Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infected erythrocytes.
Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step.
Burghaus
P A
PA
Philipps-Universität Marburg, FB Biologie/Zoologie, Abt. Parasitologie, D-35032 Marburg, Germany. burghaus@mailer.uni-marburg.de
Lingelbach
K
K
D000818:Animals; D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D004912:Erythrocytes / Q000378:metabolism* / Q000469:parasitology; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D010963:Plasmodium falciparum / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases