Multidrug Resistance: From yeast to man ( II). The molecular basis for pleiotropic drug resistance development in yeast and its relation to multidrug resistance in mammalian cells

Multidrug resistance (MDR) is defined as cross-resistance of both tumor and tissue culture cells to a wide variety of structurally and functionally unrelated cytotoxic drugs. Overexpression of the plasma membrane-bound P- glyoprotein (Pgp) and its functional homologue MRP (multidrug resistance associated protein), both of which belong to a ubiquitous superfamily of so-called ABC (ATP binding cassette) transporters, are considered principle causes of MDR development. In lower eukaryotes such as the yeast Saccharomyces cervisiae, numerous homologues of mammalian ABC drug efflux pumps have been discovered. Thus, overexpression of yeast ABC multidrug transporters strongly resembles Pgp- and MRP-mediated MDR in mammalian cells, since it results in pleiotropic drug resistance (PDR) to many structurally and functionally unrelated cytotoxic compounds. The overall objectives and specific aims of this grant proposal are centered around our attempts to (i) understand the molecular function mechanisms of selected yeast ABC transporters; (ii) to dissect the transciptional control circuits that regulate expression of yeast ABC transporters of the PDR network, and (iii) to use yeast as a model system for the structure-function analysis of mammalian P-glycoproteins such as human Mdr1. Further important goals include to unravel the normal physiological function(s) and cellular substrates of yeast ABC proteins otherwise involved in PDR development. Moreover, we want to dissect the function mechanism of the human Mdr1 drug pump implicated in MDR during cancer chemotherapy. Along this line, we are using yeast as expression host for the isolation of "tailor-made" MDR1 transports with limited drug transport specificities. Such Mdr1 gain- of-function mutants could be very useful as chemoprotective genes in vivo. Taken together, the specific aims and objectives of this proposal are: 1. to characterize selected members of the yeast ABC transporter family, including Pdr10. Pdr15, Pdr12 and Pdr16, emphasizing their biosynthesis, subcellular localization, regulation of expression, post-translational modifications and intracellular trafficking. 2. To study the biosynthesis, post-translational modifications and nuclear trafficking, proteolytic turnover and the functional interaction of Pdr1/Pdr3 and Yap1, and to identify novel ABC target genes of Pdr1/Pdr3. 3. To establish a novel RT-PCR-based assay with ABC gene-specific primers to identify physiological and adverse conditions under which individual ABC transporters are expressed; to identify novel transcription factors interacting with Pdr1/Pdr3 to selectively regulate certain yeast ABC efflux pumps under normal growth conditions and in response to stress. 4. To develop a genetic screen to identify specific inhibitors of efflux pumps such as human Mdr1, and to isolate novel Mdr1 transport mutants with limited and altered drug substrate specificity by phenotypic screening in yeast.