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 o nly weakly affected polymerization of actin to one that bound actin mo re strongly than wild-type profilin. With profilins, whose sequesterin g activity was low, the concentration of free actin monomers observed at steady-state of polymerization [A(free)], was close to that seen wi th actin alone ([A(cc)], critical concentration of polymerization). Pr ofilin mutants binding actin with an intermediate affinity like wildty pe profilin caused a lowering of [A(free)] as compared to [A(cc)], ind icating that actin monomers and profilin: actin complexes participate in polymer formation. With a mutant profilin, which bound actin more s trongly than the wild-type protein, an efficient sequestration of acti n was observed, and in this case, the [A(free)] at steady state was ag ain 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 ar e consonant with the idea that profilin:actin complexes are directly i ncorporated at the (+)-end of actively polymerizing actin filaments, w hile they do not support the view that profilin facilitates polymer fo rmation.