Abstract
Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO4(2-) binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two Pi binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO4(2-) which is subsequently titrated to H2PO4-. We speculate that beryllium fluoride and aluminum fluoride bind to the HPO4(2-) binding site. The cellular consequences of this model of phosphate release are discussed.
Highlights
The hydrolysis of ATP during actin polymerization has been postulated to alter filament structure [1,2,3, 4], change the kinetics of monomer binding [5, 6], and modulate the association of actin binding proteins [7,8,9]
We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers
We report that the actin filament severing protein gelsolin can differentiate between ADP-Pi and ADP-rich filaments, with filaments in the ADP-Pi state being severed by gelsolin much more slowly
Summary
The hydrolysis of ATP during actin polymerization has been postulated to alter filament structure [1,2,3, 4], change the kinetics of monomer binding [5, 6], and modulate the association of actin binding proteins [7,8,9]. The electron density map of the actin filament alters when Pi is bound, with domain 2 of the actin monomer moving out from the helical center of the filament and away from domain 1 of the monomer in the two start helix [3] This domain is not readily visualized in F-actin polymerized from ADP monomers, suggesting its position is variable. Binding of Pi, AlFn, or BeFn reduces the extent of severing by both intact gelsolin and its Ca2ϩ insensitive Nterminal domain These findings suggest that in cells the assembly and disassembly of filaments could be regulated by signal transduction pathways, but by the energy charge of the cell and the age of pre-existing actin filaments
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