The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

Abstract

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind . Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FKS06-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trpl his4 ku2) are FK506 sensitive, whereas prototrophic strains (TRPI his4 ku2, trpl HIS4 leu2, and trp) his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FKS06 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.Cyclosporin A (CsA), FK506, and rapamycin are potent immunosuppressants that block T-cell activation by interfering with signal transduction (41,51,74,75). CsA is a cyclic peptide, whereas FK506 and rapamycin are members of the macrolide antibiotic family. All three suppress the immune system by blocking intermediate signal transduction steps required to activate T cells. CsA and FK506 inhibit expression of genes required for response to antigen, including the genes encoding interleukin-2 and the interleukin-2 receptor (19). In contrast, rapamycin blocks a later step and prevents T-cell responses to interleukin-2 (4, 17, 18).In vitro, all three compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases or proline rotamases (immunophilins), enzymes which may fold proteins in vivo. CsA inhibits cyclophilins (22,41,75,87), a family of related prolyl isomerases, whereas rapamycin and FKS06 competitively inhibit members of another distinct family of proline isomerases, FK506-binding proteins (FKBPs) (37,41,75,79,84). The cyclophilin-CsA and FKBP-FK506 complexes bind to and inhibit the protein phosphatase calcineurin (26, 27, 57, 58), which plays a critical role in transducing signals from the T-cell antigen receptor to the nucleus (9, 68). CsA and FK506 also prevent nuclear imp...

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