The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth.
FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.
Eng
W K
WK
Department of Biomolecular Disovery, SmithKline Beecham Pharmaceuticals King of Prussia, PA 19406.
Faucette
L
L
McLaughlin
M M
MM
Cafferkey
R
R
Koltin
Y
Y
Morris
R A
RA
Young
P R
PR
Johnson
R K
RK
Livi
G P
GP
D000595:Amino Acid Sequence; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000494:pharmacology*; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D054714:Echinocandins; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D005800:Genes, Fungal; D005838:Genotype; D005964:Glucosyltransferases; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D016559:Tacrolimus / Q000494:pharmacology*
D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D054714:Echinocandins; D005656:Fungal Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D016572:Cyclosporine; D005964:Glucosyltransferases; C091618:FKS1 protein, S cerevisiae; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D016559:Tacrolimus