Choline induces transcriptional repression and proteasomal degradation of the malarial phosphoethanolamine methyltransferase.

During its intraerythrocytic life cycle, the malaria parasite Plasmodium falciparum undergoes dramatic metabolic and morphological changes and multiplies to produce up to 36 new daughter parasites. This rapid multiplication of the parasite requires an active synthesis of new membranes. The major component of these membranes, phosphatidylcholine, is synthesized via two metabolic 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. Here we provide evidence indicating that the activity of the SDPM pathway is regulated by the CDP-choline precursor, choline. We show that the phosphoethanolamine methyltransferase, Pfpmt, a critical enzyme in the SDPM pathway, is down-regulated at the transcriptional level as well as targeted for degradation by the proteasome in the presence of choline. Transcript analysis revealed that PfPMT transcription is repressed by choline in a dose-dependent manner. Immunoblotting, pulse-chase experiments, and immunoprecipitation studies demonstrated that Pfpmt degradation occurs not only in wild-type but also in transgenic parasites constitutively expressing Pfpmt. The proteasome inhibitor bortezomib inhibited choline-mediated Pfpmt degradation. These data provide the first evidence for metabolite-mediated transcriptional and proteasomal regulation in Plasmodium and will set the stage for the use of this system for conditional gene and protein expression in this organism. The most severe form of human malaria is caused by the protozoan parasite Plasmodium falciparum, causing over 2 million deaths annually (23). Within human erythrocytes, P. falciparum undergoes dramatic metabolic and morphological changes and multiplies to produce up to 36 new daughter parasites in 48 h (11). This rapid propagation of the parasite requires active synthesis of new membranes, among other components, and is fueled by lipid precursors such as choline, serine, and fatty acids derived from the host. Phosphatidylcholine accounts for the majority (about 50%) of cellular membrane phospholipids in P. falciparum (21). Recently, we demonstrated that the biosynthesis of phosphatidylcholine in P. falciparum occurs through two metabolic pathways, namely, the de novo CDP-choline pathway and the serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway (16). In the CDP-choline pathway, host choline is taken up by the parasite and phosphorylated to phosphocholine by a parasite choline kinase. The phosphocholine is subsequently converted to CDP-choline by a CDP-choline cytidylyl-transferase (PfCCT). The CDP-choline formed is then converted to phosphatidylcholine by a CDP-diacylglycerol-choline phosphotransferase (PfCEPT). On the other hand, the biosynthesis of phosphatidylcholine via the SDPM pathway utilizes host serine as a precursor (16). Serine, which is readily available in the parasite cytoplasm either via active degradation of host hemoglobin or transport from host plasma, is first decarboxylated into ethanolamine by a parasite serine decarboxylase enzyme and then phosphorylated to phosphoethanolamine by a parasite ethanolamine kinase. Phosphoethanolamine is methylated to phosphocholine by a three-step S-adenosyl-L-methioninedependent reaction, utilizing a parasite-specific enzyme, phosphoethanolamine methyltransferase (Pfpmt). The phosphocholine formed is then converted into phosphatidylcholine by the consecutive reactions of PfCCT and PfCEPT. Cell biological as well as genome-wide expression analyses suggest that Pfpmt is expressed throughout the intraerythrocytic cycle as well as during the gametocyte and sporozoite stages of the parasite (12, 16, 22). Pfpmt possesses a single catalytic domain solely responsible for the three-step S-adenosyl-L-methioninedependent methylation of phosphoethanolamine to form phosphocholine (16). Thus, P. falciparum uses two routes for the synthesis of the phosphatidylcholine precursor, phosphocholine (15). However, whether these two routes are coregulated remains unknown. Previous in vitro studies showed that Pfpmt activity is inhibited by phosphocholine (16), suggesting that exogenous choline, which is rapidly transported and phosphorylated by the parasite, could regulate Pfpmt and thus affect the contribution of the SDPM pathway to the biosynthesis of phosphatidylcholine. In this study we investigated the effects of exogenous choline on the expression of Pfpmt in wild-type and transgenic parasites constitutively expressing this enzyme. We show that exogenous choline induces Pfpmt down-regulation at the transcriptional level as well as via proteasome-mediated degradation.