Localization of the Phosphoethanolamine Methyltransferase of the Human Malaria Parasite Plasmodium falciparum to the Golgi Apparatus

Abstract

Phosphatidylcholine is the most abundant phospholipid in the membranes of Plasmodium falciparum, the agent of severe human malaria. The synthesis of this phospholipid occurs via two routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Although various components of these pathways have been identified, their cellular locations remain unknown. We have previously reported the identification and characterization of the phosphoethanolamine methyltransferase, Pfpmt, of P. falciparum and shown that it plays a critical role in the synthesis of phosphatidylcholine via the SDPM pathway. Here we provide the first evidence that the transmethylation step of the SDPM pathway occurs in the parasite Golgi apparatus. We show that the level of Pfpmt protein in the infected erythrocyte is regulated in a stage-specific fashion, with high levels detected during the trophozoite stage at the peak of parasite membrane biogenesis. Confocal microscopy revealed that Pfpmt is not cytoplasmic. Immunoelectron microscopy revealed that Pfpmt localizes to membrane structures that extend from the nuclear membrane but that it only partially co-localizes with the endoplasmic reticulum marker BiP. Using transgenic parasites expressing green fluorescent protein targeted to different cellular compartments, a complete co-localization was detected with Rab6, a marker of the Golgi apparatus. Together these studies provide the first evidence that the transmethylation step of the SDPM pathway of P. falciparum occurs in the Golgi apparatus and indicate an important role for this organelle in parasite membrane biogenesis.Malaria, the world's most important parasitic disease, is caused by intraerythrocytic protozoan parasites of the genus Plasmodium. More than 300 million clinical cases and more than 2 million deaths are reported each year with most deaths mainly caused by Plasmodium falciparum (1). Unlike other human pathogens that invade metabolically active host cells, P. falciparum invades mature erythrocytes that lack internal organelles and the metabolic pathways necessary for de novo lipid synthesis. During its intraerythrocytic life cycle, P. falciparum undergoes major metabolic and morphological changes and then divides asexually to produce up to 36 new daughter parasites (2). This rapid growth and multiplication requires active synthesis of new membranes and is fueled by lipid precursors derived from the host.Phosphatidylcholine is the major phospholipid in P. falciparum membranes, representing 50% of parasite phospholipids (for review, see Ref.3). Pharmacological studies demonstrated that inhibition of phosphatidylcholine biosynthesis is deleterious to parasite intraerythrocytic growth and multiplication, emphasizing the importance of the phospholipid metabolic pathways as possible targets for development of new antimalarial drugs (4 -10). Recent studies in P. falciparum identified two pathways of...

Disruption of the Plasmodium falciparum PfPMT Gene Results in a Complete Loss of Phosphatidylcholine Biosynthesis via the Serine-Decarboxylase-Phosphoethanolamine-Methyltransferase Pathway and Severe Growth and Survival Defects

“…As a control, the expression of the PfTPX1 gene was detected in both wild type and knock-out parasites. Analysis of the loss of PfPMT expression by immunoblotting using affinitypurified anti-PfPMT antibodies (17) showed that, although a 30 kDa PfPMT band was detectable in the wild type strain, there was no signal in the knock-out strain (Fig. 2E).…”

“…14 C]ethanolamine (top panels) or [ 14 C]-choline (bottom panels) and incubated for 12 h. The infected erythrocytes were washed twice in PBS, and lipids were extracted by the Folch method (17). The organic phase of the lipid extracts were resolved by twodimensional TLC, and signals were generated by autoradiography.…”

“…The soluble fraction was separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoblotting was done using affinity-purified antibodies against PfPMT and the P. falciparum translation elongation factor-1␣ (PfEF-1␣) as previously described (17).…”

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission

“…2C). To further validate the lack of mature gametocytes in pfpmtΔ cultures, immunofluorescence assays were performed using antibodies against PfPMT and the gametocyte-specific marker Pfg27 on culture samples from 3D7 and pfpmtΔ strains (11,12) (Fig. 2D).…”

Purification of Components of the Translation Elongation Factor Complex of Plasmodium falciparum by Tandem Affinity Purification

“…Immunoblot analyses were performed as previously described (24,26) on crude extracts of the wild-type and transgenic parasites as well as on fractions of the tandem affinity purifications using a monoclonal anti-ProtC antibody (Roche Diagnostics) as well as polyclonal anti-PfEF-1␤, anti-PfEF-1␣, and anti-Pfpmt antibodies (16,33). PAP (peroxidase-antiperoxidase soluble complex; Sigma) was used to detect the fusion proteins harboring …”