Dataset Information

Biosynthesis, export and processing of a 45 kDa protein detected in membrane clefts of erythrocytes infected with Plasmodium falciparum.

ABSTRACT: During its asexual life cycle, the human malaria parasite Plasmodium falciparum exports numerous proteins beyond its surface to its host erythrocyte. We have studied the biosynthesis, processing and export of a 45 kDa parasite protein resident in membrane clefts in the erythrocyte cytoplasm. Our results indicate that this cleft protein is made as a single tightly membrane-bound 45 kDa polypeptide in ring- and trophozoite-infected erythrocytes (0-36 h in the life cycle). Using ring/trophozoite parasites released from erythrocytes, the 45 kDa protein is shown to be efficiently transported to the cell surface. This export is specifically blocked by the drug brefeldin A, and at 15 and 20 degrees C. These results indicate that transport blocks seen in the Golgi of mammalian cells are conserved in P. falciparum. Further, the newly synthesized 45 kDa protein passes through parasite Golgi compartments before its export to clefts in the erythrocyte. In mid-to-late-ring-infected erythrocytes, a fraction of the newly synthesized 45 kDa protein is processed to a second membrane-bound phosphorylated 47 kDa protein. The t1/2 of this processing step is about 4 h, suggesting that it occurs subsequent to protein export from the parasite. Evidence is presented that, in later trophozoite stages (24-36 h), the exported 45 and 47 kDa proteins are partially converted into soluble molecules in the intraerythrocytic space. Taken together, the results indicate that the lower eukaryote P. falciparum modulates a classical secretory pathway to support membrane export beyond its plasma membrane to clefts in the erythrocyte. Subsequent to export, phosphorylation and/or conversion into a soluble form may regulate the interactions of the 45 kDa protein with the clefts during parasite development.

SUBMITTER: Das A

PROVIDER: S-EPMC1137254 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundThe intra-erythrocytic development of the malaria parasite Plasmodium falciparum depends on the uptake of a number of essential nutrients from the host cell and blood plasma. It is widely recognized that the parasite imports low molecular weight solutes from the plasma and the consumption of these nutrients by P. falciparum has been extensively analysed. However, although it was already shown that the parasite also imports functional proteins from the vertebrate host, the internalization route through the different infected erythrocyte membranes has not yet been elucidated. In order to further understand the uptake mechanism, the study examined the trafficking of human plasminogen from the extracellular medium into P. falciparum-infected red blood cells.MethodsPlasmodium falciparum clone 3D7 was cultured in standard HEPES-buffered RPMI 1640 medium supplemented with 0.5% AlbuMAX. Exogenous human plasminogen was added to the P. falciparum culture and the uptake of this protein by the parasites was analysed by electron microscopy and Western blotting. Immunoprecipitation and mass spectrometry were performed to investigate possible protein interactions that may assist plasminogen import into infected erythrocytes. The effect of pharmacological inhibitors of different cellular physiological processes in plasminogen uptake was also tested.ResultsIt was observed that plasminogen was selectively internalized by P. falciparum-infected erythrocytes, with localization in plasma membrane erythrocyte and parasite's cytosol. The protein was not detected in parasitic food vacuole and haemoglobin-containing vesicles. Furthermore, in erythrocyte cytoplasm, plasminogen was associated with the parasite-derived membranous structures tubovesicular network (TVN) and Maurer's clefts. Several proteins were identified in immunoprecipitation assay and may be involved in the delivery of plasminogen across the P. falciparum multiple compartments.ConclusionThe findings here reported reveal new features regarding the acquisition of plasma proteins of the host by P. falciparum-infected erythrocytes, a mechanism that involves the exomembrane system, which is distinct from the haemoglobin uptake, clarifying a route that may be potentially targeted for inhibition studies.

Project description:In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (?-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ?ATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ?ATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ?ATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ?ATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration.

Dataset Information

Biosynthesis, export and processing of a 45 kDa protein detected in membrane clefts of erythrocytes infected with Plasmodium falciparum.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets