Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties |
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Authors: | Guzik-Lendrum Stephanie Nagy Attila Takagi Yasuharu Houdusse Anne Sellers James R |
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Affiliation: | From the ‡Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, ;§Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599, and ;¶Structural Motility, Institut Curie CNRS, UMR144, 26 rue d''Ulm, 75248 Paris Cedex 05, France |
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Abstract: | The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-ΔPDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-ΔPDZ proteins bound to actin with K(d) values of 2.61 and 1.04 μM, respectively, but only about 50-75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein. |
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Keywords: | Actin ATPases Cytoskeleton Molecular Motors Myosin |
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