Substitution of Pro165 in Transmembrane 4 of PfCRT Abolishes Lysosome Acidification Function in Stably Transfected HEK-293F Cells

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is localised on the parasite digestive vacuole, an organelle that maintains an acidic lumen. Here we demonstrated the isolation of HEK-293F cells stably expressing wild type 3D7 and mutant Pfcrt alleles. Immuno-fluorescence staining of HEK-293F transfectants confirms the localization of Pfcrt alleles to the lysosomal vesicles. Moreover, cells expressing mut-PfCRTETSE showed greater lysosomal acidification as demonstrated by the dramatic increase in the accumulation of two weak bases, acridine orange and CytiPainter LysoOrange dyes. Furthermore, HEK-293 cells stably expressing mut-PfCRTETSE with a single substitution of proline 165 in transmembrane 4 completely inhibited the accumulation of weak bases. Taken together, our results demonstrate the role of Pro165 in PfCRT lysosomal acidification function in HEK-293F cells.