Overlap of cargo binding sites on myosin V coordinates the inheritance of diverse cargoes.
During cell division, organelles are distributed to distinct locations at specific times. For the yeast vacuole, the myosin V motor, Myo2, and its vacuole-specific cargo adaptor, Vac17, regulate where the vacuole is deposited and the timing of vacuole movement. In this paper, we show that Mmr1 functions as a mitochondria-specific cargo adaptor early in the cell cycle and that Mmr1 binds Myo2 at the site that binds Vac17. We demonstrate that Vac17 and Mmr1 compete for binding at this site. Unexpectedly, this competition regulates the volume of vacuoles and mitochondria inherited by the daughter cell. Furthermore, eight of the nine known Myo2 cargo adaptors overlap at one of two sites. Vac17 and Mmr1 overlap at one site, whereas Ypt11 and Kar9 bind subsets of residues that also bind Ypt31/Ypt32, Sec4, and Inp2. These observations predict that competition for access to Myo2 may be a common mechanism to coordinate the inheritance of diverse cargoes.