How ATP hydrolysis controls filament assembly from profilin-actin: implication for formin processivity.
Formins catalyze rapid filament growth from profilin-actin, by remaining processively bound to the elongating barbed end. The sequence of elementary reactions that describe filament assembly from profilin-actin at either free or formin-bound barbed ends is not fully understood. Specifically, the identity of the transitory complexes between profilin and actin terminal subunits is not known; and whether ATP hydrolysis is directly or indirectly coupled to profilin-actin assembly is not clear. We have analyzed the effect of profilin on actin assembly at free and FH1-FH2-bound barbed ends in the presence of ADP and non-hydrolyzable CrATP. Profilin blocked filament growth by capping the barbed ends in ADP and CrATP/ADP-Pi states, with a higher affinity when formin is bound. We confirm that, in contrast, profilin accelerates depolymerization of ADP-F-actin, more efficiently when FH1-FH2 is bound to barbed ends. To reconcile these data with effective barbed end assembly from profilin-MgATP-actin, the nature of nucleotide bound to both terminal and subterminal subunits must be considered. All data are accounted for quantitatively by a model in which a barbed end whose two terminal subunits consist of profilin-ATP-actin cannot grow until ATP has been hydrolyzed and Pi released from the penultimate subunit, thus promoting the release of profilin and allowing further elongation. Formin does not change the activity of profilin but simply uses it for its processive walk at barbed ends. Finally, if profilin release from actin is prevented by a chemical cross-link, formin processivity is abolished.
Romero
Stéphane
S
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
Didry
Dominique
D
Larquet
Eric
E
Boisset
Nicolas
N
Pantaloni
Dominique
D
Carlier
Marie-France
MF
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005326:Fetal Proteins / Q000378:metabolism*; D000080865:Formins; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D051304:Profilins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D005326:Fetal Proteins; D000080865:Formins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D051304:Profilins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate