Rapid transport of the acidic phosphoproteins of Plasmodium berghei and P. chabaudi from the intraerythrocytic parasite to the host membrane using a miniaturized fractionation procedure

Abstract

A miniaturized procedure for the separation of the host erythrocyte membrane from malarial parasites based on saponin lysis and density-gradient centrifugation with Percoll is described. The procedure requires only 20-35 microliters packed infected erythrocytes, is simple to perform, needs no sophisticated equipment, and can be completed in less than 2 h. Analysis of the isolated erythrocyte membranes and parasites using marker enzymes and electron microscopy revealed that both the purity and the yield of these fractions were relatively high. Erythrocyte membrane proteins, including spectrin, ankyrin, and band 4.1, were not found on the parasitophorous vacuolar membrane, which remained associated with some but not all of the isolated parasites. Application of this method to pulse-chase experiments indicated that the acidic phosphoproteins of Plasmodium berghei and P. chabaudi were rapidly transported from the parasite to the erythrocyte membrane immediately after their synthesis. The rapid export of these acidic phosphoproteins from the parasite distinguishes them from other proteins exported by the malarial parasite.