Investigation of the Plasmodium falciparum food vacuole through inducible expression of the chloroquine resistance transporter (PfCRT).
Haemoglobin degradation during the erythrocytic life stages is the major function of the food vacuole (FV) of Plasmodium falciparum and the target of several anti-malarial drugs that interfere with this metabolic pathway, killing the parasite. Two multi-spanning food vacuole membrane proteins are known, the multidrug resistance protein 1 (PfMDR1) and Chloroquine Resistance Transporter (PfCRT). Both modulate resistance to drugs that act in the food vacuole. To investigate the formation and behaviour of the food vacuole membrane we have generated inducible GFP fusions of chloroquine sensitive and resistant forms of the PfCRT protein. The inducible expression system allowed us to follow newly-induced fusion proteins, and corroborated a previous report of a direct trafficking route from the ER/Golgi to the food vacuole membrane. These parasites also allowed the definition of a food vacuole compartment in ring stage parasites well before haemozoin crystals were apparent, as well as the elucidation of secondary PfCRT-labelled compartments adjacent to the food vacuole in late stage parasites. We demonstrated that in addition to previously demonstrated Brefeldin A sensitivity, the trafficking of PfCRT is disrupted by Dynasore, a non competitive inhibitor of dynamin-mediated vesicle formation. Chloroquine sensitivity was not altered in parasites over-expressing chloroquine resistant or sensitive forms of the PfCRT fused to GFP, suggesting that the PfCRT does not mediate chloroquine transport as a GFP fusion protein.
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.
Florian
F
Ehlgen
James S
JS
Pham
Tania
T
de Koning-Ward
Alan F
AF
Cowman
Stuart A
SA
Ralph
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002451:Cell Compartmentation; D002738:Chloroquine / Q000494:pharmacology; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
D017931:DNA Primers; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D020126:Brefeldin A; D002738:Chloroquine