ABSTRACT
Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Carrier Proteins/metabolism , Molecular Motor Proteins/metabolism , Myosin Heavy Chains/metabolism , Animals , Carrier Proteins/genetics , Chickens , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microfilament Proteins/metabolism , Myosin Heavy Chains/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SwineABSTRACT
It is thought that Switch II of myosin, kinesin and G proteins has an important function in relating nucleotide state to protein conformation. Here we examine a myosin mutant containing an S456L substitution in the Switch II region. In this protein, mechanical activity is uncoupled from the chemical energy of ATP hydrolysis so that its gliding velocity on actin filaments is only one-tenth of that of the wild type. The mutant spends longer in the strongly bound state and exhibits a shorter step size, which together account for the reduction in in vitro velocity. This is the first single point mutation in myosin that has been found to affect step size.
Subject(s)
Dictyostelium/genetics , Myosins/genetics , Myosins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Catalytic Domain , Dictyostelium/enzymology , Dictyostelium/physiology , Kinetics , Models, Biological , Models, Molecular , Molecular Motor Proteins/metabolism , Movement , Mutation , Myosins/chemistry , Myosins/isolation & purification , Plasmids , Protein Conformation , Protein Structure, Tertiary , Spectrophotometry , Transformation, GeneticABSTRACT
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 approximately 36-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.
Subject(s)
Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/metabolism , Models, Biological , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/metabolism , Movement , Myosin Type V , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Actins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Brain , Chickens , Feedback , Kinetics , Lasers , MicrospheresABSTRACT
Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.
Subject(s)
Biophysics/methods , Calmodulin-Binding Proteins/physiology , Molecular Motor Proteins/physiology , Movement/physiology , Myosin Light Chains/physiology , Myosin Type V , Nerve Tissue Proteins/physiology , Actin Cytoskeleton/physiology , Optics and PhotonicsABSTRACT
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.
