The vac17ΔH mutation specifically impairs its interaction with Myo2. (A) In wild-type cells, Vac17 accumulates on the vacuole surface closest to the bud cortex, while Myo2 localizes to either the bud tip or the mother-bud neck. Upon vacuole arrival at the bud tip, Vac17 undergoes degradation. In the myo2(D1297N) mutant, which is defective in vacuole inheritance, Vac17 is distributed around the vacuole, whereas myo2(D1297N) maintains its normal localization at sites of polarized growth. The vac17ΔH and myo2(D1297N) double mutant does not further alter the localization pattern of Vac17 or Myo2 compared with the myo2(D1297N) mutant. Scale bars = 5 μm. (B)vac17ΔH colocalizes with Vac8-RFP (pseudo-colored yellow), indicating that its binding to Vac8 is not impaired. (C) In the absence of Vac8, Vac17 localizes presumably with Myo2 at the bud tip or the mother-bud neck, but vac17ΔH is cytosolic. (D) Deletion of the canonical Vac17(MBD) abolishes vacuole inheritance. (E) Top: SDS-PAGE analysis of apo Myo2 tail purification. The peak elutes at 15.5–16 ml. Middle: Analysis of the Myo2 tail+Vac17(MBD) complex, showing an expected peak between 15.5 and 16 ml. There is also a higher molecular weight complex with both Vac17 and Myo2 at 13.5–14 ml. This latter peak was not analyzed further. Bottom: Immunoblot analysis of the gel filtration experiment from (Fig. 1 E; top panel, lighter blue trace) shows the Myo2 tail+Vac17(H) complex at 14.5–15 ml. The membrane used to detect Myo2 was stripped and re-probed for MBP. The arrowheads correspond with the same arrowheads shown in (Fig. 1 E): Gray = apo Myo2 tail; orange = free MBP; blue = Myo2 tail+Vac17(H); green = Myo2 tail+Vac17(MBD). MBD, Myo2-binding domain. Source data are available for this figure: SourceData FS1.