Profilin Promotes Barbed-end Actin Filament Assembly without Lowering the Critical Concentration*
- From the Departments of ‡Medicine and§Biochemistry & Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0245
Abstract
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 × 106 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 μmthymosin-β4 concentration range fit a simple monomer sequestering model (1 μm K D for Thymosin-β4·Actin). The corresponding constant for thymosin-β4·pyrenyl-Actin, however, was significantly higher (∼9–10 μm), suggesting that the fluorophore markedly weakens binding to thymosin-β4. With solutions of actin (2 μm) and thymosin-β4 (2 or 4 μm), the barbed-end assembly rate rose with increasing profilin concentration (0.7–2 μm). 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.
Footnotes
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↵* This investigation was supported by National Institutes of Health Grants R01-AI23262 and R01-AI24276.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, P. O. Box 100245, Health Science Center, Gainesville, FL 32610-0245. Tel. and Fax: 352-392-1546; E-mail: dlpurich@biochem.med.ufl.edu.
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↵2 These equilibrium constants are calculated in terms of experimentally determined off- and on-rate constants. Although such derived constants are useful for testing the thermodynamic cycle, we should emphasize that the various paths in the cycle do not satisfy a rapid equilibrium condition. This fact is most directly indicated by the ability of profilin to stimulate actin polymerization.
- Abbreviations:
- Tβ4
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thymosin-β4
- A or Afree
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unpolymerized actin monomer (G-actin)
- Fn and Fn+1
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filamentous actin, respectively, containing n and (n + 1) actin subunits
- P
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profilin
- VASP
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vasodilator-stimulated phosphoprotein
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- Received May 28, 1999.
- Revision received September 22, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
