Most of the phosphatidylethanolamine (PE) in mammalian cellsis synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDPethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality. Phosphatidylethanolamine (PE)4 is an abundant phospholipid in membranes of organisms ranging from bacteria to mammals. Mammalian cells utilize two major pathways for PE biosynthesis, the CDP-ethanolamine pathway (1) and the phosphatidylserine decarboxylation pathway (2). Most of the ethanolamine that is incorporated into PE is derived from the diet but some is generated by sphingosine-1-phosphate lyase (3). In rat liver/hepatocytes (4, 5) and hamster heart (6) the majority of PE has been reported to originate from the CDP-ethanolamine pathway. In contrast, in cultured Chinese hamster ovary cells (7-9) and baby hamster kidney cells (10), the decarboxylation of phosphatidylserine (PS) produces more than 80% of PE, even when the culture medium is supplemented with ethanolamine, an obligatory substrate of the CDP-ethanolamine pathway. Many types of mammalian cells grow and divide normally when cultured in the absence of ethanolamine, suggesting that PE production from the CDP-ethanolamine pathway might not be not essential for cell growth (10), although it is likely that the relative importance of the two pathways of PE synthesis depends on the type of cell and tissue. In Escherichia coli, in which PE comprises ϳ75% of total phospholipids, all PE is derived from PS decarboxylase. Interestingly, E. coli that have been genetically manipulated to reduce the PE content to 0.007% of total phospholipids are viable when supplemented with divalent cations such as Mg 2ϩ and Ca 2ϩ (11, 12). The two major pathways for PE synthesis in mammalian cells operate in different subcellular compartments. The final reaction of the CDPethanolamine pathway, catalyzed by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, occurs primarily on endoplasmic reticulum (ER) and nuclear membranes (13, 14), whereas PS decarboxylase activity is restricted to the outer surface of mitochondrial inner membranes (15, 16). Thus, the potential exists for compartmentalization of PE pools originating from these two spatially segregated pathways. Indeed, in Chinese hamster ovary cells and in yeast, the majority of mitochondrial PE is synthesized within mitochondria by PS decar...

The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranes in vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by ϳ45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes.During assembly of very low density lipoproteins (VLDLs) 1 in the liver, triacylglycerol (TG) is concentrated within the hydrophobic core of apolipoprotein (apo) B-containing lipoprotein particles. A microsomal lumenal protein, the microsomal triacylglycerol transfer protein (MTP), has been implicated in the acquisition of TG by nascent apoB for assembly and secretion of VLDLs (reviewed in Refs. 1-4). Individuals with the rare inherited disease abetalipoproteinemia have a defect in the MTP gene and lack detectable MTP protein and MTP lipid transfer activity (5). Despite a normal apoB gene, plasma apoB is barely detectable in these patients. MTP has the ability to transfer TG and other lipids, including cholesteryl esters, diacylglycerols, and phospholipids, between membranes in vitro (6) and has been proposed to transfer TG to nascent apoBcontaining lipoproteins in vivo. This idea is consistent with immunoprecipitation studies showing that apoB and MTP interact physically at early stages of VLDL assembly (7-9). MTP is expressed primarily in the liver and intestine as a soluble heterodimer with protein-disulfide isomerase (55 kDa) (10), a ubiquitous protein of the endoplasmic reticulum (ER) lumen that catalyzes disulfide bond formation during protein folding (11). The 97-kDa MTP subunit confers all lipid transfer activity to the heterodimer (12). In in vitro assays, the lipid transfer activity of MTP displays Ping Pong Bi Bi kinetics implying that MTP transfers lipids between membranes via a "shuttle" mechanism (13). The tissu...

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a cholinesupplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDPcholine pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K m for the cytidylyltransferase. Moreover, the amount and activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.In 1998, choline was classified as an essential nutrient for humans and a minimum dietary intake was recommended (53). Choline is ubiquitously present in animals and plants and is essential for survival and normal growth of cultured cells (1-4). Choline also appears to be an essential nutrient for humans (5, 6). Upon consumption of a choline-deficient (CD) 1 diet for 3 weeks, humans develop incipient liver dysfunction, particularly when adequate levels of methionine and folate are also lacking (5). In addition, choline has been proposed to play an important function in brain development (7, 8), probably because in cholinergic neurons choline is a precursor of the neurotransmitter acetylcholine (6, 9). Choline is also a precursor of the abundant membrane phospholipids phosphatidylcholine (PC), sphingomyelin, and choline plasmalogens in eukaryotic cells. In all nucleated eukaryotic cells, PC is synthesized from choline via the CDP-choline pathway (10). In the liver, an additional PC biosynthetic pathway, the phosphatidylethanolamine N-methyltransferase (PEMT) pathway (11, 12), which utilizes S-adenosylmethionine, generates ϳ30% of hepatic PC (13-15).Choline deprivation has been widely reported to be an effective tool for specifically inhibiting the CDP-choline pathway for PC...