CALCINEURIN-DEPENDENT GROWTH OF AN FK506-HYPERSENSITIVE AND CSA-HYPERSENSITIVE MUTANT OF SACCHAROMYCES-CEREVISIAE

The immunosuppressants FK506 and cyclosporin A (CsA) bound to their re ceptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from a-mating factor arrest. Vegetative growth of yeas t does not require calcineurin, and in strains sensitive to FK506 or C sA, growth is inhibited by concentrations of drug much higher than tho se required to inhibit T cell activation or recovery from mating facto r arrest. We now describe the isolation of a mutant of Saccharomyces c erevisiae which is 100-1000-fold more sensitive to the growth inhibito ry properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca 2+ and exacerbated by EGTA. Simultaneous disruption of the two genes ( CNA1 and CNA2) encoding the alternative forms of the catalytic A subun it of calcineurin, or of the gene (CNB1) encoding the regulatory B sub unit, is lethal in an fks1 mutant. Disruption of the gene encoding FKB P12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells re sults in the loss of hypersensitivity to the relevant drug. Overexpres sion of CNA1 or CNA2, in conjunction with CNB1, results in a significa nt decrease in hypersensitivity to FK506 and CsA. The results show tha t the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The g rowth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of im mune suppression by these drugs.