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.     

Complex interplay among regulators of drug resistance genes in Saccharomyces cerevisiae

The Gal4p family of yeast zinc cluster proteins comprises regulators of multidrug resistance genes. For example, Pdr1p and Pdr3p bind as homo- or heterodimers to pleiotropic drug response elements (PDREs) found in promoters of target genes. Other zinc cluster activators of multidrug resistance genes include Stb5p and Yrr1p. To better understand the interplay among these activators, we have performed native co-immunoprecipitation experiments using strains expressing tagged zinc cluster proteins from their natural chromosomal locations. Interestingly, Stb5p is found predominantly as a Pdr1p heterodimer and shows little homodimerization. No interactions of Stb5p with Pdr3p or Yrr1p could be detected in our assays. In contrast to Stb5p, Yrr1p is only detected as a homodimer. Similar results were obtained using glutathione S-transferase pull-down assays. Importantly, the purified DNA binding domains of Stb5p and Pdr1p bound to a PDRE as heterodimers in vitro. These results suggest that the DNA binding domains of Pdr1p and Stb5p are sufficient for heterodimerization. Our data demonstrate a complex interplay among these activators and suggest that Pdr1p is a master drug regulator involved in recruiting other zinc cluster proteins to fine tune the regulation of multidrug resistance genes.     

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.