Abstract
Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca(2+) in a highly cooperative manner. We demonstrate here that Ca(2+) also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca(2+)-induced cosedimentation. The Ca(2+)-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca(2+) was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca(2+)-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5'-O-(thiotriphosphate) is induced by micromolar concentrations of Ca(2+). Since Ca(2+) regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.
Highlights
Class V myosins are widely expressed, actin-based motors that have been implicated in the transport and/or localization of a wide range of organelles as well as mRNA
A Hill plot of the data suggests the involvement of about eight Ca2ϩ-binding sites per myosin molecule. This result is strikingly similar to the activation curve of the actin-activated, MgATPase of M-Va over the same range of Ca2ϩ concentrations [12] and, suggests that calcium is affecting the ATPase activity and myosin binding to actin via a common effector
Since there are four to five calmodulin molecules bound to each neck domain of M-Va, and each calmodulin has four potential Ca2ϩ-binding sites, the data are consistent with the involvement of at least two or more molecules of calmodulin per M-Va
Summary
M-Va, myosin-Va; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis. In the experiments described here, we show that micromolar Ca2ϩ induces high affinity binding of native chick M-Va to actin filaments in the presence of ATP or its nonhydrolyzable derivative, ATP␥S. Three distinct methods have been used to characterize the actomyosin-Va complex induced by calcium: (a) cosedimentation of M-Va with F-actin, (b) quenching of pyrenelabeled F-actin by M-Va, and (c) electron microscopy imaging of the negative-stained actomyosin-Va. The results indicate that micromolar Ca2ϩ can be a switch that regulates the actin binding properties of M-Va and, may participate in the mechanism underlying processivity of this myosin
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