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 cerevisiae, expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) interaction. The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin.
Hájková
L
L
Department of Zoological Cell Biology, WGI, Stockholm University, Sweden.
Björkegren Sjögren
C
C
Korenbaum
E
E
Nordberg
P
P
Karlsson
R
R
D016475:3T3 Cells / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003285:Contractile Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008845:Microinjections; D016296:Mutagenesis / Q000502:physiology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051304:Profilins; D011392:Proline / Q000032:analysis; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; C495739:Pfn1 protein, mouse; D019269:Phosphatidylinositol 4,5-Diphosphate; D051304:Profilins; D011392:Proline