Mmr1 and Vac17(MBD) bind a shared site on Myo2 and are antiparallel to each other. (A) AlphaFold model docked within the cryo-EM map of Vac17(MBD; 127–147) bound to Myo2 tail. Vac17-binding region (blue), Mmr1-binding region (red), and overlapping region of Vac17 and Mmr1 on Myo2 (pink) (Eves et al., 2012; this study). (B) Vac17(MBD) extends along a hydrophobic groove (gold) on the Myo2 tail, which contains multiple surface residues critical for vacuole inheritance. (C) Vac17(L137) makes hydrophobic interactions with Myo2(L1301 and L1229). Dashed lines represent the distances between hydrophobic side chains, ranging from 2.1 to 4.9 Å. (D) An Mmr1 peptide (408–425) bound to the Myo2 tail (PDB accession no. 6IXP) was docked into the EM reconstruction. The Mmr1 peptide partially fits into the density of Vac17(MBD), consistent with a previous study showing that Vac17 and Mmr1 peptides compete for access to Myo2 in vitro (Eves et al., 2012). (E) Both the Vac17 and Mmr1 adaptor proteins contain serine phosphosites (depicted as space-filing models) that are important for dissociation from Myo2 (Wong et al., 2020; Obara et al., 2022). MBD, Myo2-binding domain.