MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF YEAST PDR1 MUTANTS THAT SHOW HYPERACTIVE TRANSCRIPTION OF VARIOUS ABC MULTIDRUG TRANSPORTER GENES

Mutations at the yeast PDR1 transcriptional regulator locus are respon sible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, associated with the appearance of multiple drug resistance. The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidru g resistance mutants, 1 intragenic suppressor and 6 wild types, have b een determined. Single amino acid substitutions were shown to result f rom the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1 -7 (P298A) and pdr1-8 (L1036W), whereas the intragenic suppressor muta nt pdr1-100 is deleted for the two amino acids L537 and A538. An isoge nic series of strains was constructed containing the mutant alleles pd r1-3, pdr-ld and pdr1-8 integrated into the genome. We found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N- oxide and ketoconazole were increased in all three mutants. The increa se was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mut ants. Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter a nd the pdr1-3 mutation resulted in the highest level of promoter induc tion. Concomitantly, the level of PDR5 mRNA, of Pdr5p protein, and of its associated nucleoside triphosphatase activity, was strongly increa sed in the plasma membranes of the PDR1 mutants. Again, the pdr1-3 all ele was associated with a stronger effect than the pdr1-8 and pdr1-6 a lleles. The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p tran scription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency. These gain-of-function m utations support the concept, recently proposed, that in members of th e large family of yeast Zn(2)Cys(6) transcription factors a central in hibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mu tations). This domain may interact in a locked conformation with a put ative, more C-terminally located inhibitory domain (mutated in pdr1-3) , and with the putative activation domain (mutated in pdr1-8).