ANALYSIS OF THE PROCESSING OF PLASMODIUM-FALCIPARUM RHOPTRY-ASSOCIATED PROTEIN-1 AND LOCALIZATION OF PR86 TO SCHIZONT RHOPTRIES AND P67 TO FREE MEROZOITES

The processing and localization of Plasmodium falciparum rhoptry-assoc iated protein 1 (RAP-1) products were examined using polyclonal and mo noclonal antibodies raised to a recombinant protein containing residue s 1-294 of RAP-1. Immunoblot and epitope mapping results with antibodi es that selectively bound epitopes in the RAP-1 products Pr86, p82, an d p67 showed that p82 and p67 are formed from Pr86 by progressive remo val of epitopes from the amino-terminus of the RAP-1 coding sequence. The capacity of Pr86 to form complexes was revealed after size fractio nation of parasite proteins radiolabeled in the presence of brefeldin A to prevent processing of Pr86. Fractions containing complexed Pr86 a lso contained the RAP-2 product p39 and the RAP-3 product p37, suggest ing that Pr86, p39 and p37 may form complexes similar to complexes pre viously reported for p82 and p67 with p39 or p37. Immunofluorescence l ocalization and immunoblot studies revealed that Pr86 is present in th e rhoptries, but only transiently, and that it is not detected in segm enting schizonts or extracellular merozoites. p67 and p82, on the othe r hand, were shown to be major RAP-1 components in purified merozoites . Neither p67 nor p82 were relocalized from the intracellular rhoptrie s to the merozoite surface under conditions that promoted relocalizati on of the rhoptry protein PF83/apical membrane antigen 1. These result s suggest that processing of Pr86 begins after Pr86 complexes are tran sported to the forming rhoptries and that two site-selective processin g reactions occur in the rhoptries: a rapid cleavage of Pr86 to p82 an d a delayed cleavage of p82 to p67. Since p67 is missing from ring-sta ge parasites (Howard et al., Am J Trop Med Hyg, 1984;33:1055-59), the present results indicate there is a narrow time during which p67 may p lay a role in merozoite invasion of erythrocytes. (C) 1998 Elsevier Sc ience B.V. All rights reserved.