Myosin I's are widely expressed in different tissues and across broad phylogenetic backgrounds. In intestinal epithelial brush borders, myosin I is localized in the microvillus where it bridges the gap between the membrane and the actin bundles. Myosin I is also localized near the actively ruffling edges of migrating cells. We are using the Baculovirus/Sf9 system to express full-length chicken brush border myosin I heavy chain (BBMI HC) along with calmodulin (CaM). Vertebrate myosin I's are constitutively active whereas myosin I's from low eukaryotes such as Acanthamoeba and Dictyostelium require phosphorylation at a serine located in the myosin head domain for activity. Sequence alignments of vertebrate and Acanthamoeba myosin I's reveal that most of the vertebrate proteins have a negatively charged amino acid at the position where the Acanthamoeba protein has the phosphorylatable serine. We are exploring, using site-directed mutagenesis and the Baculovirus/Sf9 system, whether a negative charge at this site is essential for actin-activated MgATPase activity and in vitro motility. Full-length chicken BBMI HC has been coexpressed with calmodulin as light chains in the Baculovirus/Sf9 cell system using pVL1393 as the transfer vector. Soluble BBMI was extracted from the infected cells using ATP and high speed centrifugation. The expressed BBMI binds to actin in an ATP-dependent manner, a property we used to aid in purification. An antibody raised against the carboxyl terminal 14 amino acids of the BBMI HC sequence was used to immunoprecipitate BBMI or capture it for in vitro motility studies. The expressed myosin I translocates actin filaments at a rate indistinguishable from that of tissue purified myosin I. The movement of actin filaments by the expressed BBMI is inhibited by Ca++ and by tropomyosin, which is characteristic of BBMI purified from tissue. We are currently expressing a carboxyl terminal, FLAG-tagged, BBMI heavy chain for further studies.
