title: Drosophila melanogaster myosin-18 represents a highly divergent motor with actin tethering properties.
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.
authors.affiliation: Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Guzik-Lendrum
authors.forename: Attila
authors.initials: A
authors.lastname: Nagy
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Takagi
authors.forename: Anne
authors.initials: A
authors.lastname: Houdusse
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
mesh_terms: D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D004331:Drosophila melanogaster / Q000378:metabolism*; D005786:Gene Expression Regulation; D007700:Kinetics; D009218:Myosins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D000199:Actins; D029721:Drosophila Proteins; D012333:RNA, Messenger; C560638:myosin-18, Drosophila; D000255:Adenosine Triphosphate; D009218:Myosins