Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments.
The Arp2/3 complex, a seven-subunit protein complex containing two actin-related proteins, Arp2 and Arp3, initiates formation of actin filament networks in response to intracellular signals. The molecular mechanism of filament nucleation, however, is not well understood. Arp2 and Arp3 are predicted to bind ATP via a highly conserved nucleotide-binding domain found in all members of the actin superfamily and to form a heterodimer than mimics a conventional actin dimer. We show here that adenosine nucleotides bind with micromolar affinity to both Arp2 and Arp3 and that hydrolyzable ATP is required for actin nucleation activity. Binding of N-WASP WA increases the affinity of both Arp2 and Arp3 for ATP but does not alter the stoichiometry of nucleotides bound in the presence of saturating concentrations of ATP. The Arp2/3 complex bound to ADP or the nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments, but addition of the phosphate analogue BeF(3) partially restores activity to the ADP-Arp2/3 complex. Bound nucleotide also regulates the affinity of the Arp2/3 complex for its upstream activators N-WASP and ActA. We propose that the active nucleating form of the Arp2/3 complex is the ADP-P(i) intermediate in the ATPase cycle and that the ATPase activity of the Arp2/3 complex controls both nucleation of new filaments and release of the Arp2/3 complex from membrane-associated activators.
Dayel
M J
MJ
Graduate Group in Biophysics and Department of Cellular and Molecular Pharmacology, University of California at San Francisco, 94143, USA.
Holleran
E A
EA
Mullins
R D
RD
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003598:Cytoskeletal Proteins; D006868:Hydrolysis; D007700:Kinetics
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D000255:Adenosine Triphosphate