Human Myosin Vc Is a Low Duty Ratio, Nonprocessive Molecular Motor

James R Sellers,Mihály Kovács,Damon Jacobs,Ikuko Fujiwara,Yasuharu Takagi,Richard E Cheney,Yi Yang

doi:10.1074/jbc.m709150200

Abstract

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

Highlights

EXPERIMENTAL PROCEDURESExpression and Purification of the Myosin Vc-S1 and heavy meromyosin (HMM) Proteins—Using a cDNA clone of the H. sapiens myosin Vc encoding the 2930 amino acids fused with an N-terminally fused enhanced GFP, we engineered a cDNA fragment that encodes the first 777 amino acids, containing the motor domain plus the first predicted light chain binding IQ motifs (myosin Vc subfragment-1; MVc-S1)
Class V myosins are part of the myosin superfamily, currently composed of as many as 37 types of myosins [1]
We report a full biochemical kinetic characterization of the Homo sapiens myosin Vc subfragment-1 (MVcS1) protein

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of the Myosin Vc-S1 and HMM Proteins—Using a cDNA clone of the H. sapiens myosin Vc encoding the 2930 amino acids fused with an N-terminally fused enhanced GFP, we engineered a cDNA fragment that encodes the first 777 amino acids, containing the motor domain plus the first predicted light chain binding IQ motifs (myosin Vc subfragment-1; MVc-S1). The SF buffer contained the following reagents: 20 mM MOPS (pH 7.0), 5 mM MgCl2, 0.05 mM EGTA, and 50 mM KCl. To determine the kinetic parameters for Pi release, double-mixing stopped-flow experiments using MDCC-PBP [55] were performed as in Ref. 54. In Vitro Actin Gliding Motility and Single Molecule TIRF Microscopy Assay—Both light microscopy assays were performed using an apparatus previously described by Sakamoto et al [57, 62] with some minor modifications. Motility assays were performed in buffer containing the following reagents: 20 mM MOPS (pH 7.4), 5 mM MgCl2, 0.1 mM EGTA, 50 mM KCl, 1 mM ATP, 5 ␮M calmodulin, 25 ␮g/ml glucose oxidase, 45 ␮g/ml catalase, 2.5 mg/ml glucose, and 20 mM dithiothreitol (final concentrations listed).

RESULTS

NADH assay NADH assay NADH assay

DISCUSSION