Polyproline binding is an essential function of human profilin in yeast.
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 (PIP2) 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 pfy1Delta. 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 PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.
Ostrander
D B
DB
Department of Microbial Molecular Biology, Pharmaceutical Research Institute, Bristol-Meyers Squibb, Princeton, NJ, USA.
Ernst
E G
EG
Lavoie
T B
TB
Gorman
J A
JA
D001483:Base Sequence; D003285:Contractile Proteins; D017931:DNA Primers; D005816:Genetic Complementation Test; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D010455:Peptides / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D003285:Contractile Proteins; D017931:DNA Primers; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D010455:Peptides; D051304:Profilins; C011083:polyproline