Site-directed mutagenesis of Asp853 in Pdr3p transcriptional activator from Saccharomyces cerevisiae.
The PDR3 gene encodes one of the main transcriptional activators involved in the control of multidrug resistance in the yeast Saccharomyces cerevisiae. Recently, it has been demonstrated that a specific D853Y mutation results in the loss of transactivation activity of Pdr3p and its conversion to multicopy suppressor of multidrug resistance. In this study, the Asp853 in Pdr3p was replaced by eight different amino acids and the function of mutated proteins was analysed. Different levels of complementation of cycloheximide hypersensitivity and expression of autoregulated PDR3 and its PDR5 target in the pdr1Deltapdr3Delta mutant strain, ranging from that of the wild-type to loss-of-function alleles, were observed in pdr3 mutants containing Pro, Glu, Arg, Asn, Ser, Leu, Phe, Ile or Tyr instead of Asp853 in Pdr3p. The introduction of the D853Y mutation into gain-of-function Pdr3p suppressed the transcription of the PDR3 and PDR5 genes and reduced both the rhodamine 6G efflux rate and the drug resistance level in corresponding double mutants. The results indicate that, while Pdr3p can tolerate several substitutions of Asp853, the occurrence of a hydrophobic amino acid at this position has an adverse effect on its function.
Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovak Republic.
Vladimira
V
Dzugasova
Silvia
S
Borecka
Monika
M
Batova
Ruzena
R
Pilisiova
Nora
N
Hervayova
Julius
J
Subik
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D025141:Drug Resistance, Fungal; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014170:Transformation, Genetic
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D004268:DNA-Binding Proteins; D050505:Mutant Proteins; C085480:PDR1 protein, S cerevisiae; C089261:PDR3 protein, S cerevisiae; C092438:PDR5 protein, S cerevisiae; D012235:Rhodamines; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003513:Cycloheximide