Abstract
Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg(2+)-ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg(2+)-ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.
Highlights
Dd myosin-5b is a dimeric motor that consists of two heavy chains, each with a molecular mass of 258
Dd myosin-5b is assumed to localize at the contractile vacuole [28], a specialized organelle for the regulation of osmotic pressure [44]
The displacement of mantADP from the ternary mantADP1⁄7acto1⁄7Dd myosin-5b complex by excess unlabeled ADP at concentrations of free Mg2ϩ-ions from 0.2 to 5 mM was monitored from the resulting decrease of mant-fluorescence
Summary
Dd myosin-5bd Hs myosin-5be Gg myosin-5af Hs myosin-5cg Dm myosin-5h sϪ1 0.069 Ϯ 0.01. 107 500 64/35 125 a Values for kcat and Kapp were calculated from fitting the data to the MichaelisMenten equation. B The apparent second order rate constant for actin binding (kcat/Kapp) was obtained from the calculated ratio of both values. E Watanabe et al [21]. G Watanabe et al [22] and Takagi et al [23], respectively. Cells transfected with EYFP-Dd myosin-5b were seeded on glass bottom Petri dishes (coverslip thickness: 160 –180 m), washed twice with Bonner’s salts solution (10 mM NaCl, 10 mM KCl, and 3 mM CaCl2), and kept in this medium during image acquisition. After 2 h cells were seeded on Petri dishes and immediately before imaging the solution containing KMG-104AM was replaced by 50 mM HEPES, pH 7.3, containing 10 mM MgCl2
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