BtaEX0023574 @ bosTau6

Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To these knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, the authors report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A_, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab ++subfamilies (Rab6, Rab14, Rab39B). Different Myo5A isoforms where tested including isoform D (includes cassette exon D (HsaEX0041195)), and isoform F (includes cassette exon F (HsaEX0041194)). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. The authors demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. The authors also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes. Note, the Myo5a isoforms show some differences in interactome.

Here the authors show 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. Moreover, 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 (HsaEX0041194), an alternatively spliced exon in the myosin-Va tail.

The authors also show that melanophilin associates with Rab27a and myosin Va on melanosomes in melanocytes, and present evidence that a domain within the carboxyl-terminal region of melanophilin interacts with the carboxyl-terminal tail of the melanocyte-specific splice isoform of myosin Va (contains cassette exon, exon F (HsaEX0041194). Thus, melanophilin can associate simultaneously with activated Rab27a and myosin Va via distinct regions, and serve as a linker between these proteins.

The mouse Myosin Va (MyoVa) is encoded by the dilute locus, which is alternatively spliced to generate several tissue specific isoforms. The tail of MyoVa is the putative cargo-binding domain. To determine the functions of different isoforms of MyoVa and the minimal cargo-binding region, the authors tagged various isoforms and different portions of the mouse MyoVa tail with a green fluorescent protein and examined their intracellular localizations in the mouse melan-a cells. The authors found that the amino acid sequence encoded by an alternatively spliced exon, exon F (HsaEX0041194), is necessary for the selective binding of MyoVa to melanosome. The MyoVa isoforms lacking this amino acid sequence are not targeted to the melanosomes, but localized to the perinuclear region instead.

Here the authors found that melanophilin directly activates the actin-activated ATPase activity of myosin Va and thus its motor activity. The actin-activated ATPase activity of the melanocyte-type myosin Va having exon-F was significantly activated by melanophilin by 4-fold. Although Rab27a binds to myosin Va/melanophilin complex, it did not affect the melanophilin-induced activation of myosin Va. Deletion of the C-terminal actin binding domain and N-terminal Rab binding domain of melanophilin resulted in no change in the activation of the ATPase by melanophilin, indicating that the myosin Va binding domain (MBD) is sufficient for the activation of myosin Va. Among MBDs, the interaction of MBD-2 with exon-F (HsaEX0041194) of myosin Va is critical for the binding of myosin Va and melanophilin, whereas MBD-1 interacting with the globular tail of myosin Va plays a more significant role in the activation of myosin Va ATPase activity. This is the first demonstration that the binding of the cargo molecule directly activates myosin motor activity. The present finding raises the idea that myosin motors are switched upon their binding to the cargo molecules, thus avoiding the waste of ATP consumption.