Calcineurin-dependent growth of an FK506- and CsA-hypersensitive mutant of Saccharomyces cerevisiae.
The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from alpha-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100-1000-fold more sensitive to the growth inhibitory properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca2+ and exacerbated by EGTA. Simultaneous disruption of the two genes (CNA1 and CNA2) encoding the alternative forms of the catalytic A subunit of calcineurin, or of the gene (CNB1) encoding the regulatory B subunit, is lethal in an fks1 mutant. Disruption of the gene encoding FKBP12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells results in the loss of hypersensitivity to the relevant drug. Overexpression of CNA1 or CNA2, in conjunction with CNB1, results in a significant decrease in hypersensitivity to FK506 and CsA. The results show that the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The growth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of immune suppression by these drugs.
Parent
S A
SA
Merck Research Laboratories, Rahway, NJ 07065.
Nielsen
J B
JB
Morin
N
N
Chrebet
G
G
Ramadan
N
N
Dahl
A M
AM
Hsu
M J
MJ
Bostian
K A
KA
Foor
F
F
D000591:Amino Acid Isomerases / Q000378:metabolism; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal; D006360:Heat-Shock Proteins / Q000378:metabolism; D009154:Mutation; D019696:Peptidylprolyl Isomerase; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D016572:Cyclosporine; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D002118:Calcium; D016559:Tacrolimus