ACTOMYOSIN KINETICS AND IN-VITRO MOTILITY OF WILD-TYPE DROSOPHILA ACTIN AND THE EFFECTS OF 2 MUTATIONS IN THE ACT88F GENE

Two missense mutations of the flight muscle-specific actin gene of Dro sophila melanogaster, Act88F, assemble into normally structured myofib rils but affect the flight ability of flies and the mechanical kinetic s of isolated muscle fibers. We describe the isolation of actin from d ifferent homozygous Act88F strains, including wild-type, an Act88F nul l mutant (KM88), and two Act88F single point mutations (E316K and G368 E), their biochemical interactions with rabbit myosin subfragment 1 (S 1), and behavior with rabbit myosin and heavy meromyosin in in vitro m otility assays. The rabbit and wild-type Drosophila actins have differ ent association rate constants with S1 (2.64 and 1.77 mu M(-1) s(-1), respectively) and in vitro motilities (2.51, 1.60 mu m s(-1)) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increas ing from 0.11 to 0.17 mu M) and a reduced velocity in vitro (reduced b y 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with re spect to the recently published atomic models for the actomyosin struc ture and our findings that G368E fibers show a reduced rate constant f or delayed tension development and increased fiber stiffness. We inter pret these results as possibly caused either by effects on Al myosin l ight chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed wit h respect to its likely position within the tropomyosin binding site o f actin.