Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete Ϸ36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity. C lass-V myosins, two-headed actin-based motors (1), have been implicated in several forms of organelle transport (2). The various roles of molecular motors require special kinetic adaptations (3). Unlike muscle myosin-II, which assembles in large arrays, myosin-V is a processive motor (4), meaning that one molecule can undergo multiple productive catalytic cycles and associated mechanical steps before it detaches from its track. To understand the mechanism for chemomechanical transduction, one must decipher the kinetic scheme underlying ATP turnover and movement. Presteady-state kinetic studies have helped clarify such mechanisms in many motor proteins (5). In the case of myosin-V, kinetic characterization of truncated single-headed constructs in bulk studies has contributed important insights into the myosin-V ATPase cycle (6, 7). However, to understand the mechanism for myosin-V processivity, it is essential to study the full-length double-headed dimer through the course of its movement. In the present study, we used a force feedback-enhanced laser trap to measure the stepping rate of myosin-V molecules purified from brain. This allowed us to characterize the rate-limiting transition in the turnover cycle. Materials and MethodsBead Preparation. One-microliter Polystyrene beads (Ø 356 nm, Polysciences, 2.5% solid) were incubated for 15 min in 99 l of buffer (25 mM imidazole HCl, pH 7.4͞25 mM KCl͞1 mM EGTA͞10 mM DTT͞4 mM MgCl 2 ) containing 10 g͞ml BSA (to preblock the surface), 1 g͞ml tetramethyl rhodaminelabeled BSA, and 30 pM tissue-purified chick-brain myosin-V [purification as described in (8) Optical Trap. Beads were optically trapped and positioned near a fluorescently labeled biotinylated actin filament immobilized onto an avidin-coated coverslip. Imaging and trap steering were as described (9-11). A feedback loop (M44 DSP-board, Innovative Integration, West Lake Village, CA) maintains a constant separation between the bead and trap centers. This distance scales with the load experienced by the molecule as it steps along the actin filament. The trap stiffness was calibrated for each trapped bead from the amplitude of the thermal diffusion. For some beads, it was also calibrated by measurement of the bead rise time in response to sudden trap displacement and by the 3-dB corner frequency in the diffusion power spectrum. The three methods gave consistent results. Results and DiscussionPolystyrene beads, sparsely coated with myosin-V molecules, were optically trapped ...