After a decade of painstaking work, Wellems and colleagues recently identified the gene that appears to be responsible for conferring chloroquine resistance (CQR) to the malarial parasite Plasmodium falciparum. This gene, pfcrt, encodes what appears to be a polytopic integral membrane protein with 10 putative transmembraneous helices. Pfcrt protein is localized to the digestive vacuolar membrane of the intraerythrocytic parasite. Several distinct patterns of mutations in Pfcrt confer CQR, but the function of mutant proteins is not yet known, nor is the endogeneous physiology catalyzed by the wild type protein understood. Deciphering Pfcrt function at the molecular level is central to defining CQR, to anticipating additional drug resistance pathways, and for devising improved therapy. Analysis of molecular level function in vivo is extraordinarily challenging since the protein is localized to an endo membrane of an intracellular parasite. Thus, over the past several years we have endeavored to engineer high level overexpression of malarial integral membrane proteins in yeast, and have recently reported the high level overexpression of mutant and wild type Pfcrt proteins in both S. cerevisiae and P. pastoris. To our knowledge, this represents the first successful overexpression of malarial polytopic integral membrane proteins in a heterologous system. Based upon this success, we now propose a detailed vesicle and proteoliposome based study to decipher the function of Pfcrt. We also propose to extend our success with this approach to analysis of another key membrane protein involved in antimalarial drug resistance, Pf mdr 1. The data to be generated are vital for understanding drug resistance in this and other systems, and will provide a template for design of additional in depth studies of apicomplexan membrane transport proteins.
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