Drosophila Myosin VIIA Is a High Duty Ratio Motor with a Unique Kinetic Mechanism

Watanabe, Shinya; Ikebe, Reiko; Ikebe, Mitsuo

doi:10.1074/jbc.m511592200

Cited by 35 publications

(29 citation statements)

References 48 publications

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“…Previous in vitro studies of Drosophila myosin VIIA have suggested that myosin VIIA can be a cargo-transporting motor because it is a high duty ratio motor (13) and the forced dimer construct of the tail-truncated Drosophila myosin VIIA moves processively in vitro (14). In the present study, we showed that human myosin VIIA can transport its cargo complex in ARPE-19 cells, thus functioning as a cargo transporter.…”

Section: Discussionsupporting

confidence: 48%

“…These results suggest that human myosin VIIA does not form a stable dimer in cells. It was reported previously that Drosophila myosin VIIA is a high duty ratio motor suitable for a processive movement (13), and the forced dimer of the tail-truncated Drosophila myosin VIIA can move processively in vitro (14). Therefore, we asked whether human myosin VIIA may move on actin filaments in vivo once it forms a dimer.…”

Section: Resultsmentioning

confidence: 99%

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Cargo binding activates myosin VIIA motor function in cells

Sakai

Umeki

Ikebe

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Myosin VIIA, thought to be involved in human auditory function, is a gene responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Recent studies have suggested that it can move processively if it forms a dimer. Nevertheless, it exists as a monomer in vitro, unlike the well-known two-headed processive myosin Va. Here we studied the molecular mechanism, which is currently unknown, of activating myosin VIIA as a cargo-transporting motor. Human myosin VIIA was present throughout cytosol, but it moved to the tip of filopodia upon the formation of dimer induced by dimer-inducing reagent. The forced dimer of myosin VIIA translocated its cargo molecule, MyRip, to the tip of filopodia, whereas myosin VIIA without the forced dimer-forming module does not translocate to the filopodial tips. These results suggest that dimer formation of myosin VIIA is important for its cargo-transporting activity. On the other hand, myosin VIIA without the forced dimerization module became translocated to the filopodial tips in the presence of cargo complex, i.e., MyRip/Rab27a, and transported its cargo complex to the tip. Coexpression of MyRip promoted the association of myosin VIIA to vesicles and the dimer formation. These results suggest that association of myosin VIIA monomers with membrane via the MyRip/Rab27a complex facilitates the cargo-transporting activity of myosin VIIA, which is achieved by cluster formation on the membrane, where it possibly forms a dimer. Present findings support that MyRip, a cargo molecule, functions as an activator of myosin VIIA transporter function.

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Section: Discussionsupporting

confidence: 48%

Section: Resultsmentioning

confidence: 99%

Cargo binding activates myosin VIIA motor function in cells

Sakai

Umeki

Ikebe

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The actin-dependent MgATPase activity of myosin 7a-FL shows important differences compared with that of the S1 fragment reported previously (14,15). The V max of both constructs is Ϸ1 s Ϫ1 , but myosin 7a-FL has a high K ATPase of 30 Ϯ 12 M, unlike S1 where the K ATPase is 1.1 Ϯ 0.1 M (Fig.…”

Section: Expression and Purification Of Full-length Myosin 7a With Camentioning

confidence: 72%

A FERM domain autoregulates Drosophila myosin 7a activity

Yang

Baboolal

Siththanandan

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

126

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Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of Vmax Ϸ 1s ؊1 and KATPase Ϸ 1 M actin. We find that myosin 7a-FL has Vmax similar to S1 but KATPase Ϸ 30 M. Thus, at low actin concentrations (5 M), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the KATPase is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces KATPase to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with Kd Ϸ 30 M regardless of ATP, similar to the KATPase value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the Cterminal FERM domain binds to actin.regulation ͉ electron microscopy ͉ ATPase activity

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“…To express the recombinant myosin Vc proteins, Sf9 cells were co-infected with two viruses expressing myosin Vc heavy chain and calmodulin. The expressed proteins were purified through an anti-FLAG M2 affinity column (Sigma) as previously described (29). The purified proteins were dialyzed against buffer A (25 mM KCl, 20 mM MOPS-KOH (pH 7.5), 2 mM MgCl 2 , 1 mM EGTA, and 1 mM DTT).…”

Section: Methodsmentioning

confidence: 99%

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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Drosophila Myosin VIIA Is a High Duty Ratio Motor with a Unique Kinetic Mechanism

Cited by 35 publications

References 48 publications

Cargo binding activates myosin VIIA motor function in cells

Cargo binding activates myosin VIIA motor function in cells

A FERM domain autoregulates Drosophila myosin 7a activity

Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor

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