D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

Hiroaki Kubota,Sergey V Mikhailenko,Harumi Okabe,Hideki Taguchi,Shin'Ichi Ishiwata

doi:10.1074/jbc.m109.013565

Hiroaki Kubota, Sergey V Mikhailenko + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.013565

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Abstract

To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V "walked" with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP.P(i) state of both myosins.

Highlights

EXPERIMENTAL PROCEDURESPlasmid Construction—The pBIG plasmid, which contained a neomycin-resistant gene and the complete Dictyostelium actin 15 sequence [14] with the E360H mutation, was a gift from Prof
The D-loop is highly conserved between different classes of actin, and its sequence in rabbit skeletal and Dictyostelium actins is identical with the only exception that Gln41 of rabbit skeletal actin is replaced with Thr
The effects of subtilisin cleavage and the M47A mutation can be directly compared even when studied with different actin classes, and the obtained results should be applicable for the cytoplasmic actin on which myosin V travels in cells

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—The pBIG plasmid, which contained a neomycin-resistant gene and the complete Dictyostelium actin 15 sequence [14] with the E360H mutation, was a gift from Prof. For an in vitro motility assay and a single-molecule myosin V movement assay, both wild-type and modified rabbit skeletal and Dictyostelium G-actins were polymerized by the addition of 100 mM KCl, 2 mM MgCl2, 2 mM MOPS (pH 7.0), and 1.5 mM NaN3. Measurement of the Actin-activated ATPase of MMV-HMM— Actin-activated ATPase of MMV-HMM was determined by monitoring the absorbance of NADH at 340 nm in the solution containing 56 nM MMV-HMM, 2.35 ␮M calmodulin, 20 mM imidazole-HCl (pH 7.4), 4 mM MgCl2, 40 mM KCl, 1 mM EGTA, 53 ␮M phalloidin, 400 ␮M NADH (Wako (Osaka, Japan)), 800 ␮M phosphoenolpyruvate (Wako), 47 ␮g/ml pyruvate kinase (Oriental Yeast (Tokyo, Japan)), 12 ␮g/ml lactate dehydrogenase (Oriental Yeast), 1 mM ATP, 1 mM dithiothreitol, and various concentrations of actin (2.2–39.7 ␮M). Cosedimentation Assay—The apparent affinity of MMV-S1 for actin was determined by cosedimentation assay, performed in the solution containing 20 mM imidazole-HCl (pH 7.4), 4 mM MgCl2, 1 mM EGTA, 53 ␮M phalloidin, 1 mM dithiothreitol, and various concentrations of KCl (40 –150 mM), as follows. The supernatant and the pellet were applied to an SDS-polyacrylamide gel, and the amount of MMV-S1 was determined by densitometry using CS analyzer software (ATTO (Tokyo, Japan))

RESULTS

Dictyostelium wild typea

DISCUSSION