Saccharomyces cerevisiae cells possess the ability to simultaneously acquire resistance to an array of drugs with different cytotoxic activities. The genes involved in this acquisition are referred to as pleiotropic drug resistant (PDR) (14,20,22,25,30,49

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporterencoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1-and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K m(ATP) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.Multidrug transporters, including members of the ATPbinding cassette (ABC) 2 family, show unusual flexibility toward their substrate cargo. They efflux structurally diverse xenobiotic compounds and confer broad-spectrum hyperresistance when they are overexpressed, a property that impedes chemotherapeutic treatment of pathogens and cancer. Several fungi, including the clinically relevant human pathogenic species Candida albicans and Cryptococcus neoformans, contain major multidrug transporters that are close homologues to the Saccharomyces cerevisiae transporter Pdr5 (1). It is well established that many clinical isolates of C. albicans overproduce the Pdr5 homologue Cdr1 (2). Multidrug-resistant fungi are an increasing problem in the treatment of immunocompromised patients with AIDS and cancer (3).The Pdr5 subfamily of multidrug transporters shows significant differences in molecular architecture from their mammalian counterparts such as P-glycoprotein (P-gp) or Mrp1. The nucleotide-binding domains (NBDs) of these important fungal transporters precede the transmembrane domains (TMDs) and thus their orientation is the reverse of P-gps and Mrp1s. Furthermore, the Walker A, B, and signature motifs are degenerate when compared with nonfungal ABC counterparts. For example, a cysteine replaces a lysine in Walker A of the N-...

The network of genes which mediates multiple drug resistance in yeast includes, among others, the PDR1 gene, which encodes a putative regulator of gene expression, and PDR5, a locus whose amplification leads to resistance. We demonstrate that disruption of PDR5 causes marked hypersensitivity not only to cycloheximide but also to sulphometuron methyl and the mitochondrial inhibitors chloramphenicol, lincomycin, erythromycin and antimycin. Genetic analysis of double mutants containing an insertion in PDR5 (pdr5:Tn5), which renders cells hypersensitive to cycloheximide, and a pdr1 mutation, which confers resistance to this inhibitor, indicates that the expression of resistance requires a functional PDR5 gene. The same interdependency is observed for chloramphenicol, but not for oligomycin, lincomycin, erythromycin or sulphometuron methyl. Northern analysis of PDR1 and PDR5 transcripts reveals that the 5.2 kbp PDR5 transcript is overexpressed in pdr1 (resistant) mutants, but underexpressed in a disruption of PDR1. These observations provide strong experimental support for our former proposal that the PDR5 gene is a target for regulation by the PDR1 gene product.