Molecular mechanism of terbinafine resistance in Saccharomyces cerevisiae.
Ten mutants of the yeast Saccharomyces cerevisiae resistant to the antimycotic terbinafine were isolated after chemical or UV mutagenesis. Molecular analysis of these mutants revealed single base pair exchanges in the ERG1 gene coding for squalene epoxidase, the target of terbinafine. The mutants did not show cross-resistance to any of the substrates of various pleiotropic drug resistance efflux pumps tested. The ERG1 mRNA levels in the mutants did not differ from those in the wild-type parent strains. Terbinafine resistance was transmitted with the mutated alleles in gene replacement experiments, proving that single amino acid substitutions in the Erg1 protein were sufficient to confer the resistance phenotype. The amino acid changes caused by the point mutations were clustered in two regions of the Erg1 protein. Seven mutants carried the amino acid substitutions F402L (one mutant), F420L (one mutant), and P430S (five mutants) in the C-terminal part of the protein; and three mutants carried an L251F exchange in the central part of the protein. Interestingly, all exchanges identified involved amino acids which are conserved in the squalene epoxidases of yeasts and mammals. Two mutations that were generated by PCR mutagenesis of the ERG1 gene and that conferred terbinafine resistance mapped in the same regions of the Erg1 protein, with one resulting in an L251F exchange and the other resulting in an F433S exchange. The results strongly indicate that these regions are responsible for the interaction of yeast squalene epoxidase with terbinafine.
Leber
Regina
R
Institute of Molecular Biology, Biochemistry and Microbiology, Karl-Franzens-Universität Graz, Graz, Austria.
Fuchsbichler
Sandra
S
Klobucníková
Vlasta
V
Schweighofer
Natascha
N
Pitters
Eva
E
Wohlfarter
Kathrin
K
Lederer
Mojca
M
Landl
Karina
K
Ruckenstuhl
Christoph
C
Hapala
Ivan
I
Turnowsky
Friederike
F
D000483:Alleles; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D025141:Drug Resistance, Fungal; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009281:Naphthalenes / Q000494:pharmacology*; D010105:Oxygenases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics*; D050603:Squalene Monooxygenase; D000077291:Terbinafine
D000935:Antifungal Agents; D004271:DNA, Fungal; D009281:Naphthalenes; D010105:Oxygenases; D050603:Squalene Monooxygenase; D000077291:Terbinafine