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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    • "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.
    The Journal of Cell Biology 05/2015; 209(3). DOI:10.1083/jcb.201411010 · 9.83 Impact Factor
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    • "Auto-inhibition to limit the consumption of ATP/GTP by motors not bound to cargos is conserved in Myosins (Jung et al., 2008; Li et al., 2006; Umeki et al., 2009) and Kinesins (Al-Bassam et al., 2003; Imanishi et al., 2006; Seiler et al., 2000). As both protein families share a common ancestor, it is not unexpected that there is a common mechanism to this auto-inhibition, in which the tail folds back to the motor domain. "
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    ABSTRACT: The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis we have gained an understanding of how KHC achieves specificity in its transport and how it is regulated. This is, to our knowledge, the first in vivo structural/functional analysis of the tail in animal Kinesins. We show that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, our data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs.
    Development 11/2013; 141(1). DOI:10.1242/dev.097592 · 6.46 Impact Factor
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    • "The globular tail domain (GTD) of MyoVa has been used as an inhibitor of myosin function since it lacks the motor domain and acts as a dominant negative construct (Li et al., 2006). "
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    ABSTRACT: Directed transport of the mRNA binding protein, zipcode binding protein1 (ZBP1), into developing axons is believed to play an important role in mRNA localization and local protein synthesis. The role of molecular motors in this process is unclear. We elucidated a role for myosin Va (MyoVa) to modulate the axonal localization and transport of ZBP1 in axons. Using cultured rat hippocampal neurons, ZBP1 colocalized with MyoVa in axons and growth cones. Interaction of MyoVa with ZBP1 was evident by coimmunoprecipitation of endogenous and overexpressed proteins. Inhibition of MyoVa function with the globular tail domain (GTD) of MyoVa protein or short hairpin RNA led to an accumulation of ZBP1 in axons. Live cell imaging of mCherryZBP1 in neurons expressing GTD showed an increase in the number of motile particles, run length, and stimulated anterograde moving ZBP1 particles, suggesting that MyoVa controls availability of ZBP1 for microtubule-dependent transport. These findings suggest a novel regulatory role for MyoVa in the transport of ZBP1 within axons.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2012; 32(43):15133-41. DOI:10.1523/JNEUROSCI.2006-12.2012 · 6.34 Impact Factor
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