Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum.
The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4-5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems.
Klonis
Nectarios
N
Department of Biochemistry, La Trobe University, Melbourne, VIC 3086, Australia.
Tan
Olivia
O
Jackson
Katherine
K
Goldberg
Daniel
D
Klemba
Michael
M
Tilley
Leann
L
D000818:Animals; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014617:Vacuoles / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
D006454:Hemoglobins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
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