Mechanoenzymatic Characterization of Human Myosin Vb

Watanabe, Shinya; Mabuchi, Katsuhide; Ikebe, Reiko; Ikebe, Mitsuo

doi:10.1021/bi051682b

Cited by 41 publications

(33 citation statements)

References 59 publications

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“…First, the ADP release rate from actomyosin Vc was significantly larger than the overall ATPase cycle rate; therefore, it does not solely determine the rate of the ATP hydrolysis cycle. This is quite different from myosin Va and myosin Vb, in which the ADP release step explains 80 -90% of the entire cycle rate (13,22). Second, unlike myosin Va, the P i burst size is only 0.3, suggesting that the ATP hydrolysis step (K 3 ) is largely shifted to the prehydrolyzed form (MT).…”

Section: Discussionmentioning

confidence: 77%

“…The observed rate of dissociation of acto-M5CIQ1 is explained by the ADP dissociation step (kЈ ϩ5 ) from acto-M5CIQ1, since the ATP binding step does not limit the dissociation rate at these ATP concentrations. The obtained ADP dissociation rate from acto-M5CIQ1 was significantly larger than the V max of the steady-state ATPase cycle rate of M5CIQ1, in contrast to myosin Va and Vb, whose rates of ADP dissociation explain the overall actin-activated ATPase cycling rate (13,22). These results suggest that the ADP dissociation step is not the rate-limiting step in the ATPase cycle of myosin Vc, which is quite different from myosin Va and Vb.…”

mentioning

confidence: 71%

“…Multimolecule in Vitro Motility Assay-The actin gliding velocity was measured by an in vitro actin gliding assay as described previously (22,30). The experiment was done with a buffer containing 25 mM KCl, 5 mM MgCl 2 , 1 mM EGTA, 25 mM MOPS-KOH (pH 7.5), 0.2 mg/ml calmodulin, 1 mM ATP, 10 mM DTT, and oxygen scavenger system (216 g/ml glucose oxidase, 36 g/ml catalase, 4.5 mg/ml glucose).…”

Section: Methodsmentioning

confidence: 99%

“…The results indicate that calmodulin has a much higher affinity for the first IQ motif of myosin Vc than LC17b. It should be noted that the light chains of mammalian myosin Va and Vb are calmodulin but not LC17 (22,36). In the present study, we used M5CIQ1 and GFP-M5CHMM with calmodulin for all experiments.…”

Section: Expression and Purification Of Myosin Vcmentioning

confidence: 99%

“…The question is whether all three myosin V isoforms expressed in vertebrates are processive motors and support vesicular transportation in cells. Quite recently, we reported that human myosin Vb is a high duty ratio and processive motor like myosin Va (22). In the present study, we analyzed the detailed ATP hydrolysis mechanism of human myosin Vc for the first time.…”

mentioning

confidence: 92%

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Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor

Watanabe

Satō

et al. 2008

Journal of Biological Chemistry

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There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. K ATPase of the actin-activated ATPase was 62 M, which is much higher than that of myosin Va (ϳ1 M). The rate of ADP release from actomyosin Vc was 12.7 s ؊1 , which was 2 times greater than the entire ATPase cycle rate, 6.5 s ؊1 . P i burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms.Class V myosins function as actin-based motors for various cargo transportations. Class V myosins have been widely found in species from lower organisms, such as yeast, Caenorhabditis elegans, and Drosophila, to vertebrates. In vertebrates, there are three distinct subclasses of myosin V. Among them, most of the research has focused on myosin Va, which plays a critical role in membrane trafficking, including melanosome transport (1, 2) and endoplasmic reticulum transport (3-6). In contrast to myosin Va expressed mainly in brain and melanocytes, myosin Vb and Vc are widely expressed in a variety of tissues, although the relative expression level is distinct from each other. For instance, myosin Vb is most abundant in kidney, whereas myosin Vc is significantly expressed in epithelial and glandular tissues, including pancreas, colon, and stomach (7).Evidence has shown that myosin V is a cargo transporter and serves as a membrane transporter in various biological processes. Myosin Va is involved in melanosome transportation in melanocytes (1, 2) and synaptic vesicle movement in neuronal cells (3-6). On the other hand, myosin Vb is involved in the plasma membrane recycling systems of transferrin receptor (8), chemokine receptor CXCR2 (9), and M4 muscarinic acetylcholine receptor (10). Although the tissue distribution of the expression of myosin Vb and Vc is overlapped, it is thought that they have a distinct physiological relevance. Myosin Vb directly interacts with the small GTP-binding protein, Rab11a (8), whereas myosin Vc colocalizes with Rab8, but not Rab11a (7), suggesting that each myosin V isoform has specific target cargo molecules and is involved in different membrane trafficking pathways.Th...

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Section: Discussionmentioning

confidence: 77%

mentioning

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Section: Expression and Purification Of Myosin Vcmentioning

confidence: 99%

mentioning

confidence: 92%

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Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor

Watanabe

Satō

et al. 2008

Journal of Biological Chemistry

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A Myosin V Inhibitor Based on Privileged Chemical Scaffolds

et al. 2010

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Switching the motor off: Privileged chemical scaffolds were used as a starting point for the development of a specific chemical inhibitor 1 of myosin V, a key motor protein required for intracellular transport (see picture; ADP=adenosine diphosphate, ATP=adenosine triphosphate). The potency of 1, which does not compete directly with nucleotide binding, is comparable to that of other known motor‐protein inhibitors used as probes in chemical biology.

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