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-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, 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 defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically 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 phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.
Lee
S S
SS
Adolf-Butenandt-Institut für Zellbiologie, Ludwig-Maximilians-Universität, München/Germany.
Karakesisoglou
I
I
Noegel
A A
AA
Rieger
D
D
Schleicher
M
M
D000818:Animals; D003285:Contractile Proteins; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics*; D017354:Point Mutation; D051304:Profilins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D011994:Recombinant Proteins