Abstract
Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.
Highlights
Sin IIIA is concentrated in the distal ends of rod and cone ellipsoid and colocalizes with the plus-distal ends of inner segment actin filament bundles, where actin forms the microvilli-like calycal processes [4]
The major cytoskeletal structure of filopodia is the actin bundles, and the plus ends of the actin filaments are localized at the tip; the localization of myosin IIIA at the tip of filopodia suggests that this myosin traveled on actin filaments and accumulated at the end of the actin track
The results suggest that myosin IIIA is not a conventional high duty ratio motor, but it spends a majority of its ATP hydrolysis cycle time on actin
Summary
CaM, calmodulin; mantATP, methylanthraniloyl-ATP; mantADP, methylanthraniloyl-ADP; HuM3MD, human myosin III motor domain; M3KD, the kinase domain of human myosin III; GST, glutathione S-transferase. The actin binding site in the tail was identified as the motif DFRXXL that was originally identified in myosin light chain kinase [20] in the C-terminal end of the tail, and a point mutation of the conserved Arg abolished the interaction with actin in vitro. This raises a hypothesis that the actin binding site at the tail serves as a tethering site and plays a critical role in preventing myosin IIIA from dissociating from the actin track during the movement, supporting processive movement as is shown for the processive movement of kinesin [21]. The results suggest that myosin IIIA is not a conventional high duty ratio motor, but it spends a majority of its ATP hydrolysis cycle time on actin
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