Abstract
Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 μM). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg(2+) modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.
Highlights
Molecular properties of class VIII myosin are not characterized
ATM1 Exhibits Low Enzymatic Activity and High Affinity for Actin—In this study, we reported the first analysis of the enzymatic properties of plant-specific class VIII myosin
We showed that actin-sliding velocity (Fig. 3) and actin-activated ATPase activity of Arabidopsis class VIII myosin, ATM1 (Fig. 2), were lower by a factor of 10 –100 compared with those of class plant-specific XI myosins [15, 17, 18] and were similar to those of animal slow myosins [30, 51, 52]
Summary
Results: Arabidopsis class VIII myosin, ATM1, has low enzymatic activity and high affinity for actin and is primarily localized at the cell cortex. ADP dissociation from the actin1⁄7ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. GFP-fused fulllength ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. The dissociation rates of ADP from acto-class XI myosins are extremely fast and account for their high actin-sliding velocities [15, 19, 20]. Molecular properties such as ATPase activity and actin-sliding velocity of class VIII myosin have not been characterized. We expressed Arabidopsis class VIII myosin, ATM1, in a baculovirus system and uncovered its molecular. Its subcellular localization was determined by expressing GFP-fused full-length ATM1 in Arabidopsis under the control of its native promoter
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