Protein trafficking in Plasmodium falciparum

Many proteins are secreted during the intraerythrocytic stage of the malaria parasite Plasmodium falciparum lifecycle. Yet protein trafficking and secretion remains poorly understood in this organism, in part due to the atypical nature of its secretory process. For instance, there is no detectable Golgi stack and little or no N-glycosylation. In addition, homologues of Golgi markers are localized to unusual sites and cis and trans Golgi markers are segregated. In light of this remarkable cell biology, three hypotheses have been proposed for protein trafficking. The first suggests the complete absence of a Golgi complex; the second, a rudimentary Golgi with unstacked cisternae; and the last, a novel compartment for protein secretion beyond the parasite membrane.; To gain insight into the trafficking machinery of this parasite, Plasmodium ADP-Ribosylation Factor (plARF), a cis-Golgi marker, and a related ADP-ribosylation Factor-Like protein (plARL) have been cloned and characterized. Both genes appear to be single copy and are highly expressed in the schizont stage. Recombinant plARF is capable of binding GTP. To localize plARF and plARL, antibodies were developed but none recognized the native proteins. Transient and stable transfection studies were conducted to localize plARF and to understand its function in both a heterologous system and the Plasmodium context. Transient transfection experiments in Plasmodium showed that plARF is most likely a cytoplasmic protein with no single compartment association. Disruption of the GTP binding domain of plARF resulted in two phenotypes. The T31N mutant, a constitutively inactive plARF, showed no difference from the cytoplasmic distribution of the wild type. The Q71L mutant, a constitutively active GTP bound plARF, occasionally showed a more punctate localization within the parasite cytoplasm.; In the second part of the thesis, an unusual Histidine Rich Protein II (HRPII) was studied. Biochemical studies revealed that HRPII is secreted de novo into the extracellular milieu in all three intracellular stages within 10 minutes of synthesis. Furthermore, HRPII possesses a putative leader peptide which is not cleaved during synthesis, and the protein is neither N- nor O-glycosylated. Additionally, HRPII is insensitive to BFA and oryzalin. HRPII may have isoforms which are not phosphorylated. Erythrocytes resealing experiments suggest that HRPII may require a parasite component to be secreted. The HRPII study suggests that there is a rapid transport of secreted proteins which do not require glycosylation for sorting, are BFA insensitive, and do not require microtubules. This novel type of secretion most likely requires as of yet unknown resident components and exists in parallel with a more classical type protein trafficking for parasite resident proteins and secreted proteins which are BFA sensitive.