Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology, By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-binding)-proline-bi nding activity respectively, W3N profilin is no longer able to bind to poly -(L)-proline concomitant with a slight reduction in actin binding, The K114 E profilin exhibited a profound decrease in its ability to interact with ac tin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin, The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D, discoideum mutants which have defe cts in the F-actin content, cytokinesis and development (Haugwitz et al., C ell 79, 303- 314, 1994). Expression of K114E or W3N displayed a reduction i n the F-actin content, normal cell morphology, and the transformants were c apable of undergoing complete development. Interestingly, only cells that d rastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in p hagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppr ession of the mutant phenotype than the G-actin binding activity of profili n, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities .