Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg 2؉ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a Gactin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.Living cells undergo changes in shape and motile behavior by spatially and temporally controlled rearrangements of the actin cytoskeleton. In the physiological ionic conditions, F-actin is assembled at steady state in the cell medium. Changes in the F-actin/G-actin ratio, which occur in response to stimuli, are made possible by shifts in steady state, i.e. changes in the critical concentration for filament assembly. These changes are elicited by capping proteins and profilin (1, 2) and amplified by G-actin-binding proteins (3). A high level of capping of barbed ends maintains the high critical concentration of pointed ends in the cytoplasm. A steep energetic gradient is therefore created between the cell medium and the loci where uncapped barbed ends are nucleated at the plasma membrane. We understand that in this way capping of barbed ends in the cytoplasm is required for a more efficient local actin assembly. In support of this view, recent evidence indeed indicates that the level of motility in fibroblasts (4) and Dictyostelium (5) correlates with the level of barbed end capping. Similarly, the actinbased propulsive movement of Listeria results from the local creation and maintenance of new uncapped barbed ends at the bacterium surface, while filaments are capped in the bulk cytoplasm (6).Profilin has unique properties among G-actin-binding proteins. Under physiological ionic conditions (Mg-actin, 0.1 M KCl), it binds G-actin tightly (K ϭ 10 7 M Ϫ1 for vertebrate profilin I) and participates in the establishment of the pool of unassembled actin when barbed ends are capped. In contrast, when barbed ends are uncapped, the participation of profilinactin complex in...

The interaction of bovine spleen profilin with ATP- and ADP-G-actin and poly(L-proline) has been studied by spectrofluorimetry, analytical ultracentrifugation, and rapid kinetics in low ionic strength buffer. Profilin binding to G-actin is accompanied by a large quenching of tryptophan fluorescence, allowing the measurement of an equilibrium dissociation constant of 0.1-0.2 microM for the 1:1 profilin-actin complex, in which metal ion and nucleotide are bound. Fluorescence quenching monitored the bimolecular reaction between G-actin and profilin, from which association and dissociation rate constants of 45 microM-1 s-1 and 10 s-1 at 20 degrees C could be derived. The tryptophan(s) which are quenched in the profilin-actin complex are no longer accessible to solvent, which points to W356 in actin as a likely candidate, consistent with the 3D structure of the crystalline profilin-actin complex [Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. W., & Lindberg, U. (1993) Nature 365, 810-816]. Upon binding poly(L-proline), the fluorescence of both tyrosines and tryptophans of profilin is enhanced 2.2-fold. A minimum of 10 prolines [three turns of poly(L-proline) helix II] is necessary to obtain binding (KD = 50 microM), the optimum size being larger than 10. Binding of poly(L-proline) is extremely fast, with k+ > 200 microM-1 s-1 at 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymosin ␤ 4 is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of T␤ 4 in actin assembly in the high range of concentrations (up to 300 M) at which T␤ 4 is found in highly motile blood cells. T␤ 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 M). As the concentration of T␤ 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The T␤ 4 -actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and T␤ 4 -actin complex, the critical concentration is the sum of free G-actin and T␤ 4 -G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by T␤ 4 -G-actin complex. The incorporation of T␤ 4 -actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, T␤ 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of T␤ 4 -actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing ␤-thymosins.

The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50-fold, respectively. On the profilin-actin complex as well as on G-actin alone, nucleotide exchange is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion. 2) Under physiological ionic conditions, nucleotide exchange on G-actin is 1 order of magnitude faster than at low ionic strength. The rate of MgATP dissociation is increased by profilin from 0.05 s-1 to 2 s-1, the rate of MgADP dissociation is increased from 0.2 s-1 to 24 s-1. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 x 10(6) to 10(7) M-1) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-G-actin. Profilin binding to actin lowers the affinity of metal-nucleotide by about 1 order of magnitude. These results restrain the possible roles of profilin in actin assembly in vivo.