Profilin Promotes Barbed-end Actin Filament Assembly without Lowering the Critical Concentration

• Published in: The Journal of biological chemistry 1999-12, Vol.274 (52), p.36963-36972
• Main Authors: Kang, F, Purich, D L, Southwick, F S
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Animals; Contractile Proteins; Humans; Light; Microfilament Proteins - pharmacology; Polymers - chemistry; Profilins; Rabbits; Scattering, Radiation; Thermodynamics; Thymosin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.52.36963

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-β4, both actin and Profilin·Actin (P·A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant (∼10 × 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 μ m thymosin-β4 concentration range fit a simple monomer sequestering model (1 μ m K D for Thymosin-β4·Actin). The corresponding constant for thymosin-β4·pyrenyl-Actin, however, was significantly higher (∼9–10 μ m ), suggesting that the fluorophore markedly weakens binding to thymosin-β4. With solutions of actin (2 μ m ) and thymosin-β4 (2 or 4 μ m ), the barbed-end assembly rate rose with increasing profilin concentration (0.7–2 μ m ). Actin assembly in presence of thymosin-β4 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-β4·Actin and ushers actin as a Profilin·Actin complex onto growing barbed filament ends.