Abstract
Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (Kd = 0.1 microM) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments.
Highlights
The original protein of this type, ADF,1 got its name “actin depolymerizing factor” by virtue of its ability to depolymerize actin filaments
Using a new fluorescence anisotropy assay, we confirm the higher affinity of actophorin for Mg-ADP-actin than Mg-ATPactin and characterize in detail the effect of both actophorin and profilin on the exchange of the bound nucleotide
We show that actophorin bound to ADP- or ATP-actin does not inhibit actin filament elongation at either the barbed or pointed end of filaments
Summary
The original protein of this type, ADF, got its name “actin depolymerizing factor” by virtue of its ability to depolymerize actin filaments. Carlier et al [21] questioned the severing activity of the ADF/ cofilin family and proposed a “dynamizing” mechanism for plant cofilin Their interpretation was that plant cofilin promotes actin filament turnover by increasing the rates of ATPactin association at the barbed end and ADP-actin dissociation at the pointed end. Either by severing or enhancing dissociation of subunits from the pointed end, ADF/cofilin proteins should release MgADP-actin monomers, which bind actophorin and cofilin with higher affinity than ATP-actin [21, 22]. Both porcine brain and yeast cofilin inhibit the exchange of Mg-ATP bound to actin [23, 24]. We show that actophorin bound to ADP- or ATP-actin does not inhibit actin filament elongation at either the barbed or pointed end of filaments
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