Isolation and molecular characterization of the carboxy-terminal pdr3 mutants in Saccharomyces cerevisiae

Multidrug resistance in Saccharomyces cerevisiae mainly results from the ov erexpression of genes coding for the membrane efflux pumps, the major facil itators and the ABC binding cassette transporters, under the control of key transcription regulators encoded by the PDR1 and PDR3 genes. Pdr3p transcr iptional activator contains a weak activation domain near the N-terminal zi nc finger, a central regulatory domain, and a strong activation domain near the carboxyl terminus. Here we report the results of the mutational analys is of the C-terminal region of Pdr3p. After in vitro mutagenesis of the PDR 3 gene six single amino acid substitutions were identified and resulted in resistance to cydoheximide, sulfomethuron methyl, 4-nitroquinoline oxide, f luconazole, mucidin, chloramphenicol and oligomycin. All the C-terminal pdr 3 mutant alleles also conferred multidrug resistance in the presence of the wild-type PDR3 gene. The prlr3 mutations resulted in overexpression of bot h the PDR3 and PDR5 genes as revealed by transactivation experiments involv ing the PDR3-lacZ and PDR5-lacZ fusion genes and Western blot analyses usin g antibodies against Pdr5p. Most of the C-terminal pdr3 mutations were foun d in two sequence stretches exhibiting a high degree of amino acid identity with Pdr1p indicating that they might play a significant role in protein-p rotein interactions during the initiation of transcription of genes involve d in multidrug resistance.