Abstract
Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.
Highlights
Class I myosins are produced by a wide range of organisms and cell types [1]. They share a conserved motor domain, a light chain-binding domain, and a tail region that contains a polybasic region that directly binds to membranes via electrostatic interactions [2, 3]
To directly measure the motor activity of myosin-ID, we generated a motor domain fragment with an artificial lever arm consisting of D. discoideum ␣-actinin repeats 1 and 2 [15, 16]
The direct functional characterization of the vast majority of myosins is greatly impeded because their respective light chains have not been identified
Summary
Reagents—Standard chemicals were purchased from Sigma, and restriction enzymes, polymerases, and DNA-modifying enzymes were from Roche Applied Science. For the production of motor domain constructs fused to two D. discoideum ␣-actinin repeats (2R), a DNA fragment encoding 2R, a Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly linker, enhanced yellow fluorescent protein, and a His tag was obtained as XhoI/SphI fragment from pM790 –2R-EYFP [25] and inserted in the XhoI/SphIdigested myosin-ID motor domain expression plasmid. TEDS site phosphorylation was performed by mixing 1 mg/ml D692–2R with 0.027 mg/ml activated kinase and incubation in the presence of 1 mM EGTA, 3 mM MgCl2, and 2 mM ATP at 30 °C for 20 min. A. castellanii myosin-I heavy chain kinase was activated by autophosphorylation at 30 °C for 20 min in a buffer containing 100 mM imidazole, pH 7.0, 4 mM ATP, 6 mM MgCl2, and 2 mM EGTA [12]. The transient kinetics data were interpreted as described previously [27, 29, 34, 35]
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