Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infectederythrocytes

Plasmodium falciparum, a unicellular parasite that causes human malaria, in fects erythrocytes where it develops within a vacuole. The vacuolar membran e separates the parasite from the erythrocyte cytosol, Some secreted parasi te proteins remain inside the vacuole, and-others are transported across th e vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent prot ein and luciferase as reporters in transiently transfected parasites. Becau se of the higher sensitivity of the enzymatic assay, luciferase was quantif ied 3 days after transfection, whereas reliable detection of green fluoresc ent protein required prolonged drug selection. Luciferase was confined to t he parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-t erminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar conte nt and the erythrocyte cytosol. The results suggest that no specific protei n sequences are required for translocation across the vacuolar membrane. Th e high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involv e a rate-limiting step.