Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance.

Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors. To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor. Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance. The basic components of this system, the P(GAL1)-PDR3 and P(PDR5)-pma1(D378N) fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive S. cerevisiae strain deleted in the PDR1 and PDR3 genes. This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously. In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance.

Loss-of-function pdr3 mutations convert the Pdr3p transcription activator to a protein suppressing multidrug resistance in Saccharomyces cerevisiae.

The PDR1 and PDR3 genes encode the main transcription activators involved in the control of multidrug resistance in Saccharomyces cerevisiae. To identify the amino acids essential for Pdr3p function, the loss-of-function pdr3 mutants were isolated and characterized. Two plasmid-borne pdr3 alleles, pdr3-E902Ter and pdr3-D853Y, which failed to complement drug hypersensitivity in the Deltapdr1Deltapdr3 mutant strain, were isolated. The E902Ter mutation resulted in a truncated protein lacking the C-terminal activation domain. The D853Y mutation allowed the expression of entire Pdr3p, but its transactivation function was lost. When overexpressed from the P(GAL1) promoter, the two mutant alleles increased the sensitivity of wild-type cells to cycloheximide and fluconazole and suppressed drug resistance in gain-of-function pdr1 and pdr3 mutant strains. The drug-sensitizing effect of overexpressed loss-of-function pdr3 mutant alleles correlated with their ability to suppress PDR5 transcription and rhodamine 6G accumulation in transformants of the wild-type and Deltapdr1 mutant strains. These results demonstrate that amino acid residue Asp853 is essential for Pdr3p function, and indicate that specific loss-of-function pdr3 mutations can convert the Pdr3p transcription activator to a multicopy suppressor of multidrug resistance.