Investigating the Plasmodium falciparum chloroquine resistance transporter in a heterologous expression system
Liebelt, David A.
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Malaria is a devastating global problem, with an estimated 300 to 500 million cases occurring annually. Chloroquine (CQ has been used globally as a cheap and efficacious antimalarial drug. Development of chloroquine-resistant (CQR) strains of Plasmodium falciparum, the most widespread and dangerous causative agent of malaria, has rendered CQ all but useless in many regions. Mutations in a transmembrane protein, Plasmodium falciparum Chloroquine Resistance Transporter (Pf CRT), have been shown to be the determinant of CQ resistance. In the parasites, PfCRT is localized in the digestive vacuole (DV) membrane. PfCRT is a 424 amino acid protein. Based on sequence and hydropathy analysis it is predicted to have a cytoplasmic N- and C-terminus and ten membrane-spanning segments. Despite being responsible for CQ resistance and vital to the parasite, we lack information about PfCRT's structure, its endogenous function, and its role in CQ resistance.;In order to study the structure and function of PfCRT in a more experimentally tractable system than in intraerythrocytic parasites we have expressed PfCRT heterologously in mammalian HEK293T cells by transient transfection. We demonstrated that PfCRT localizes subcellularly and is structurally intact on the limiting membrane of the lysosomes. PfCRT was not seen on the plasma membrane by immunofluorescence microscopy. Furthermore, in order to study the functional consequences of PfCRT expression we have measured intralysosomal pH using pH-sensitive, fluorescent dyes loaded into lysosomes. Expression of chloroquine-sensitive (CQS) PfCRT decreased lysosomal pH relative to transfected control cells, and CQR PfCRT decreased intralysosomal pH relative to CQS PfCRT. Furthermore, CQS PfCRT with a K76T mutation acidified lysosomes just as CQR PfCRT did. CQR PfCRT with a T76K mutation acidified lysosomes to a similar or lesser extent than CQS PfCRT. These results show an effect of PfCRT expression in a heterologous system, and confirm that the residue at position 76 (which is critical for CQ resistance) is important for the lysosomal acidification by CQR PfCRT. To further investigate effects of PfCRT on lysosomal function we used calcium imaging to show that PfCRT does not cause a significant calcium leak from lysosomes.;We also used this heterologous expression system in attempts to (1) identify lysosomal targeting signals in PfCRT and (2) map the membrane topology of PfCRT. We created several different mutant and deletion constructs to disrupt the known lysosomal targeting signals in PfCRT, however all of these constructs retained lysosomal targeting. In our attempts to disrupt localization of PfCRT, we discovered that a yellow fluorescent protein (YFP) fused to the N-terminus of PfCRT caused PfCRT to express on the plasma membrane. We attempted to use this construct to study the transmembrane topology of PfCRT but experiments to date suggest that the topology of this N-terminal YFP-fusion construct is significantly different than the topology of PfCRT expressed in the lysosomal membrane. The mechanism by which the N-terminal YFP fusion alters the lysosomal sorting and transmembrane topology of PfCRT remains unknown.
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