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 Kd 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.

Mammalian Myosin-18A, a Highly Divergent Myosin

“…Mammalian myosin-18A␣-S1 in the absence of nucleotide binds actin with an affinity (K d ϳ5.0 M) similar to that previously reported for the isoforms of D. melanogaster myosin-18 (15). Mammalian myosin-18A␤, however, exhibits a lower actin affinity around 50 M. The higher affinity of the myosin-18A␣ isoform is probably related to the actin binding sequence located in the extended N-terminal region between the PDZ domain and the KE-rich region of that isoform (14).…”

“…Class-18 myosins represent the extreme edge of this diversity. Previous work demonstrated that D. melanogaster myosin-18 bound ATP poorly and showed no evidence for an ATPase activity and that it bound actin in an ATP-insensitive manner (15). Earlier biochemical studies using myosin-18A fragments expressed in mammalian cells suggested that it may lack the enzymatic activity that is usually associated with myosins and may function more like an actin-binding protein than as a motor protein, although enzymatic measurements were not directly performed (13,14).…”

“…Biochemical analysis of murine myosin-18A␣ suggested an ATP-insensitive actin binding site in the KE-rich region or the region between the KE-rich and PDZ domains, but myosin-18A␤ did not bind to actin even in the absence of ATP in vitro and did not strongly co-localize with actin in vivo (13,14). A previous report analyzing the biochemical properties of Drosophila melanogaster myosin-18 isoforms also reported ATP-insensitive actin binding and suggested that myosin-18 may act as a dynamic actin tether (15).…”

“…Analysis was performed using custom software written in LabVIEW 8.5 (National Instruments, Corp., Austin, TX), and histograms were plotted using Origin 8.5 (OriginLab Corp., Northampton, MA). Detachment rate and power stroke histograms were compiled from data collected from seven pedestals each for both myosin-18A-HMMs (15).…”

“…12A), which is predicted to protrude into the nucleotide binding site and may sterically reduce nucleotide accessibility (15). In myosins and kinesins, conformational changes in the ␥-phosphate sensor switch-1 upon ATP binding are coupled to changes in the track affinity of the motor.…”