The major sites of cellular phospholipid synthesis and molecular determinants of fatty acid and lipid head group specificity

Phosphatidylcholine and phosphafidylethanolamine are the two main phospholipids in eukaryotic cells comprising similar to50 and 25% of phospholipid mass, respectively. Phosphatidylcholine is synthesized almost exclusively through the CDP-choline pathway in essentially all mammalian cells. Phosphatidylethanolamine is synthesized through either the CDP-ethanolamine pathway or by the decarboxylation of phosphatidylserine, with the contribution of each pathway being cell type dependent. Two human genes, CEPT1 and CPTI, code for the total compliment of activities that directly synthesize phosphatidylcholine and phosphafidylethanolamine through the CDP-alcohol pathways. CEPT1 transfers a phosphobase from either CDP-choline or CD17-ethanolamine to diacylglycerol to synthesize both phosphatidylcholine and phosphatidylethanolamine, whereas CPTI synthesizes phosphatidylcholine exclusively. We show through immunofluorescence that brefeldin A treatment relocalizes CPTI, but not CEPT1, implying CPTI is found in the Golgi. A combination of coimmunofluorescence and subcellular fractionation experiments with various endoplasmic reticulum, Golgi, and nuclear markers confirmed that CPT1 was found in the Golgi and CEPT1 was found in both the endoplasmic reticulum and nuclear membranes. The rate-limiting step for phosphatidylcholine synthesis is catalyzed by the amphitropic CTP:phosphocholine cytidylyltransferase a, which is found in the nucleus in most cell types. CTP:phosphocholine cytidylyltransferase alpha is found immediately upstream cholinephosphotransferase, and it translocates from a soluble nuclear location to the nuclear membrane in response to activators of the CDP-choline pathway. Thus, substrate channeling of the CDP-choline produced by CTP:phosphocholine cytidylyltransferase a to nuclear located CEPT1 is the mechanism by which upregulation of the CDP-choline pathway increases de novo phosphatidylcholine biosynthesis. In addition, a series of CEPT1 site-directed mutants was generated that allowed for the assignment of specific amino acid residues as structural requirements that directly alter either phospholipid head group or fatty acyl composition. This pinpointed glycine 156 within the catalytic motif as being responsible for the dual CDP-alcohol specificity of CEPT1, whereas mutations within helix 214-228 allowed for the orientation of transmembrane helices surrounding the catalytic site to be definitively positioned.