Abstract
Myosin X is expressed in a variety of cell types and plays a role in cargo movement and filopodia extension, but its mechanoenzymatic characteristics are not fully understood. Here we analyzed the kinetic mechanism of the ATP hydrolysis cycle of acto-myosin X using a single-headed construct (M10IQ1). Myosin X was unique for the weak "strong actin binding state" (AMD) with a K(d) of 1.6 microm attributed to the large dissociation rate constant (2.1 s(-1)). V(max) and K(ATPase) of the actin-activated ATPase activity of M10IQ1 were 13.5 s(-1) and 17.4 mum, respectively. The ATP hydrolysis rate (>100 s(-1)) and the phosphate release rate from acto-myosin X (>100 s(-1)) were much faster than the entire ATPase cycle rate and, thus, not rate-limiting. The ADP off-rate from acto-myosin X was 23 s(-1), which was two times larger than the V(max). The P(i)-burst size was low (0.46 mol/mol), indicating that the equilibrium is significantly shifted toward the prehydrolysis intermediate. The steady-state ATPase rate can be explained by a combination of the unfavorable equilibrium constant of the hydrolysis step and the relatively slow ADP off-rate. The duty ratio calculated from our kinetic model, 0.6, was consistent with the duty ratio, 0.7, obtained from comparison of K(m ATPase) and K(m motility). Our results suggest that myosin X is a high duty ratio motor.
Highlights
It has been reported that myosin X is present at the edge of lamellipodia, membrane ruffles, and the tip of filopodia in cultured cells, suggesting that myosin X plays a role in regions where actin is in dynamic reorganization [6]
Because Ena/ VASP antagonizes the ability of capping proteins to inhibit actin polymerization at the barbed ends in vitro [9], it is thought that the distribution of Ena/VASP at specific cellular locations may regulate the elongation of actin filaments that is correlated with the change in cytoskeletal structure and cell shape
For myosin X, a previous biochemical study has suggested that myosin X is not a processive motor the duty ratio of myosin X, the fraction of time that myosin spends in a strong actin binding form, may be higher than conventional myosin based upon the surface density dependence of actin gliding velocity [16]
Summary
It has been reported that myosin X is present at the edge of lamellipodia, membrane ruffles, and the tip of filopodia in cultured cells, suggesting that myosin X plays a role in regions where actin is in dynamic reorganization [6]. Because Ena/ VASP antagonizes the ability of capping proteins to inhibit actin polymerization at the barbed ends in vitro [9], it is thought that the distribution of Ena/VASP at specific cellular locations may regulate the elongation of actin filaments that is correlated with the change in cytoskeletal structure and cell shape Taken together, these findings have suggested myosin X is a cargo transporter. For myosin X, a previous biochemical study has suggested that myosin X is not a processive motor the duty ratio of myosin X, the fraction of time that myosin spends in a strong actin binding form, may be higher than conventional myosin based upon the surface density dependence of actin gliding velocity [16]. The results suggest that myosin X is a high duty ratio motor
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