Analysis of the antimalarial drug resistance protein Pfcrt expressed in yeast.
Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000) Mol. Cell 6, 861-871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiae and Pichia pastoris yeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild type versus CQR-conferring mutant Pfcrt function, which may include effects on H(+) transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000) Mol. Biochem. Parasitol. 110, 107-124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of important P. falciparum transport proteins and membrane associated vaccine candidates.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D000962:Antimalarials / Q000494:pharmacology*; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D003062:Codon; D004351:Drug Resistance / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D008565:Membrane Proteins / Q000494:pharmacology*; D026901:Membrane Transport Proteins; D008957:Models, Genetic; D009154:Mutation; D010843:Pichia / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011424:Propionibacterium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013997:Time Factors
D000962:Antimalarials; D003062:Codon; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001710:Biotin; D002738:Chloroquine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases