Profilin promotes barbed-end actin filament assembly without lowering the critical concentration

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin . 4.1 . Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta 4, both actin and Profilin . Actin (P . A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant (similar to 10 x 10(6) M-1 S-1) for actin monomer or Profilin . Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 mu M, thymosin-beta 4 concentration range fit a simple monomer sequestering model (1 mu M K-D for Thymosin-beta 4 . Actin). The corresponding constant for Thymosin-beta 4 . pyrenyl-Actin, however, was significantly higher (similar to 9-10 mu M), suggesting that the fluorophore markedly weakens binding to thymosin-beta 4. With solutions of actin (2 mu M) and thymosin-beta 4 (2 or 4 mu M), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 mu M). Actin assembly in presence of thymosin-beta 4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M. F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta 4 . Actin and ushers actin as a Profilin . Actin complex onto growing barbed filament ends.