DOMINANT MISSENSE MUTATIONS IN A NOVEL YEAST PROTEIN RELATED TO MAMMALIAN PHOSPHATIDYLINOSITOL 3-KINASE AND VPS34 ABROGATE RAPAMYCIN CYTOTOXICITY

Rapamycin is a macrolide antifungal agent that exhibits potent immunos uppressive properties. In Saccharomyces cerevisiae, rapamycin sensitiv ity is mediated by a specific cytosplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP 1) results in recessive drug resistance, and expression of hFKBP12 res tores rapamycin sensitivity. These data support the idea that FKBP12 a nd rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rap amycin-resistant mutants both in wild-type haploid and diploid cells a nd in haploid rbp1=URA3 cells engineered to express hFKBP12. Genetic a nalysis indicated that the dominant mutations are nonallelic to mutati ons in RBP1 and define two genes, designated DPR1 and DRR2 (for domina nt rapamycin resistance). Mutant copies of DRR1 and DPR2 were cloned f rom genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele rev ealed a long open reading frame predicting a novel 2470-amino-acid pro tein with several motifs suggesting an involvement in intracellular si gnal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significan t sequence similarity to the 110-kDa catalytic subunit of both yeast ( VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption o f DRR1 in a mutant haploid strain restored drug sensitivity and demons trated that the gene encodes a nonessential function. DNA sequence com parison of seven independent drr1dom alleles identified single base pa ir substitutions in the same codon within the phosphatidylinositol 3-k inase domain, resulting in a change of Ser-1972 to Arg or Asn. We conc lude either that DRR1 (alone or in combination with DRR2) acts as a ta rget of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.