BIOGENESIS OF RHOPTRY ORGANELLES IN PLASMODIUM-FALCIPARUM

Biogenesis of the rhoptry organelle of Plasmodium falciparum was studied by examining the synthesis and assembly of rhoptry proteins at different stages of intraerythrocytic development. Rhoptry proteins examined in this study were those of the high molecular weight complex of 140/130/1 10 kDa and referred to as Rhop-H1,2,3 and the low molecular weight complex of 80 and 42 kDa referred to as Rhop-L1,2. Co-ordinate, stage-specific expression of three proteins, Rhop-H3, Rhop-LI and Rhop-L2, was observed; maximum levels of mRNA at the 8 nucleus stage correlated with the onset of protein synthesis. In contrast, mRNA levels for DNA polymerase-alpha, a marker for DNA replication during schizogony, was maximum just prior to the onset of the first nuclear division, indicating that rhoptry biogenesis is not co-ordinate with nuclear division. The assembly of newly synthesized rhoptry proteins was followed by subcellular fractionation of schizonts at different stages of development. At the four-nucleus stage a vesicle could be isolated by sucrose gradient fractionation which had a peak density of 1.12 g ml-1 and contained only Rhop-H2 and Rhop-H3 proteins. This vesicle could represent an intermediate or pre-rhoptry compartment. At the 8-nucleus stage, the Rhop-L1 protein was also detected in a vesicle of low density. At the 16-nucleus stage, the proteins were present in vesicles having a significantly greater density in sucrose, 1. 16 g ml-1, similar to that of the mature organelle. The study suggested that the rhoptry proteins first accumulate in a low density vesicle and that assembly into this compartment is staggered. Immunoelectronmicroscopy studies indicated that the Rhop-H3 protein is first present in small granular compartments that becomes more electron dense and enlarges due to the stage-dependent incorporation of proteins.