Studies with Novel Pdr5p Substrates Demonstrate a Strong Size Dependence for Xenobiotic Efflux

Abstract

The yeast (Saccharomyces cerevisiae) multidrug transporter Pdr5p effluxes a broad range of substrates that are variable in structure and mode of action. Previous work suggested that molecular size and ionization could be important parameters. In this study, we compared the relative sensitivity of isogenic PDR5 and pdr5 strains toward putative substrates that are similar in chemical structure. Three series were used: imidazolecontaining compounds, trialkyltin chlorides, and tetraalkyltin compounds. We demonstrate that the Pdr5p transporter is capable of mediating transport of substrates that neither ionize nor have electron pair donors and that are much simpler in structure than those transported by the human MDR1-encoded P-glycoprotein. Furthermore, the size of the substrate is critical and independent of any requirement for hydrophobicity. Substrates have surface volumes greater than 90 Å 3 with an optimum response at ϳ200 -225 Å 3 as determined by molecular modeling. Assays measuring the efflux from cells of [ 3 H]chloramphenicol and [ 3 H]tritylimidazole were used. A concentration-dependent inhibition of chloramphenicol transport was observed with imidazole derivatives but not with either the organotin compounds or the antitumor agent doxorubicin. In contrast, several of the organotin compounds were potent inhibitors of tritylimidazole efflux, but the Pdr5p substrate tetrapropyltin was ineffective in both assays. This argues for the existence of at least three substrate-binding sites on Pdr5p that differ in behavior from those of the mammalian P-glycoprotein. Evidence also indicates that some substrates are capable of interacting at more than one site. The surprising observation that Pdr5p mediates resistance to tetraalkyltins suggests that one of the sites might use only hydrophobic interactions to bind substrates.In Saccharomyces cerevisiae, broad spectrum resistance to structurally and mechanistically diverse inhibitors is mediated by several members of the ATP-binding cassette (ABC) 1 superfamily of transporters, including Pdr5p, a 160-kDa ABC transporter found in the plasma membrane. Pdr5p is essential for basal levels of resistance to a broad array of substrates, including antifungal and antitumor agents, hormones, and ionophores (1-4). Compared with isogenic wild-type strains, pdr5 loss-of-function mutants exhibit hypersensitivity because of their inability to efflux inhibitors (3, 5).Although an important study (3) provided strong indirect evidence for the existence of more than one substrate-binding site, the chemical basis of Pdr5p substrate specificity is unclear. One reason is that many of the compounds analyzed to date are complex in chemical structure. As an alternative approach, we searched for a series of relatively simple compounds that were systematically related to each other in structure but varied in their ability to serve as Pdr5p substrates. By comparing several series, we hoped to identify shared properties that are used in the Pdr5p-substrate interaction. In the initial study, we comp...

ELM1 Is Required for Multidrug Resistance in Saccharomyces cerevisiae

“…Doxorubicin is a known substrate of many ABC transporters (Eytan 2005) and deletion of PDR5 leads to hypersensitivity to doxorubicin and many other drugs (Rogers et al 2001;Golin et al 2003). Transport activities for doxorubicin were determined by analytically detecting the fluorescence of the drug in acid soluble supernatants extracted from cells and separated by HPLC ( Figure 2A).…”

Functional Characterization of Candida albicans ABC Transporter Cdr1p

“…The expression of ⌬Cdr1p led to decreased resistance to nystatin, while resistance to miconazole was retained. Recently, by using a variety of novel sub- (14) have reported that this ABC drug transporter from S. cerevisiae has at least three drugbinding sites and suggested that some substrates might even interact at more than one site. Taken together, our results suggest that Cdr1p harbors different drug-binding sites, which recognize structurally dissimilar drugs.…”

The Transmission Interface of the Saccharomyces cerevisiae Multidrug Transporter Pdr5: Val-656 Located in Intracellular Loop 2 Plays a Major Role in Drug Resistance

“…Overexpression leads to hyperresistance for all known substrates (13)(14)(15). This efflux pump has a basic structure found in full-length members of this superfamily.…”