Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein

Mol Cell Biol. 1991 Mar;11(3):1718-23. doi: 10.1128/mcb.11.3.1718-1723.1991.

Abstract

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Isomerases / genetics*
  • Amino Acid Isomerases / isolation & purification
  • Amino Acid Sequence
  • Base Sequence
  • Carrier Proteins / genetics*
  • Carrier Proteins / isolation & purification
  • Cloning, Molecular
  • Drug Resistance, Microbial
  • Fungal Proteins / genetics
  • Genetic Complementation Test
  • Molecular Sequence Data
  • Molecular Weight
  • Peptidylprolyl Isomerase
  • Polyenes / pharmacology*
  • Recombinant Proteins / genetics
  • Restriction Mapping
  • Saccharomyces cerevisiae / drug effects*
  • Saccharomyces cerevisiae / genetics
  • Sirolimus
  • Tacrolimus Binding Proteins

Substances

  • Carrier Proteins
  • Fungal Proteins
  • Polyenes
  • Recombinant Proteins
  • Amino Acid Isomerases
  • Tacrolimus Binding Proteins
  • Peptidylprolyl Isomerase
  • Sirolimus

Associated data

  • GENBANK/M35697
  • GENBANK/M35698
  • GENBANK/M35699
  • GENBANK/M35700
  • GENBANK/M63892
  • GENBANK/M63981
  • GENBANK/M63982
  • GENBANK/M73693
  • GENBANK/M73694
  • GENBANK/M73695