Cargo adaptors use a handhold mechanism to engage with myosin V for organelle transport

Myo2, a myosin V motor, is essential for organelle transport in budding yeast. Its attachment to and detachment from cargo are mediated by adaptor molecules. Vac17, a vacuole-specific adaptor, links Myo2 to Vac8 on the vacuole membrane, and plays a key role in the formation and dissociation of the Myo2-Vac17-Vac8 complex. Using genetics, cryo-electron microscopy and structure prediction, we find that Vac17 interacts with Myo2 through two distinct sites rather than a single interface. Similarly, the peroxisome adapter Inp2 engages two separate regions of Myo2, one of which overlaps with Vac17. These findings support a “handhold” model, in which cargo adaptors occupy multiple sites on the Myo2 tail, enhancing motor-cargo interactions and likely providing additional regulatory control over motor recruitment. Summary This study provides insights into how cargo adaptors bind myosin V. Genetics, cell-based assays, cryo-EM, and AlphaFold, reveal that the vacuole-specific adaptor uses a handhold mechanism to attach to two areas on the myosin V tail. Moreover, evidence is presented that other adaptors use a similar strategy.