The role of profilin in actin polymerization and nucleotide exchange

Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using Ply-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [A(free)], was close to that seen with actin alone ([A(cc)], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wildtype profilin caused a lowering of [A(free)] as compared to [A(cc)], indicating that actin monomers and profilin: actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [A(free)] at steady state was again close to [A(cc)], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.