The motor domain determines the large step of myosin-V

Tanaka, Hiroto; Homma, Kazuaki; Iwane, Atsuko H.; Katayama, Eisaku; Ikebe, Reiko; Saito, Junya; Yanagida, Toshio; Ikebe, Mitsuo

doi:10.1038/415192a

Cited by 125 publications

(86 citation statements)

References 27 publications

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“…The attachment lifetime at 2 mM ATP (0.12 s) is shorter than that at 1 M ATP concentration (0.52 s). The value at 2 mM ATP is consistent with the V max of the actin-activated Mg-ATPase and the expected dwell time (0.11 s) from Tanaka et al (20) if S1 is not a processive molecule. At 1 M ATP the lifetime is longer as expected for a second order reaction at low substrate concentration.…”

Section: Characterization Of Expressed Myosin Iq Mutant-to Exam-supporting

confidence: 69%

Neck Length and Processivity of Myosin V

Sakamoto

Wang

Schmitz³

et al. 2003

Journal of Biological Chemistry

146

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Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 M or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.The myosin superfamily consists of at least 18 classes of actin-dependent molecular motors (1). Class V myosins transport cargos such as endoplasmic reticulum in neurons, melanosomes in melanocytes, and mRNA in yeast (2). Similar to other myosins, they are composed of a head that binds ATP and actin and a neck region consisting of calmodulin (CaM) 1 molecules bound to an ␣-helical segment of the myosin heavy chain. The C-terminal tail domain of myosin V has coiled-coil forming motifs that dimerize and create two-headed molecules but do not self-associate into filamentous structures. The IQ motifs of the heavy chain, implicated in the binding of CaM or light chain subunits, have the consensus sequence, IQXXXRGXXXR, where X is any amino acid (3). The neck of mouse myosin V has six IQ motifs, each of which binds CaM, making its neck longer than that of most myosins (4). The six IQ motifs of all myosin V superfamily members are separated by 25-23-25-23-25 amino acids.The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term "low duty cycle" motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpede...

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Section: Characterization Of Expressed Myosin Iq Mutant-to Exam-supporting

confidence: 69%

Neck Length and Processivity of Myosin V

Sakamoto

Wang

Schmitz³

et al. 2003

Journal of Biological Chemistry

146

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“…This value is similar to the step size (36 nm) of myosin Va (13,15). Therefore, it is reasonable to assume that myosin Vb moves on actin filaments with large steps in a manner similar to that of myosin Va.…”

Section: Discussionsupporting

confidence: 56%

“…This processive behavior is consistent with the function of myosin Va in the cell as a cargo transporter (9). A finding critical to understand the mechanism of processive movement of myosin Va is the extremely large step size of myosin Va (13,15,16). Because myosin Va has an extremely long neck domain, it was proposed that myosin Va moves with a handover-hand mechanism in which myosin heads span a helical repeat of the actin filament with its long neck and walk straight along it with one head attached and the other head dissociated (17)(18)(19).…”

mentioning

confidence: 99%

Mechanoenzymatic Characterization of Human Myosin Vb

et al. 2006

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There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V max and K actin of the actin-activated ATPase activity of HuM5B were 9.7 ( 0.4 s -1 and 8.5 ( 0.1 µM, respectively. K actin and K ATP of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 ( 0.5 s -1 , which was comparable to the V max of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 ( 0.03 µm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.Myosin is a mechanoenzymatic protein that interacts with actin and converts the chemical energy from ATP hydrolysis to the mechanical work. In addition to well-characterized conventional, filament-forming, two-headed myosin II of muscle and non-muscle cells, a number of myosin-like proteins have been discovered, and it is known that myosin constitutes a diverse superfamily divided into at least 18 classes (1-7). Class V myosins are one of the most ancient myosins of the superfamily, and their genes have been identified in species from yeast, Caenorhabditis elegans, and Drosophila to humans (7). Dictyostelium MyoJ and plant class XI and VIII myosins are structurally and functionally related to class V myosins. Yeast contains two class V myosins, while C. elegans and Drosophila contain a single class V myosin. In mammals, there are three distinct subclasses of myosin V. Mammalian class V myosin was first discovered from dilute mice that have a lighter color coat due to the lack of proper pigment transport (8). This myosin V is now classified as myosin Va, and most of the biochemical and cell biological works for class V myosin have been done with this myosin V isoform (see ref 9 for a review). There are two additional myosin V isoforms reported, i.e., myosin Vb and myosin Vc (10, 11), yet their properties at a molecular level have not been studied.The structure of myosin Va was visualized by electron microscopy, and it was shown that myosin Va is the twoheaded structure with the long neck connecting the head and the coiled-coil domain. On the basis of the amino acid sequence homology with myosin Va (10, 11), it is thought that, overall, the structure of myosin Vb and myosin Vc resembles that of myosin Va. The heavy chain of myosin Vb has a molecular mass of 214 kDa and composed of the N-terminal conserved m...

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“…Yanagida and co-workers created a myosin V construct with a shorter lever arm and they observed that the step size does not change with a decreasing length of the lever arm. 47 (The same experiment was then repeated by using similar constructs and it was shown that the step size depends on the length of the lever arm 19 ). 4.…”

Section: The Challengementioning

confidence: 99%

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

2005

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We introduce the technique of FIONA, fluorescence imaging with one nanometer accuracy. This is a fluorescence technique that is able to localize the position of a single dye within ∼1 nm in the x-y plane. It is done simply by taking the point spread function of a single fluorophore excited with wide field illumination and locating the center of the fluorescent spot by a two-dimensional Gaussian fit. We motivate the development of FIONA by unraveling the walking mechanism of the molecular motors myosin V, myosin VI, and kinesin. We find that they all walk in a hand-over-hand fashion.

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The motor domain determines the large step of myosin-V

Cited by 125 publications

References 27 publications

Neck Length and Processivity of Myosin V

Neck Length and Processivity of Myosin V

Mechanoenzymatic Characterization of Human Myosin Vb

Fluorescence Imaging with One Nanometer Accuracy: Application to Molecular Motors

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