Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

Abstract

In the Drosophila flight muscle actin mutant E93K there is a charge reversal on the surface of actin close to the proposed position of tropomyosin when it is in the off state. Using a quantitative in vitro motility assay we have found that the wild type Drosophila ACT88F actin behaved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upon the E93K mutant actin filament movement was completely different from wild type and resembled the response of wild type with tropomyosin؉troponin at pCa9 (i.e. the filaments were switched off). Velocity of E93K actin did not increase, and the fraction of filaments motile was reduced 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 the 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 regulation.

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

“…Actin mutants provide an alternative approach to assess specific interactions between tropomyosin and actin. In one such study, tropomyosin suppressed the in vitro motility of E93K actin filaments, leading to the suggestion that tropomyosin binding to the outer domain of actin is affected by charged surface residues on subdomain 2 (16). The present D56A/E57A binding data support the conclusion that acidic residues on subdomain 2 normally act to weaken tropomyosin binding.…”

“…In the case of the Ca 2ϩ G-actin, polymerization by Mg 2ϩ addition causes a heterogeneous population of Ca 2ϩ or Mg 2ϩ F-actin, here termed conventional F-actin. Similar to other actins in which the amino acid residues of subdomain 2 have been modified (17,19) or mutated (16), polymerization of D56A/E57A actin was altered under some conditions. Polymerization was normal in the presence of 3 mM MgCl 2 and low ionic strength, but it was necessary to add phalloidin to prevent depolymerization of the mutant filaments under the high salt conditions of the co-sedimentation binding assays (data not shown).…”

“…For example, a Drosophila melanogaster actin subdomain 2 mutant, E93K, which inhibits tropomyosin-actin sliding in isolated protein preparations, has been utilized to investigate tropomyosin-based regulation (16). These and other data (17)(18)(19)(20) suggested that actin subdomain 2 may be important for tropomyosin and troponin function.…”

“…Helical reconstruction was carried out using standard methods (38 -40) as described previously (8,36). (16,17,19) suggest that modification or mutation of actin subdomain 2 can alter interactions of actin with tropomyosin (16). To explore this quantitatively, we examined the binding of tropomyosin to yeast actin mutant D56A/E57A (Fig.…”

Alternative Exon-encoded Regions of Drosophila Myosin Heavy Chain Modulate ATPase Rates and Actin Sliding Velocity

“…The effect varies according to myosin type and isoform. Smooth muscle tropomyosin increases actin sliding velocity ϳ10-fold with phosphorylated Limulus striated muscle myosin (54) and 2-4-fold on smooth muscle myosin (55), but only slightly increases velocities on rabbit skeletal HMM (56). On this basis the Emb 18 isoform acts like a smooth muscle myosin, while the IFM myosin behaves more like rabbit skeletal myosin.…”