Abstract 2364 Cargo adaptors bind myosin V with a hand-hold mechanism

Molecular motors are critical contributors to regulated cellular processes, by mediating cargo trafficking and positioning throughout the cellular milieu. Here, we focus on Myo2, an essential myosin V motor in Saccharomyces cerevisiae, which is the primary driver of organelle localization. While at least nine cargo-specific adaptors for Myo2 have been identified, how they regulate/co-regulate cargo motility is unclear. Previously, we reported that Vac17(112-157), vacuole-specific adaptor, directly competes for access to Myo2 tail. By combining genetic studies with single-particle cryo-electron microscopy (cryo-EM), we examine the molecular basis of how Myo2 engages with cargoes. Through genetic screen and secondary structure analysis, we identified a new functional region, Vac17(18-108). The vac17Δ18-108 mutant displays a defect in vacuole inheritance, whereby small buds do not initially inherit vacuoles, but large buds eventually receive them. The vac17Δ18-108 mutant is mislocalized and found throughout the vacuole membrane rather than in polarized puncta. Interestingly, Vac17(1-109) and Vac17(110-157) each independently binds Myo2 tail in vitro. We hypothesized that both Vac17 regions are important for stable vacuole transport. To understand how the interaction occurs, we purified the extended Vac17(1-157) peptide and Myo2 tail complex for cryo-EM structure determination. Our structural analysis reveals a strong density for Vac17 on one side of the Myo2 tail, in agreement with the established Myo2 surface residues important for the vacuole inheritance pathway. We also observed additional fuzzy density outside of Myo2 tail, which is currently limited to 2D class averages. In parallel to cryo-EM studies, we used AlphaFold to predict complex structures of Myo2 tail with Vac17(1-157). The predictions of Vac17 agree with the binding site identified in our density map. Surprisingly, a second Vac17(1-109) binding site, not originally found in cryo-EM structure, was also identified in a distal region of Myo2 tail that is characterized to be important for trafficking other cargoes. In each binding region, we identified interacting residues for both Myo2 and Vac17, and showed that charge complementation of selected mutants rescues the vacuole inheritance defects, which supports the hypothesis that these are bona fide contact sites. Together, the genetic and structural studies suggest a model whereby the adaptor's binding to two distinct sites on motor tail provides stable cargo transport complex. These findings further suggest that other myosin V adaptors may employ a similar cargo adaptor attachment mechanism.