The ABC transporter Pdr5p mediates the efflux of nonsteroidal ecdysone agonists in Saccharomyces cerevisiae.

We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH-5992) is actively excluded by resistant cells of insects. To identify the transporter that could be involved in the efflux of RH-5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter-deletion mutants of yeast Saccharomyces cerevisiae. PDR5 (pleiotropic drug resistance 5) deletion mutants (Deltapdr5 and Deltapdr5Deltasnq2) retained significantly higher levels of 14C-radiolabeled RH-5992 within the cells when compared to wild-type strain or single deletion mutants of SNQ2 (Deltasnq2) and YCF1 (Deltaycf1). Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of [14C]RH-5992 from these cells as efficiently as the wild-type cells. These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of [14C]RH-5992 in yeast. This exclusion was temperature-dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process. The mutants with the PDR5 deletion can also selectively accumulate [14C]RH-0345 and [14C]RH-2485, but not [14C]RH-5849, indicating that these three compounds share the same transporter Pdr5p for efflux.     

Casein kinase I-dependent phosphorylation and stability of the yeast multidrug transporter Pdr5p

The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane of Saccharomyces cerevisiae. Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells. Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residues in vitro. Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile. We showed Pdr5p to be ubiquitylated when overexpressed. However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism. Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface. In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation.     

The yeast Pdr15p ATP-binding cassette (ABC) protein is a general stress response factor implicated in cellular detoxification

ATP-binding cassette (ABC) transporters play important roles in drug efflux, but some may also function in cellular detoxification. The Pdr15p ABC protein is the closest homologue of the multidrug efflux transporter Pdr5p, which mediates pleiotropic drug resistance to hundreds of unrelated compounds. In this study, we show that the plasma membrane protein Pdr15p displays limited drug transport capacity, mediating chloramphenicol and detergent tolerance. Interestingly, Pdr15p becomes most abundant when cells exit the exponential growth phase, whereas its closest homologue, Pdr5p, disappears after exponential growth. Furthermore, in contrast to Pdr5p, Pdr15p is strongly induced by various stress conditions including heat shock, low pH, weak acids, or high osmolarity. PDR15 induction bypasses the Pdr1p/Pdr3p regulators but requires the general stress regulator Msn2p, which directly decorates the stress response elements in the PDR15 promoter. Remarkably, however, Pdr15p induction bypasses upstream components of the high osmolarity glycerol (HOG) pathway including the Hog1p and Pbs2p kinases as well as the dedicated HOG cell surface sensors. Our data provide evidence for a novel upstream branch of the general stress response pathway activating Msn2p. In addition, the results demonstrate a cross-talk between stress response and the pleiotropic drug resistance network.     

A new function of isonitrile as an inhibitor of the Pdr5p multidrug ABC transporter in Saccharomyces cerevisiae

Pdr5p in Saccharomyces cerevisiae is a functional homologue of mammalian P-glycoprotein implicated in multidrug resistance (MDR). In order to obtain useful inhibitors to overcome MDR in clinical tumors, screening of Pdr5p inhibitors has been carried out. We isolated a fungal strain producing Pdr5p inhibitors using our original assay system, and it was classified as Trichoderma sp. P24-3. The purified inhibitor was identified as isonitrile, 3-(3'-isocyano-cyclopent-2'-enylidene)-propionic acid, a compound whose carboxyl residue is essential for the inhibitory activity. A non-toxic concentration of the isonitrile (41.5 microg/ml, 255 microM) inhibited Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. In addition, addition of the isonitrile led to accumulation of rhodamine 6G, a substrate of Pdr5p, in the Pdr5p-overexpressing cells. The inhibitory profiles of the isonitrile against S1360 mutants (S1360A and S1360F) of Pdr5p were different from those of FK506 and enniatin. The isonitrile did not influence PDR5 gene expression and the amount of Pdr5 protein, nor did it inhibit the function of Snq2p, a homologue of Pdr5p. Interestingly, the isonitrile inhibited the function of Cdr1p and Cdr2p, Pdr5p homologues in pathogenic yeast Candida albicans. Thus, it was found that the isonitrile shows a different inhibitory spectrum from that of FK506 and enniatin as a potent inhibitor for Pdr5p, Cdr1p, and Cdr2p.     

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily.