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Figure 5.
Identification of a second Vac17–Myo2 interface. (A) 2D class averages of Vac17–Myo2 without or with the fuzzy density (blue arrowhead). These were observed in the same dataset. Location of the Vac17(MBD) (green arrowhead). (B) A second reconstruction of the Vac17–Myo2 interaction includes the additional density (blue) obtained through particle subtraction. Focused alignment-free 3D classification was performed on the subtracted particles. The additional Vac17 density is located near the Myo2 region, crucial for transporting various cargoes, including peroxisomes, secretory vesicles, astral microtubules, and others (Eves et al., 2012). Underlined residues are important for vacuole inheritance, some of which were previously implicated in the transport of other cargoes, while others had not been tested (cyan). Among them, E1338 is predicted by AlphaFold to adopt a short helix (black), but it remains unresolved in the cryo-EM map and other myosin V tail structures. (C) Vacuole inheritance was tested for sites on Myo2 near the Vac17 density, Vac17(H) (n = 3 independent experiments; ≥35 cells per group per n). Significance was determined using ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent SD. ns = not significant. (D) Representative blot of an in vitro pull-down assay of the Strep II-Myo2 tail with Vac17(H) to assess the impact of Myo2 mutations on the interaction. Wild-type and Myo2 mutants were co-expressed with either MBP or MBP-Vac17(H) and immobilized on Strep-TactinXT beads. (E) Binding efficiency from D was analyzed by normalizing to Vac17 and Myo2 soluble input levels, respectively, to account for their variable expression levels. The intensity ratio of Vac17 pulled down with Myo2 mutants was then compared as a fold change from wild-type complex. Data were analyzed using ordinary one-way ANOVA with a multiple comparisons test (n = 3 independent purifications). **P < 0.01; *P < 0.05. (F) A suppressor mutation within Vac17(H), N60Y, restores vacuole inheritance in the myo2(E1333R and V1448A) double mutant by engaging a newly identified Myo2 site. In a separate experiment, introducing a partial vac17(F132Y) mutation—located in the canonical Myo2-binding domain—into the myo2(E1333R and V1448A) background abolished vacuole inheritance. These results suggest that interactions at both sites are important for vacuole transport. n = 3 independent experiments from separate yeast colonies, with ≥35 cells per group per experiment. Data were analyzed using ordinary one-way ANOVA with a multiple comparisons test. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns = not significant. (G) An AlphaFold model of the Vac17(H)–Myo2 interaction. This region of Vac17 forms an antiparallel three-helix bundle. The predicted direct contact between Vac17 h2, residues 42–79, and Myo2 is supported by the observation that the vac17(N60Y) mutant in h2 rescues the vacuole inheritance defect of the myo2(E1338R and V1448A) mutant at this newly identified site. Note that Myo2(E1338) is absent from all resolved structures, and its precise location remains unknown. Green = Vac17(MBD) peptide; red = suppressor residues that may interact with Myo2; cyan = newly identified Myo2 residues; orange = Myo2 residue, previously characterized in other cargo transport, also binds Vac17(H) and functions in vacuole inheritance. MBD, Myo2-binding domain. Source data are available for this figure: SourceData F5.

Identification of a second Vac17–Myo2 interface. (A) 2D class averages of Vac17–Myo2 without or with the fuzzy density (blue arrowhead). These were observed in the same dataset. Location of the Vac17(MBD) (green arrowhead). (B) A second reconstruction of the Vac17–Myo2 interaction includes the additional density (blue) obtained through particle subtraction. Focused alignment-free 3D classification was performed on the subtracted particles. The additional Vac17 density is located near the Myo2 region, crucial for transporting various cargoes, including peroxisomes, secretory vesicles, astral microtubules, and others (Eves et al., 2012). Underlined residues are important for vacuole inheritance, some of which were previously implicated in the transport of other cargoes, while others had not been tested (cyan). Among them, E1338 is predicted by AlphaFold to adopt a short helix (black), but it remains unresolved in the cryo-EM map and other myosin V tail structures. (C) Vacuole inheritance was tested for sites on Myo2 near the Vac17 density, Vac17(H) (n = 3 independent experiments; ≥35 cells per group per n). Significance was determined using ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Error bars represent SD. ns = not significant. (D) Representative blot of an in vitro pull-down assay of the Strep II-Myo2 tail with Vac17(H) to assess the impact of Myo2 mutations on the interaction. Wild-type and Myo2 mutants were co-expressed with either MBP or MBP-Vac17(H) and immobilized on Strep-TactinXT beads. (E) Binding efficiency from D was analyzed by normalizing to Vac17 and Myo2 soluble input levels, respectively, to account for their variable expression levels. The intensity ratio of Vac17 pulled down with Myo2 mutants was then compared as a fold change from wild-type complex. Data were analyzed using ordinary one-way ANOVA with a multiple comparisons test (n = 3 independent purifications). **P < 0.01; *P < 0.05. (F) A suppressor mutation within Vac17(H), N60Y, restores vacuole inheritance in the myo2(E1333R and V1448A) double mutant by engaging a newly identified Myo2 site. In a separate experiment, introducing a partial vac17(F132Y) mutation—located in the canonical Myo2-binding domain—into the myo2(E1333R and V1448A) background abolished vacuole inheritance. These results suggest that interactions at both sites are important for vacuole transport. n = 3 independent experiments from separate yeast colonies, with ≥35 cells per group per experiment. Data were analyzed using ordinary one-way ANOVA with a multiple comparisons test. ****P < 0.0001; ***P < 0.001; **P < 0.01; ns = not significant. (G) An AlphaFold model of the Vac17(H)–Myo2 interaction. This region of Vac17 forms an antiparallel three-helix bundle. The predicted direct contact between Vac17 h2, residues 42–79, and Myo2 is supported by the observation that the vac17(N60Y) mutant in h2 rescues the vacuole inheritance defect of the myo2(E1338R and V1448A) mutant at this newly identified site. Note that Myo2(E1338) is absent from all resolved structures, and its precise location remains unknown. Green = Vac17(MBD) peptide; red = suppressor residues that may interact with Myo2; cyan = newly identified Myo2 residues; orange = Myo2 residue, previously characterized in other cargo transport, also binds Vac17(H) and functions in vacuole inheritance. MBD, Myo2-binding domain. Source data are available for this figure: SourceData F5.

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