Characterization of a Membrane-associated Rhoptry Protein of Plasmodium falciparum

Ross L. Coppel,Hisashi Fujioka,Dirk Truman,Agnieszka E. Topolska,Angela Lidgett

doi:10.1074/jbc.m307859200

Abstract

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.

Highlights

Plasmodium falciparum malaria is one of the most important infectious diseases of humans, accounting for ϳ2 million deaths each year
We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptryassociated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion
We present a characterization of the protein encoded by this gene, designated the rhoptryassociated membrane antigen (RAMA), and show that it encodes an unusual rhoptry protein that is made early in parasite

Summary

Introduction

Plasmodium falciparum malaria is one of the most important infectious diseases of humans, accounting for ϳ2 million deaths each year. Rhoptries of Plasmodium parasites are paired club-shaped organelles located at the apical end of merozoites, the form of the parasite that invades RBCs. Following the attachment of merozoites to the RBC surface, rhoptries discharge their contents onto the RBC membrane [1]. Most of our knowledge is based on microscopic examinations, which suggest that rhoptry biogenesis follows the secretory pathway route, with rhoptry organelles being formed by sequential fusion of post-Golgi vesicles [2, 3], why particular vesicles are selected is unclear. Rhoptry contents include both protein and lipid components, which assemble to form membrane-like structures. RAP1 is involved in transport of RAP2 to the rhoptry, but neither RAP1 nor RAP2 are required for invasion [12], whereas the gp rhoptry protein is a serine protease that cleaves RBC membrane components band 3 and glycophorin A after being discharged from rhoptries [13]

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