Little is known about how molecular motors bind to their vesicular cargo. We have now shown that myosin Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor-protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin Va. Melanophilin creates this link by binding to Rab27a in a GTP-dependent fashion through its amino terminus, and to myosin Va through its carboxy terminus. This latter interaction, similar to the ability of myosin Va to colocalize with melanosomes and influence their distribution in vivo, is absolutely dependent on the presence of exon-F, an alternatively spliced exon in the myosin Va tail. In vitro reconstitution experiments using purified myosin Va, melanophilin, and GFP-tagged Rab27a, coupled with TIRF microscopy to visualize myosin Va-dependent movement on actin, show that the complex of Rab27a and melanophilin is both required and sufficient to form a functional myosin Va receptor. Finally, introduction of dominant negative versions of Rab27a into living cells, coupled with FRAP microscopy to determine the residence time of melanophilin and myosin Va on melanosomes, shows that regulation of the nucleotide state of Rab27a in vivo controls the recruitment of myosin Va onto the melanosome. Togther, these results have provided the first molecular description of an organelle motor for an actin-based motor, illustrated how alternate exon usage can be used to specify cargo, further expanded the functional repertiore of Rab GTPases and their effectors, and revealed a novel regulatory mechanism for the association of motors to organelles. CARMIL, also known as Acan125, is a multi-domain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. We have now obtained biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL co-purified extensively with two polypeptides that were shown by microsequencing to be the a- and b-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical crosslinking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ~0.4 mM or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl terminal, ~200 residue, proline-rich domain of CARMIL. Finally, chemical crosslinking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer:dimer equilibrium with an association constant of 1.0 x 106 M-1. Together, these results indicate that CARMIL self associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (~1 and 2 mM for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.
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