CHARACTERIZATION OF A MUTANT PROFILIN WITH REDUCED ACTIN-BINDING CAPACITY - EFFECTS IN-VITRO AND IN-VIVO

We are investigating structure-function relationships in profilin and actin by site specific mutagenesis using a yeast, Saccharomyces cerevi siae, expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are loca ted close to the major actin binding site on profilin, did not signifi cantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) inte raction. The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly in creased profilin-enhanced nucleotide exchange on the actin, When micro injected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelia l cells, the mutant profilin did not change the organization of the mi crofilament system like the wild-type profilin did. This provides furt her evidence that profilin controls microfilament organization in the cell by interacting directly with actin. (C) 1997 Academic Press.