MODULATION OF ACTIN FILAMENT SLIDING BY MUTATIONS OF THE SH2 CYSTEINEIN DICTYOSTELIUM MYOSIN-II

The cysteine residue called SH2 in the skeletal myosin heavy chain is conserved among various species. Cys 678 in Dictyostelium myosin II is equivalent to SH2 in skeletal myosin, Using the Dictyostelium myosin II heavy chain gene, SH2 was mutated to Gly, Ala, Ser, or Thr. These m utant myosins were expressed in Dictyostelium myosin-null cells, To in vestigate how these mutations affect the motor functions of myosin, we examined the phenotypes of the transformed cells. We also purified th e mutant myosins, and characterized them by measuring the actin-activa ted MgATPase activity, sliding velocity of actin filaments and force l evel. All of these mutant myosins complemented the myosin-specific def ects of the myosin-null cells, Consistent with these observed phenotyp es, all of the purified mutant myosins retained similar actin-activate d MgATPase activities and force levels to those of the wild-type myosi n (WT). However, the sliding velocities of actin filaments were signif icantly different (WT greater than or equal to Ser>Ala much greater th an Thr>Gly). In particular, the Gly and Thr mutants exhibited a striki ng decrease in velocity, while the Ser mutant exhibited velocity compa rable to that of the wild-type myosin. Thus, mutations of SH2 resulted in uncoupling of ATP hydrolysis and the sliding. (C) 1997 Academic Pr ess.