The globular tail domain of myosin Va Functions as an inhibitor of the myosin Va motor

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2006; 281(31):21789-98. DOI: 10.1074/jbc.M602957200
Source: PubMed


The actin-activated ATPase activity of full-length mammalian myosin Va is well regulated by Ca2+, whereas that of truncated myosin Va without the C-terminal globular tail domain (GTD) is not. Here, we have found that exogenous GTD is capable of inhibiting the actin-activated ATPase activity of GTD-deleted myosin Va. A series of truncated constructs of myosin Va further showed that the entire length of the first coiled-coil (coil-1) of the tail domain is critical for GTD-dependent regulation of myosin Va and that deletion of 58 residues from the C-terminal end of coil-1 markedly hampered regulation. Negative staining electron microscopy revealed that GTD-deleted myosin Va formed a "Y"-shaped structure, which was converted to a triangular shape, similar to the structure of full-length myosin Va in the inhibited state, by addition of exogenous GTD. In contrast, the triangular shape was not observed when the C-terminal 58 residues of coil-1 were deleted, even in the presence of exogenous GTD. Based on these results, we propose a model for the formation of the inhibited state of myosin Va. GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated with the end of coil-1. Once the head interacts with the GTD, the triangular inhibited conformation is stabilized. Consistent with this model, we found that shortening of the neck of myosin Va by two IQ motifs abolished the regulation by GTD, whereas regulation was partially restored by shortening of coil-1 by an amount comparable to that of the two IQ motifs.

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    • "are estimated from the actin filament to the center of the Qdot. Overall length of MyoVa- FL was estimated based on the average value of reported distal tail lengths [4] [27] "
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    ABSTRACT: Myosin Va (MyoVa) is a processive molecular motor involved in intracellular cargo transport on the actin cytoskeleton. The motor’s processivity and ability to navigate actin intersections are believed to be governed by the stiffness of various parts of the motor’s structure. Specifically, changes in calcium may regulate motor processivity by altering the motor’s lever arm stiffness and thus its interhead communication. In order to measure the flexural stiffness of MyoVa subdomains, we use tethered particle microscopy, which relates the Brownian motion of fluorescent quantum dots, which are attached to various single- and double-headed MyoVa constructs bound to actin in rigor, to the motor’s flexural stiffness. Based on these measurements, the MyoVa lever arm and coiled-coil rod domain have comparable flexural stiffness (0.034 pN/nm). Upon addition of calcium, the lever arm stiffness is reduced 40% as a result of calmodulins potentially dissociating from the lever arm. In addition, the flexural stiffness of the full-length MyoVa construct is an order of magnitude less stiff than both a single lever arm and the coiled-coil rod. This suggests that the MyoVa lever arm-rod junction provides a flexible hinge that would allow the motor to maneuver cargo through the complex intracellular actin network.
    Full-text · Article · Nov 2015 · Journal of Biophysics
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    • "Therefore, an interaction between the head and tail domains is necessary and sufficient to regulate myosin V in vivo to achieve a normal distribution. The two internal loops present in mammalian myosin V coiled-coil stalks have been shown in vitro to be required for autoinhibition, likely because of the flexibility gained to bring the head and tail domains together (Li et al., 2006). Myo2p lacks these internal loops, though the length of the lever arm and coiled coil domains are nearly identical (21 and 24 nm, respectively). "
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    ABSTRACT: Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor. © 2015 Donovan and Bretscher.
    Full-text · Article · May 2015 · The Journal of Cell Biology
    • "Under basal conditions, both isoforms display a predominantly cytosolic distribution, likely due to the fusion protein adopting the closed, " inactive " conformation (see later discussion of Figure 5, A and B). Micromolar levels of calcium and/or cargo binding induce myosin Va to undergo a conformational switch to its open, active conformation (Liu et al., 2006; Thirumurugan et al., 2006). Indeed, subcellular fractionation experiments demonstrated that the majority of endogenous myosin Va partitioned in the membrane fraction when A431 cell lysates were prepared with CaCl 2 , whereas when calcium was chelated by ethylene glycol tetraacetic acid (EGTA), the major pool of myosin Va was found in the cytosolic fraction (Figure 2A). "
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    ABSTRACT: Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli's Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid "living chip" assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation.
    No preview · Article · Mar 2015 · Methods in molecular biology (Clifton, N.J.)
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