Filament Assembly from Profilin-Actin*
- From the Laboratoire d’Enzymologie et Biochimie Structurales,‡Centre de Génétique Moléculaire, CNRS, 91198 Gif-sur-Yvette, France and §Institut Curie, Physics Section, 11 rue Pierre et Marie Curie, 75005 Paris, France
Abstract
Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 μm under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 μm under physiological conditions. The PAcovpolymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.
Footnotes
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↵* This work was supported in part by the Association Francaise contre les Myopathies, the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, and a grant from the European Economic Community (Contract CHRX-CT94-0652).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. Fax: 33 01 69 82 31 29; E-mail: carlier{at}lebs.cnrs-gif.fr.
- Abbreviations:
- PA
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profilin-MgATP-actin complex
- NBD
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7-chloro-4-nitrobenzene-2-oxa-1,3-diazole, EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
- S1
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myosin subfragment-1
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- Received September 18, 1998.
- Revision received December 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
