Dictyostelium Myosin-5b Is a Conditional Processive Motor

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.Class 5 myosins are dimeric actin-based motors that are involved in various forms of intracellular trafficking (1). Dependent on the isoform and cell type, class 5 myosins have been implicated in the movement of membranes and organelles (2-4), the transport of synaptic and secretory vesicles (5, 6), and the active delivery of receptors and mRNA-protein complexes to their place of action (7,8). The unique modular structure of class 5 myosins is essential for these specialized transport functions (9, 10). Each heavy chain of a dimeric myosin-5 molecule consists of a motor domain that binds actin and hydrolyzes ATP (11), followed by a long neck region to which up to six light chains can bind (12, 13). Parts of the adjacent tail region form a coiled-coil and the C terminus consists of a globular domain that mediates the binding to cargo and regulates activity of the motor (14, 15).Despite the high sequence similarity between myosin-5 isoforms, the individual members display differences in their mechanoenzymatic properties, which characterize them either as processive or non-processive motors. Processive myosins, like vertebrate myosin-5a, are capable of taking successive steps along actin as single molecules before detaching (16). The overall movement has been described as a coordinated stepping process of both heads in a hand-over-hand mechanism that is driven by intramolecular strain (17, 18). In contrast, non-processive myosins bind to the actin filament perform just one step and then dissociate rapidly. A notable difference between processive and non-processive myosins is displayed in the duty ratio, i.e. the fraction of the total ATPase cycle time a motor spends in the strong actin binding states. Processive myosins have a high duty ratio (Ͼ0.5), whereas non-processive myosins display a low duty ratio...

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

“…Mg 2+ ions only affect the long-lived dwell times in myosin-5b. To further elucidate the molecular nature of the two dwell time populations, we used the delay of ADP release by Mg 2+ that is characteristic for D. discoideum myosin-5b (22). If for both dwell time populations the fluorescence signal ends via release of ADP, the time constants of both populations should increase with free Mg 2+ .…”

“…Singleheaded D. discoideum myosin-2 (M761-2R) (21), D. discoideum myosin-5b (J829-2R) (22), and D. discoideum myosin-1B (B698-2R) (27) were expressed in D. discoideum. 2R refers to two α-actinin subunits that replaced the native light chain binding domain (21).…”

The SEC6 protein is required for function of the contractile vacuole inChlamydomonas reinhardtii

“…1A) and similar cytosolic electron dense material has also been detected in another green alga Mesostigma viride (Buchmann and Becker, 2009). (3) A role for cytoskeletal elements during the CV cycle could only be demonstrated in Dictyostelium (Taft et al, 2008), indicating that force generation during systole by cytoskeletal elements does not play any role in most systems. Changes in membrane structure have been implicated in water expulsion during systole in Paramecium , but whether this is a general mechanism remains to be seen.…”

Functional Characterization of Human Myosin-18A and Its Interaction with F-actin and GOLPH3

“…Transient kinetic experiments were performed at 20°C with either a Hi-tech Scientific SF-61 DX single mixing stopped-flow system (TgK Scientific Ltd., Bradford on Avon, UK) or an Applied Photophysics PiStar 180 instrument in assay buffer (20 mM MOPS (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT) using procedures and kinetic models described previously (Scheme 1) (20,21). Error bars represent S.E.…”