Functional and biochemical analysis of the yeast multidrug resistance transporters Qdr2 and Aqr1

Principal Investigator: Miguel Cacho Teixeira
Contract:
PTDC/BIA-MIC/72577/2006 

 

The therapeutic potential of drugs is seriously limited by the manifestation of cellular drug resistance. Among the mechanisms underlying the multidrug resistance (MDR) phenomenon in various organisms, is the action of transmembrane transport proteins that presumably catalyse the active expulsion of structurally and functionally unrelated cytotoxic compounds out of the cell or their intracellular partitioning.
On the basis of the complete genome sequence of Saccharomyces cerevisiae, it was found at least 23 genes that have largely escaped characterization by classical approaches, encode proteins belonging to the major facilitator superfamily (MFS), required for resistance to multiple drugs, the alleged drug:H+ antiporters. With the participation in the European network EUROFAN for the functional analysis of novel yeast genes discovered by systematic sequencing, our laboratory has started, in 1996, the functional analysis of this poorly characterized family of proteins. We proved the involvement of seven of these transporters in yeast resistance to fungicides, weak  acids food preservatives, herbicides, antitumour agents, antimalarial drugs, and other growth inhibitory compounds that are usually not present in the yeast cell natural environment. This observation appears to suggest that the physiological function of these transporters may have nothing to do with broad chemoprotection but rather with the transport of a still unidentified specific physiological substrate and that they may transport drugs or other compounds only fortuitously or opportunistically. We also localized these transporters at the plasma membrane and studied the transcription regulation of the encoding genes.
The mechanisms behind the apparent promiscuity of the multidrug efflux pumps in different organisms and the presence of a multitude of different efflux pumps that may provide protection against compounds that are not usually present in their natural environment remains elusive and a topic of debate. Recently, we obtained evidence supporting the notion that the expression of Qdr2p, a member of the MFS-MDR family of transporters, plays an important role in the maintenance of physiological levels of K+ in the cell. We also found that this biological function becomes crucial under environmental conditions leading to K+-limited growth, either due to the presence of limiting levels of K+ in the growth medium or to the presence of quinidine. This
antimalarial and antiarrhythmic drug increases the requirement for potassium in yeast cells by decreasing K+ uptake rate and K+ accumulation, especially in the deltaqdr2 mutant. Our data are consistent with the hypothesis that the putative drug:H+ antiporter Qdr2p may also couple K+ movement with substrate(s) export, presumably with quinidine These observations led us to consider the function of this protein as an alternative K+ transporter in yeast and to examine the other homologous MDR-MFS proteins. From these very preliminary results, Aqr1p emerged as a good candidate since the deltaaqr1 mutant exhibits a deficient growth under low potassium conditions, when compared to the wild-type strain. In S. cerevisiae, the Trk1p and Trk2p are the
plasma membrane proteins responsible for the high or moderate affinity potassium uptake. However, the viability of deltatrk1deltatrk2 cells has indicated the existence of additional forms to transport potassium. Although the nature of this transport remains unclear, it is usually accepted that several non-specific transporters may be involved in the low-affinity transport of K+ observed in the deltatrk1deltatrk2 mutant. The present project will continue our recent studies on the biochemical and physiological function of Qdr2 and Aqr1 MFS-MDR transporters. In particular, we intent to elucidate the involvement of Qdr2p and Aqr1p in potassium homeostasis and the link between this hypothesized biological role and their involvement in conferring resistance to a number of drugs and other chemical stresses in yeast. These studies will also
involve the analysis of the traffic of the proteins in response to biologically relevant conditions and the postulated regulation of protein activity by post-translation events. The role of covalent modifications of Qdr1p and Aqr1p by phosphorylation and ubiquitylation will be examined. These studies, using the fundamental model eukaryote S. cerevisiae, will accelerate the understanding of the molecular mechanisms governing multidrug resistance, currently one of the most exciting and puzzling topics in cell biology, with important implications for human health, and also in Biotechnology, Agriculture and in the Environment