Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization.

M F Carlier,D Pantaloni

doi:10.1016/s0021-9258(19)35428-6

Abstract

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.

Highlights

MATERIALS ANDMETHODSChemicals-All reagents used to prepare the buffers were Merck analytical grade. Dithiothreitol, ATP, and ADP came from Boehringer Mannheim; EGTA,' A p d , NBD-Cl, sodium orthovanadate, cytochalasin D, and DNase I (grade IV) were from Sigma; N-pyrene iodoacemide was from Molecular Probes; 32Piand [y3'P]ATP were from Amersham Corp
Marie-France Carlier and Dominique Pantaloni From the Laboratoire d'Enzymobgie, Centre National de la Recherche Scientifique,91190 Gif-sur-Yuette,France
The above equation suggests that, depending on the combination of rates of growth, ATP hydrolysis, and Pi dissociation, either ATP-actin, ADP-Pi-actin, or ADP-actin can be the terminal subunits of the filament, andthe kinetic parameters of filament growth could be different in each case

Summary

MATERIALS ANDMETHODS

Chemicals-All reagents used to prepare the buffers were Merck analytical grade. Dithiothreitol, ATP, and ADP came from Boehringer Mannheim; EGTA,' A p d , NBD-Cl, sodium orthovanadate, cytochalasin D, and DNase I (grade IV) were from Sigma; N-pyrene iodoacemide was from Molecular Probes; 32Piand [y3'P]ATP were from Amersham Corp. Measurement of Rate of Filament Elongation-Initial rates of filament elongation or dissociation and theirdependence on the concentration of G-actin (J(c)plot) were measured, as described previously [3], by diluting an aliquot of 50 pl of F-actin seeds solution into 750 pl of polymerization buffer containing G-actina t different concentrations. Evolution of the Rate Constant for Actin Dissociation from Filaments during the Time Course of Polymerization-Mg-actin at high concentration (38-40 p ~w)as fully polymerized within a very short period of time (45-60 s ) by addition of 2 mM MgClz followed immediately by a 15-9sonication. At different times following the onset of polymerization, aliquots of this solution were diluted 20-fold in the same polymerization buffer containing 0.1 or 0.2 mg/ml DNase I. Aliquots of Factin were removed at different times following the onset of polymerization and diluted in a fluorescence cuvette containing 2.5 p~ Gactin in polymerization buffer. The hydrolysis of ATP was monitored by extraction of the 32P-labeledphosphomolybdic complex as described previously [3]

RESULTS

Binding of Inorganic Phosphate to Actin Filaments a

YI c

DISCUSSION