TROPOMYOSIN AND TROPONIN REGULATION OF WILD-TYPE AND E93K MUTANT ACTIN-FILAMENTS FROM DROSOPHILA FLIGHT-MUSCLE - CHARGE REVERSAL ON ACTIN CHANGES ACTIN-TROPOMYOSIN FROM ON TO OFF STATE

In the Drosophila flight muscle actin mutant E93K there is a charge re versal on the surface of actin close to the proposed position of tropo myosin when it is in the off state. Using a quantitative in vitro moti lity assay we have found that the wild type Drosophila ACT88F actin be haved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upo n the E93K mutant actin filament movement was completely different fro m wild type and resembled the response of wild type with tropomyosin+t roponin at pCa9 (i.e. the filaments were switched off), Velocity of E9 3K actin did not increase, and the fraction of filaments motile was re duced to less than 15% by adding up to 30 nM tropomyosin. When myosin subfragment-1 modified by N-ethylmaleimide was mixed with mutant E93K actin-tropomyosin filaments we observed that it restored motility of t he filaments to the level observed with E93K actin alone. We conclude that electrostatic charge on the surface of domain 2 of actin plays a critical role in determining the state of actin-tropomyosin that is a central feature of the steric blocking mechanism of actin filament reg ulation.