THE TYROSINE89 RESIDUE OF YEAST FKBP12 IS REQUIRED FOR RAPAMYCIN BINDING

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that e xhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for F K506- and Rm-binding proteins, respectively). These proteins possess p eptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae , Rm sensitivity (Rm(S)) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null muta tions in the yeast RBP1 gene result in a recessive drug resistance phe notype. To identify missense mutations that define amino acid (aa) res idues in RBP1 involved in drug sensitivity, we selected and geneticall y characterized over 250 independent Rm(R) rbp1 mutants and screened t hem for both RBP1-specific mRNA and protein expression. Whereas all rb p1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independen t clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some allele s contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conse rved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydroph obic drug-binding pocket for FK506 and Rm. Analysis of [H-3]Rm binding in rbp1 mutant vs. wild-type yeast cell extracts indicated that the T yr89-->Asp (Y89D) change results in an approx. 95% loss of binding act ivity. These data confirm the assignment of this Tyr residue as being involved in Rm binding.