The purification and in vitro motility analysis of Drosophila melanogaster ACT88F mutants.

The indirect flight muscle (IFM) specific ACT88F actin isoform o f Drosophila melanogaster was purified from flies on both a large and small scale. The development o f a mini-actin purification protocol allowed the isolation o f pure ACT88F actin from ten pairs o f dissected IFMs in sufficient quantities, (approximately 5pg), for many in vitro motility assays. An actin preparation, starting with 10,000 flies, (10g), was also developed, using anion exchange chromatography to isolate the ACT88F isoform from the five other Drosophila isoforms. Milligram quantités of ACT88F, containing a 10% “contamination” of an unknown type III actin isoform, provide sufficient material for the future in vitro biochemical and kinetic characterisation of ACT88F mutants. The in vitro motility o f four ACT88F mutants, G368E, E316K, E334K and E93K was investigated using a rabbit skeletal muscle HMM. A significant 35% reduction in G368E filament velocity under standard assay conditions (SAC) was also seen when under various ionic and ATP concentrations. E316K only showed a significant 36% reduction in its filament velocity at limiting ATP concentrations. Under all conditions in the motility assay, E334K mutant filaments displayed no in vitro movement. Where wild-type (WT) actin washed off the surface at 50mM KC1 in the motility assay, E334K actin dissociated at 30mM KC1. Three copolymers o f E334K and WT actin, each representing a different proportion and distribution o f mutant monomers in the cofilament, were all able to move under SAC. As the percentage fraction of E334K actin in the cofilament was increased, filament velocity decreased. Although E93K actin filaments washed off the surface under standard assay conditions, binding to, and movement o f this mutant over the surface was seen at lower ionic strengths, albeit with a significant 50% reduction in filament velocity. These results are discussed with respect to the atomic structure o f actin and the model o f the actin-Sl reconstruction. ACT88F was expressed in Saccharomyces cerevisiae. The expression o f WT and six ACT88F mutants was confirmed by two-dimensional gel electrophoresis and/or Western blotting with actin specific antibodies. However, the levels o f recombinant protein were not great enough to support in vitro biochemical studies.