Interaction of a Class V Myosin from Budding Yeast with its Adapter Protein

Like their mammalian counterparts, class V myosins in S. cerevisiae (Myo2p and Myo4p) bind to various adapter proteins to target a particular cargo for transport. Myo4p uses the adapter proteins She3p and She2p in order to transport mRNA from the mother cell to the bud. She3p binds to the rod of Myo4p, and prevents it from dimerizing, thus forming a single-headed motor complex (Hodges et al., 2008; Bookwalter et al., 2009). Because the Myo4p/She3p complex is single-headed, the question arises as to whether enough motors can bind to a single She2p to enable continuous cargo transport. The She2p crystal structure suggested that She2p exists as a dimer (Niessing et al., 2004). In contrast, our sedimentation equilibrium measurements of She2p were consistent with formation of a tetramer in solution, in principle allowing for binding of four motor heads. We showed that Myo4p/She3p forms a complex with tetrameric She2p in the absence of mRNA, based on sedimentation velocity experiments and co-purification. Mutation of Ser 120 to Tyr converts She2p to a dimer. The ability of the motor complex to bind to this and other She2p mutants is being tested in order to map the binding interface. Total internal reflection fluorescence microscopy is being used to test whether the native She2p tetramer can bind enough single-headed motors to support continuous movement on actin. The ability of She2p mutants to support correct bud tip localization of ASH1 mRNA in living yeast cells will also be assessed. These studies will help elucidate how a non-processive single-headed motor can act as a cargo transporter.