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The current status of the biochemistry of phospholipid biosynthesis is presented. The review focuses on the identification and characterization of molecular tools such as purified enzymes and cloned genes and cDNAs for those enzymes. The enzymes discussed are those involved in the biosynthesis of the major phospholipid classes, namely, phosphatidate, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylinositol and its phosphorylated derivatives, and cardiolipin. The review centers on the pathways in mammals and yeast. Novel genetic approaches used to delineate pathways and clone cDNAs are discussed. The regulatory roles played by some of the enzymes involved in controlling the biosynthetic pathways are presented.

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.

Structure superpositions relate GCT to the catalytic domains from class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidylyltransferases. GCT and aminoacyl-tRNA synthetases catalyze analogous reactions, bind nucleotides in similar U-shaped conformations, and depend on histidines from analogous HXGH motifs for activity. The structural and other similarities support proposals that GCT, like the synthetases, catalyzes nucleotidyl transfer by stabilizing a pentavalent transition state at the alpha-phosphate of CTP.

To probe the mechanism of lipid activation of CTP: phosphocholine cytidylyltransferase (CCT␣), we have characterized a catalytic fragment of the enzyme that lacks the membrane-binding segment. The kinetic properties of the purified fragment, CCT␣236, were characterized, as well as the effects of expressing the fragment in cultured cells. CCT␣236 was truncated after residue 236, which corresponds to the end of the highly conserved catalytic domain. The activity of purified CCT␣236 was independent of lipids and about 50-fold higher than the activity of wild-type CCT␣ assayed in the absence of lipids, supporting a model in which the membrane-binding segment functions as an inhibitor of the catalytic domain. The k cat /K m values for CCT␣236 were only slightly lower than those for lipid-activated CCT␣. The importance of the membrane-binding segment in vivo was tested by expression of CCT␣236 in CHO58 cells, a cell line that is temperature-sensitive for growth and CCT␣ activity. Expression of wild-type CCT␣ in these cells complemented the defective growth phenotype when the cells were cultured in complete or delipidated fetal bovine serum. Expression of CCT␣236, however, did not complement the growth phenotype in the absence of serum lipids. These cells were capable of making phosphatidylcholine in the delipidated medium, so the inability of the cells to grow was not due to defective phosphatidylcholine synthesis. Supplementation of the delipidated medium with an unsaturated fatty acid allowed growth of CHO58 cells expressing CCT␣236. These results indicate that the membranebinding segment of CCT␣ has an important role in cellular lipid metabolism. CTP:phosphocholine cytidylyltransferase (CCT)1 is a critical participant in the CDP-choline pathway, catalyzing the synthesis of CDP-choline for the biosynthesis of phosphatidylcholine (PC), a major component of eukaryotic cell membranes (1, 2). CCT is rate-limiting for the CDP-choline pathway and is extensively regulated at the enzymatic level. Mammalian CCT is present as both soluble and membrane-associated forms.Activation of CCT often occurs simultaneously with the translocation of the enzyme from a soluble form to membrane-associated form, resulting in an increase in the rate of PC synthesis. Consistent with in vivo membrane activation, the soluble form of CCT is activated by the addition of certain lipids in vitro. There are two isoforms of mammalian CCT; the most extensively studied, CCT␣, is nuclear and apparently ubiquitously expressed (2). The recently discovered CCT␤ is cytoplasmic and exhibits tissue-specific expression (3).Mammalian CCT␣ contains several functional regions: an N-terminal nuclear localization signal, a central catalytic domain, a membrane/lipid (M/L) activation segment, and a Cterminal phosphorylation region (see Fig. 1). There is a high degree of sequence similarity within the catalytic domain of all known forms of CCT, with the yeast catalytic domain being 56% identical to that of mammalian CCT␣, and the catalytic domains of CCT␣ and CCT␤ bei...

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Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.

The function of the putative amphipathic helices between residues 236 and 314 of CTP:phosphocholine cytidylyltransferase was examined by constructing two truncation mutants; CT314 was missing the entire phosphorylation segment, whereas CT236 was missing both the region with the putative amphipathic helices and the phosphorylation segment. Stable cells lines expressing these truncation mutants in Chinese hamster ovary 58 cells were isolated and characterized. CT314 was predominantly soluble in control cells but became membrane-associated in cells treated with oleate, which also causes translocation of wild-type cytidylyltransferase. CT236 was found to be soluble both in control cells and in cells treated to cause translocation. These results strongly suggest that the membrane-binding site is located within residues 237-314. When assayed for activity in vitro, the mutant forms were catalytically active in the presence of exogenous lipids. CT236, moreover, was as active in the absence of lipids as in their presence, whereas CT314 required lipids for activity. The rate of phosphatidylcholine synthesis in cells expressing CT236 was considerably higher than in wild-type cells, consistent with the enzyme being constitutively active in the cells. These results indicate that residues 237-314 constitute an inhibitory segment; when this segment is removed from the catalytic domain by truncation or by binding to membranes, an inhibitory constraint is removed and cytidylyltransferase is activated.