title: Kinetics of the interaction of myo1c with phosphoinositides.
abstract: myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c(IQ-tail)) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c(IQ-tail) with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P(2), or by situating PtdIns(4,5)P(2) in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c(IQ-tail) dissociated from PtdIns(4,5)P(2) at a slower rate (2.0 s(-1)) than the pleckstrin homology domain of phospholipase C-delta (13 s(-1)). The presence of additional anionic phospholipid reduced the myo1c(IQ-tail) dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-delta, suggesting that additional electrostatic interactions in myo1c(IQ-tail) help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c(IQ-tail) from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c(IQ-tail) as it remains bound to the membrane.
authors.affiliation: Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
authors.forename: Jennine M Dawicki
authors.initials: JM
authors.lastname: McKenna
authors.forename: E Michael
authors.initials: EM
authors.lastname: Ostap
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D000838:Anions; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008956:Models, Chemical; D024461:Myosin Type I; D009218:Myosins / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D000199:Actins; D000838:Anions; D008055:Lipids; C470422:Myo1c protein, mouse; D010716:Phosphatidylinositols; D024461:Myosin Type I; D009218:Myosins; D008274:Magnesium
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