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Myosin Va (myoV) is a processive molecular motor that transports intracellular cargo along actin tracks with each head taking multiple 72-nm hand-over-hand steps. This stepping behavior was observed with a constitutively active, truncated myoV, in which the autoinhibitory interactions between the globular tail and motor domains (i.e., heads) that regulate the full-length molecule no longer exist. Without cargo at near physiologic ionic strength (100 mM KCl), full-length myoV adopts a folded (approximately 15 S), enzymatically-inhibited state that unfolds to an extended (approximately 11 S), active conformation at higher salt (250 mM). Under conditions favoring the folded, inhibited state, we show that Quantum-dot-labeled myoV exhibits two types of interaction with actin in the presence of MgATP. Most motors bind to actin and remain stationary, but surprisingly, approximately 20% are processive. The moving motors transition between a strictly gated and hand-over-hand stepping pattern typical of a constitutively active motor, and a new mode with a highly variable stepping pattern suggestive of altered gating. Each head of this partially inhibited motor takes longer-lived, short forward (35 nm) and backward (28 nm) steps, presumably due to globular tail-head interactions that modify the gating of the individual heads. This unique mechanical state may be an intermediate in the pathway between the inhibited and active states of the motor.

Myosin Va is a well known processive motor involved in transport of organelles. A tail-inhibition model is generally accepted for the regulation of myosin Va: inhibited myosin Va is in a folded conformation such that the tail domain interacts with and inhibits myosin Va motor activity. Recent studies indicate that it is the C-terminal globular tail domain (GTD) that directly inhibits the motor activity of myosin Va. In the present study, we identified a conserved acidic residue in the motor domain (Asp-136) and two conserved basic residues in the GTD (Lys-1706 and Lys-1779) as critical residues for this regulation. Alanine mutations of these conserved charged residues not only abolished the inhibition of motor activity by the GTD but also prevented myosin Va from forming a folded conformation. We propose that Asp-136 forms ionic interactions with Lys-1706 and Lys-1779. This assignment locates the GTD-binding site in a pocket of the motor domain, formed by the N-terminal domain, converter, and the calmodulin in the first IQ motif. We propose that binding of the GTD to the motor domain prevents the movement of the converter/lever arm during ATP hydrolysis cycle, thus inhibiting the chemical cycle of the motor domain.

MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.