Myosin IXb is a single-headed minus-end-directed processive motor

Inoue, Akira; Saito, Junya; Ikebe, Reiko; Ikebe, Mitsuo

doi:10.1038/ncb774

Cited by 85 publications

(78 citation statements)

References 30 publications

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“…The mean actin translocating velocity was 0.11 Ϯ 0.05 m/s that was significantly slower than other myosin superfamily members (31, 35, 38 -42). Interestingly, high Ca 2ϩ concentration did not abolish the motility activity of human myosin III in contrast to other calmodulin binding myosins such as myosin V (31,35), myosin I (27, 43), and myosin IX (41).…”

Section: Figmentioning

confidence: 85%

“…It was originally hypothesized that the unique large insertion of myosin VI between the motor domain and the neck domain was responsible for the reverse directionality of motility, thus making the class VI myosin the only minus-directed myosin. However, recent studies have revealed that this is not the case and that there are additional members in the myosin superfamily that move in the minus direction (29,41). To determine the direction of movement of human myosin III, we utilized F-actin filaments in the in vitro motility assay that were labeled throughout with fluorescein and labeled with a rhodamine cap at the filament's pointed end (see "Experimental Procedures").…”

Section: Figmentioning

confidence: 99%

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Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Komaba

Inoue

Maruta

et al. 2003

Journal of Biological Chemistry

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The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actinbased motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with K actin of 112 M and V max of 0.34 s ؊1 , indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 ؎ 0.05 m/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.Myosin III is a member of the myosin superfamily, which consists of at least 18 classes (1-5). The class III myosin represents the most divergent member of the myosin superfamily. The motor domain of myosin III shows ϳ24 -27% sequence identity to myosin Is and ϳ22-26% sequence identity to myosin-IIs (6). Of particular interest is that myosin III has an amino terminus domain that resembles to protein kinases. This domain contains the characteristic motifs for protein kinases such as a glycine-rich loop, an invariable Lys residue required for ATP binding, and a catalytic loop. Given its high degree of divergence, if myosin III evolved at the same rate as other myosins, the myosin III lineage would predate the divergence of yeast. Class III myosin was originally found in Drosophila photoreceptor cells and subsequently found in vertebrates including human myosin III (7,8).The function of Myosin III is best studied in Drosophila photoreceptor cells. Each photoreceptor cell has a specialized organelle consisting of a stack of microvilli known as a rhabdomere. The phototransduction machinery is localized in the rhabdomere (9). Drosophila photoreceptors undergo a prolonged depolarization afterpotential that persists after cessation of the light stimulus. Prolonged depolarization afterpotential results from the stable conversion of rhodopsin to the light-activated form, metarhodopsin, in response to blue light. During a prolonged depolarization afterpotential, photoreceptor cells become refractory to subsequent prolonged depolarization afterpotential-inducing stimuli and are inactivated. Mutants that are defective for both inactivation and the prolonged depolarization afterpotential are known as neither inactivation nor afterpotential (nina) mutants (10, 11), and the myosin III was identified (12) as one of eight nina complementation gr...

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Section: Figmentioning

confidence: 85%

Section: Figmentioning

confidence: 99%

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Komaba

Inoue

Maruta

et al. 2003

Journal of Biological Chemistry

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“…In class VI myosin, calmodulin was found to be associated with a unique insert between the converter and the IQ-motif that repositions the lever arm and reverses directional movement of myosin VI toward the minus-end of actin filaments (37,38). A human recombinant Myo9b construct that was truncated in the tail region was reported to move toward the minus-end of actin filaments (6). By contrast, native full-length Myo9b was reported to be a plus-, and not a minus-end directed motor, suggesting that the tail domain of Myo9b regulates motor directionality (24).…”

Section: Discussionmentioning

confidence: 99%

“…Native Myo9b immunoadsorbed from human leukocyte extracts moved toward the plus-end of the actin filament (24). However, a truncated recombinant Myo9b was reported to move toward the minusend of the actin filament (6). The reason(s) for this difference in Myo9b directionality is not known.…”

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confidence: 99%

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

Liao¹,

Elfrink²,

Bähler³

2010

Journal of Biological Chemistry

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Mammalian myosin IXb (Myo9b) has been shown to exhibit unique motor properties in that it is a single-headed processive motor and the rate-limiting step in its chemical cycle is ATP hydrolysis. Furthermore, it has been reported to move toward the minus-and the plus-end of actin filaments. To analyze the contribution of the light chain-binding domain to the movement, processivity, and directionality of a single-headed processive myosin, we expressed constructs of Caenorhabditis elegans myosin IX (Myo9) containing either the head (Myo9-head) or the head and the light chain-binding domain (Myo9-head-4IQ). Both constructs supported actin filament gliding and moved toward the plus-end of actin filaments. We identified in the head of class IX myosins a calmodulin-binding site at the N terminus of loop 2 that is unique among the myosin superfamily members. Ca 2؉ /calmodulin negatively regulated ATPase and motility of the Myo9-head. The Myo9-head demonstrated characteristics of a processive motor in that it supported actin filament gliding and pivoting at low motor densities. Quantum dot-labeled Myo9-head moved along actin filaments with a considerable run length and frequently paused without dissociating even in the presence of obstacles. We conclude that class IX myosins are plus-end-directed motors and that even a single head exhibits characteristics of a processive motor.Myosins form a large superfamily of actin-based molecular motors that is composed of at least 35 classes (1). Class IX myosins arose in metazoa after the separation of the fungi (1). Invertebrates contain a single myosin class IX gene with the exception of the Drosophila species that have lost their class IX myosin. Bony fishes contain four myosin IX genes and other vertebrates, including mammalia two genes. The two class IX myosins in mammals, Myo9a 2 and Myo9b, exist in multiple splice variants (2). Myo9a has been shown to play a role in epithelial differentiation and morphology whereas Myo9b regulates the migration of macrophages and possibly other immune cells (3, 4). Class IX myosins share a similar structure with the myosins of the other classes, containing a head region, a calmodulin/light chain-binding domain, and a tail region.Additionally, class IX myosins carry some unique features, including a large N-terminal extension preceding the head domain and a long insertion within the head domain in loop 2. The tail region comprises a C1 zinc-binding domain and a RhoGAP domain. Because of this RhoGAP domain, class IX myosins are involved in signal transduction regulating the dynamics of the actin cytoskeleton (2, 5).Mammalian Myo9b, the only class IX myosin studied so far in vitro, exhibits unique mechano-chemical properties. It has been reported to take multiple successive steps along actin filaments without dissociating, indicating that it is a processive motor (6 -8). This is remarkable because Myo9b is a singleheaded myosin. Other myosins that move processively on actin filaments, such as myosin V, dimeric myosin VI, and myosin VII, are two-...

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“…The minus-end of an actin filament or a microtubule can be fluorescently labeled to distinguish it from the plus-end. Using this technique, it was discovered that, different from conventional kinesin, the kinesin-related protein motors ncd [26] and Kar3 [27] are minus-end-directed microtubulebased motors and, different from conventional myosin, myosin VI [28,29] and myosin IX [30] are minus-end directed actin-based motors.…”

Section: Backwards Directed Movementmentioning

confidence: 99%

How Linear Motor Proteins Work

Oiwa

Manstein

Controlled Nanoscale Motion

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Most animals perform sophisticated forms of movement such as walking, running, flying and swimming using their skeletal muscles. Although directed movement is not generally associated with plants, cytoplasmic streaming in plant cells can reach velocities greater than 50 µm/s and thus constitutes one of the fastest forms of directed movement. Unicellular eukaryotic organisms and prokaryotes display diverse mechanisms by which they are able to actively move towards a food source, light or other sensory stimuli. On the cellular level active transport of vesicles and organelles is required, since the cytoplasm resembles a gel with a mesh size of approximately 50 nm, which makes the passive transport of organelle-sized particles impossible. For elongated cells such as neurons, even proteins and small metabolites have to be actively transported.Linear motor proteins, moving on cytoskeletal filaments such as actin filaments and microtubules, are predominantly responsible for the motile activity in eukaryotic cells. They are chemo-mechanical enzymes that use the chemical energy from adenosinetriphosphate (ATP) hydrolysis to generate force and to move cargoes along their filament tracks. Under physiological conditions, the energy input per molecule of ATP corresponds to the chemical free energy liberated by its hydrolysis to adenosinediphosphate (ADP) and inorganic phosphate (P i ), ca. 10 −19 J (100 pNnm). The thermodynamic efficiency of motor proteins varies between 30 and 60%. As machines, motor proteins are unique since they convert chemical energy to mechanical work directly, rather than through an intermediate such as heat or electrical energy. Structural Features of Cytoskeletal Motor ProteinsThree families of linear, cytoskeletal motor proteins have been described: kinesin, dynein, and myosin ( Fig. 3.1). Kinesin and dynein family members K. Oiwa and D

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Myosin IXb is a single-headed minus-end-directed processive motor

Cited by 85 publications

References 30 publications

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

How Linear Motor Proteins Work

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