Vac17(MBD) contains an unstructured peptide that extends along the Myo2 tail. (A) Cryo-EM reconstruction of the Vac17 peptide (green) bound to the Myo2 tail (gray). (B) AlphaFold model of the Vac17(1–157) and Myo2 tail complex was trimmed to fit into the density and refined in real space using Phenix. Vac17(MBD) density (green) is located near the Myo2 helix (H6), which was previously characterized to contain residues critical for vacuole inheritance (dark blue) and for the inheritance of both vacuole and mitochondria (pink) (Eves et al., 2012; Pashkova et al., 2006). (C) Vac17 residues predicted to form contacts with known Myo2 residues. Close contacts are shown in green dashed lines, and interactions over longer distances of 3.8–5 Å are in yellow lines. An asterisk represents one of the Vac17 suppressors, I140V, identified in the screen. This further supports the hypothesis that this region of Vac17 directly interacts with Myo2. (D) Vacuole inheritance in small buds of the indicated Vac17 mutants (n = 3 experiments from independent yeast colonies, with ≥35 cells per n). Note that in the vac17(L137A) mutant, only 6% of large buds contained a vacuole, whereas vacuoles were present in large buds in the other mutants reported here (not shown). Significance was determined by an unpaired two-tailed Student’s t test. Error bars are SD. ****P < 0.0001; ***P < 0.001; **P < 0.005; ns = not significant. (E) Close-up view showing predicted interaction of Vac17(R142) with Myo2(E1293) and Myo2(E1222). (F) Charge reversal mutations of vac17(R142E) and myo2(E1222K) partially restore vacuole inheritance (n = 3; ≥35 cells per n for each bud size). Significance was determined using ordinary one-way ANOVA with a multiple comparisons test. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns = not significant. Error bars are SD. MBD, Myo2-binding domain.