Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast.

Since the isolation of the pfcrt gene 10 years ago [1], much has been learned regarding the encoded PfCRT protein and its role in conferring chloroquine resistance (CQR) in P. falciparum malarial parasites [2, 3]. Geographically distinct patterns of amino acid substitutions in PfCRT are associated with a variety of different CQR phenotypes in P. falciparum isolates from around the globe. The different phenotypes are further characterized by cross resistance patterns to other drugs and variable contributions from additional allele inheritances, gene mutations or gene over-expression events that work in concert with PfCRT mutations to cause antimalarial multidrug resistance [4-6]. One additional gene that appears to be involved in multidrug resistance is pfmdr1, which encodes a human P- glycoprotein orthologue that is mutated and / or alternately expressed in many CQR isolates [7-9]. Further elucidation of the PfCRT molecular mechanism is essential for controlling current and newly evolving drug resistance phenomena, and for developing second tier drug therapies. As one example of the former, CQR has been found in the related malarial parasite P. vivax, similar to P. falciparum [10,11], albeit in a different geographically disposed pattern. However, unlike the case for PfCRT and P. falciparum, amino acid substitutions in the PvCRT orthologue are apparently not required for P. vivax CQR [10]. It would be helpful to translate concepts elucidated for P. falciparum CQR towards progress in understanding P. vivax CQR. Unfortunately, many approaches for studying CQR in P. vivax are inaccessible since there are no convenient laboratory – based methods available for culturing P. vivax. Heterologous expression of key P. vivax proteins, compared side – by – side with their P. falciparum orthologues, might provide one avenue for elucidating some aspects of P. vivax biology. For example, a model for why PvCRT mutations are apparently not related to P. vivax CQR could conceivably be generated from such experiments. Previously, we have shown that both wild type (“HB3” isoform) and mutant CQR associated (“Dd2” [S.E. Asian] and “7G8” [S. American]) isoforms bind CQ [12,13]. Surprisingly, affinity constants for equilibrium binding of 3H-CQ were found to be similar for HB3 and Dd2 PfCRT [12]. Subsequent use of a CQ photoaffinity analogue and mass spectrometry allowed us to map a single specific quinoline binding site for HB3 PfCRT [13]. The predicted site lies proximal to PfCRT residues that have been found to be mutated in CQR conferring isoforms. At low external doses of CQ, infected red blood cells harboring CQR parasites accumulate less CQ relative to CQS [14-16]. Also, CQ is known to concentrate within the parasite digestive vacuole (DV) [16,17], and subcellular PfCRT protein localization includes high levels within the DV membrane [1,18]. It has thus been proposed that PfCRT mediates CQ transport from the DV in some fashion, in order to lower CQ accumulation within the DV. As summarized elsewhere [2], there is considerable support for this overall model, yet a number of issues remain to be resolved. Two recent studies have provided direct evidence for PfCRT mediated CQ transport. One uses oocytes injected with pfcrt cDNA to measure slow, but heightened accumulation of 3H-CQ into the oocytes vs time [19] and the other uses purified PfCRT protein reconstituted into proteoliposomes (PLs) and a novel fluorescent CQ reporter to measure pH gradient (ΔpH) and electrical potential (ΔΨ) stimulated efflux from the PLs [20]. Some differences in the two studies are found, in particular, estimates of turnover (drug transported per PfCRT per second) are much slower for the oocyte experiments relative to PL, and the PL study reports transport by both HB3 and Dd2 PfCRT isoforms, whereas the oocyte study suggests that only Dd2 PfCRT is capable of drug transport (see [2] for a more detailed discussion). For these reasons and others, we have endeavored to devise additional convenient model systems for the study of PfCRT isoforms as well as PvCRT and other malarial transport proteins. Metabolic induction of transporters in S. cerevisiae growing under ambient conditions is in theory another such system, but high AT bias and unusual gene structure for some P. falciparum genes, as well as cellular targeting issues, often complicates application of these techniques. In this study, we have perfected pYES2 based galactose-inducible expression of PfCRT and PvCRT. We use this tool to further analyze questions related to PfCRT-mediated CQ transport, and to also test for putative CQ transport by the important PfCRT orthologue PvCRT.