Kinetic Characterization of the Mitochondrial Transporter Human Myosin XIX

Abstract

Human myosin XIX is a novel class of actin-based motor implicated in the transport and localization of mitochondria. We expressed myosin XIX using the baculovirus/SF9 cell expression system. The truncated MYO19-3IQ construct was co-expressed with the calmodulin light chain and chaperones Unc45 and Hsp90. The chaperone proteins significantly increased the level of myosin XIX expression 20-fold. To determine the mechanochemical properties that underlie cellular MYO19 activity we carried out transient kinetic experiments and measured elementary rate constants. ADP is released in two steps (k-AD1 = 4.5 s−1 and k-AD2 = 1.5 s−1) from the actomyosinADP complex. Phosphate dissociation is fast and has a kmax of 47 s−1 . The rate of hydrolysis (M·T = M·D·P) determined by quenched flow is >16 s−1 and is favorable with an equilibrium constant of (KH = 2). The maximal rate of ATP induced dissociation of myosin from the actomyosin (k-TA) is 670s−1 . We conclude that the rate-limiting step of the ATPase cycle is the slower step of the ADP release because it's rate is the same as the actin activated steady-state ATPase (Vmax = 1.6 s−1). The human Myosin XIX has a high duty ratio (0.9) which is consistent with its role in transporting mitochondria.