Open AccessPolyproline binding is an essential function of human profilin in yeast
Author(s) -
Ostrander Darin B.,
Ernst Eileen G.,
Lavoie Thomas B.,
Gorman Jessica A.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00354.x
Subject(s) - polyproline helix , mutant , profilin , acetylation , biology , gene , saccharomyces cerevisiae , biochemistry , yeast , mutation , microbiology and biotechnology , cell , peptide , actin cytoskeleton , cytoskeleton
Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli . The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) binding remained unchanged. In many strains of Saccharomyces cerevisiae , disruption of the gene encoding profilin, PFY1 , is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline‐binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1 Δ. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline‐binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP 2 . These data strongly suggest that polyproline binding is an essential function of profilin.