic of Vac17. Indicated residues are suppressors identified in a mutagenesis screen that restore vacuole inheritance in the myo2(N1304S) mutant. h1–3 = helix 1–3 predicted by all AlphaFold models to adopt an antiparallel three-helix bundle (H); MBD = Myo2-binding domain; VBD = Vac8-binding domain. (B) Vacuole inheritance in Δvac17 cells transformed with a low-copy plasmid that expresses the indicated Vac17-3xEnvy constructs (green) or empty vector. Vacuole membranes (magenta) were pulse-chase labeled with FM 4–64 and incubated for one doubling time prior to imaging. Dashed lines indicate the cell perimeters. Scale bar = 5 μm. (C) Quantification of vacuole inheritance. Scoring was based on the presence or absence of labeled vacuoles at the indicated bud sizes. Data from four independent yeast cultures (n = 4) were analyzed, with a minimum of 30 cells per culture measured for each bud size. Bud size was determined based on the ratio of the diameters of the bud cell to the mother cell. Values ≤0.3 was classified as small. Significance was calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons; ****P < 0.0001; ns = not significant. Error bars indicate SEM. (D) Vac17 localization relative to the vacuole in cells where vacuole inheritance occurred (n = 4 independent experiments; ≥30 cells per n for each bud size). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001; ns = not significant. Error bars are SD. (E) Size-exclusion chromatography traces of the Myo2 tail (gray), either alone or co-expressed with N-terminally MBP-fused Vac17 peptides: Vac17(H) (light blue; top panel) and Vac17(MBD) (green; bottom panel). The orange arrowhead indicates free maltose-binding protein (MBP; 44 kDa), which was cleaved from the Vac17 peptides using TEV protease following MBP affinity purification. In a separate experiment, the Myo2 tail co-expressed with MBP-Vac17(H) was first purified via amylose resin, treated with TEV protease, and subsequently subjected to Strep-Tactin pulldown to isolate Myo2. Gel filtration confirmed that Vac17 remains associated with Myo2 (dark blue; top panel). Note that Vac17 peptides alone were unstable in the absence of Myo2.

The N-terminal region of Vac17 is important for vacuole inheritance and binds to Myo2. (A) Schematic of Vac17. Indicated residues are suppressors identified in a mutagenesis screen that restore vacuole inheritance in the myo2(N1304S) mutant. h1–3 = helix 1–3 predicted by all AlphaFold models to adopt an antiparallel three-helix bundle (H); MBD = Myo2-binding domain; VBD = Vac8-binding domain. (B) Vacuole inheritance in Δvac17 cells transformed with a low-copy plasmid that expresses the indicated Vac17-3xEnvy constructs (green) or empty vector. Vacuole membranes (magenta) were pulse-chase labeled with FM 4–64 and incubated for one doubling time prior to imaging. Dashed lines indicate the cell perimeters. Scale bar = 5 μm. (C) Quantification of vacuole inheritance. Scoring was based on the presence or absence of labeled vacuoles at the indicated bud sizes. Data from four independent yeast cultures (n = 4) were analyzed, with a minimum of 30 cells per culture measured for each bud size. Bud size was determined based on the ratio of the diameters of the bud cell to the mother cell. Values ≤0.3 was classified as small. Significance was calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons; ****P < 0.0001; ns = not significant. Error bars indicate SEM. (D) Vac17 localization relative to the vacuole in cells where vacuole inheritance occurred (n = 4 independent experiments; ≥30 cells per n for each bud size). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001; ns = not significant. Error bars are SD. (E) Size-exclusion chromatography traces of the Myo2 tail (gray), either alone or co-expressed with N-terminally MBP-fused Vac17 peptides: Vac17(H) (light blue; top panel) and Vac17(MBD) (green; bottom panel). The orange arrowhead indicates free maltose-binding protein (MBP; 44 kDa), which was cleaved from the Vac17 peptides using TEV protease following MBP affinity purification. In a separate experiment, the Myo2 tail co-expressed with MBP-Vac17(H) was first purified via amylose resin, treated with TEV protease, and subsequently subjected to Strep-Tactin pulldown to isolate Myo2. Gel filtration confirmed that Vac17 remains associated with Myo2 (dark blue; top panel). Note that Vac17 peptides alone were unstable in the absence of Myo2.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal