Alignment of amino acid sequences from various acyltransferases [sn-glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I-IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I-IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases. Using Escherichia coli GPAT (PlsB) as a model acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I-IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine and aspartate in block I, the glycine in block III, and the proline in block IV all play a role in E. coli GPAT catalysis. The phenylalanine and arginine in block II and the glutamate and serine in block III appear to be important in binding the glycerol 3-phosphate substrate. Since blocks I-IV are also found in LPAAT, DHAPAT, and LPEAT, we believe that these conserved amino acid motifs are diagnostic for the acyltransferase reaction involving glycerol 3-phosphate, 1-acylglycerol 3-phosphate, and dihydroxyacetone phosphate substrates.

Although triacylglycerol stores play the critical role in an organism's ability to withstand fuel deprivation and are strongly associated with such disorders as diabetes, obesity, and atherosclerotic heart disease, information concerning the enzymes of triacylglycerol synthesis, their regulation by hormones, nutrients, and physiological conditions, their mechanisms of action, and the roles of specific isoforms has been limited by a lack of cloned cDNAs and purified proteins. Fortunately, molecular tools for several key enzymes in the synthetic pathway are becoming available. This review summarizes recent studies of these enzymes, their regulation under varying physiological conditions, their purported roles in synthesis of triacylglycerol and related glycerolipids, the possible functions of different isoenzymes, and the evidence for specialized cellular pools of triacylglycerol and glycerolipid intermediates.

SummaryFour homologous isoforms of glycerol-3-phosphate acyltransferase (GPAT), each the product of a separate gene, catalyze the synthesis of lysophosphatidic acid from glycerol-3-phosphate and longchain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-offunction and loss-of-function studies in mice and in cell culture strongly suggests that each isoform contributes to the synthesis of triacylglycerol. Much work remains to fully delineate the regulation of each GPAT isoform and its individual role in triacylglycerol synthesis. KeywordsGlycerolipid; phospholipid; membrane; lipid droplet; lysophosphatidic acid; diacylglycerol Glycerol-3-phosphate acyltransferases are members of the pfam 01553 family of acyltransferasesAfter Eugene Kennedy and his colleagues showed that the esterification of glycerol-3-phosphate with a long-chain acyl-CoA was the initial step in the synthesis of phospholipids [1] and triacylglycerol (TAG) [2], Pullman's group reported that this sn-glycerol-3-phosphate acyltransferase activity (GPAT; EC 2.3.1.15) was comprised of what appeared to be two isoforms, one located in the mitochondrial outer membrane and the other in the endoplasmic reticulum [3]. The endoplasmic reticulum (microsomal) activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and exhibited no preference for particular acyl-CoA species, whereas the mitochondrial activity was resistant to NEM inactivation and preferred to use saturated acyl-CoAs like 16:0-CoA and 18:0-CoA [4]. With the identification of four genes encoding separate GPAT isoenzymes [5-11], we now know that GPAT mediated regulation of glycerolipid synthesis is more complex than anyone had previously thought; investigators are currently struggling with the question as to why four separate isoforms are required for glycerolipid biosynthesis.Gpat1, the first mammalian GPAT isoform cloned [5,6], resides in the outer mitochondrial membrane, is resistant NEM inactivation and prefers to use saturated acyl-CoAs [4]. A second mitochondrial GPAT, GPAT2, also resides in the outer mitochondrial membrane, but its activity is inhibited by NEM and it has no long-chain acyl-CoA preference [12]. The NEM-

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC 6.2.1.3) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, noncross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Refeeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.The first step in long chain fatty acid use in mammals requires the ligation of fatty acid with coenzyme A (CoA). This reaction, catalyzed by acyl-CoA synthetase (ACS, 1 EC 6.2.1.3), produces acyl-CoAs, which are primary substrates for energy use via ␤-oxidation and for the synthesis of triacylglycerol, phospholipids, cholesterol esters, and sphingomyelin, and are the source of signaling molecules like ceramide, diacylglycerol, and arachidonic acid (1, 2). Acyl-CoAs up-regulate uncoupling protein in brown fat and key enzymes of glycolysis, gluconeogenesis, and ␤-oxidation; are essential for vesicle trafficking; and play a critical role in the transport of fatty acids into cells by making transport unidirectional. Protein esterification with myristate and palmitate anchors proteins to specific membranes and enables them to function correctly (3). Thus, acylCoAs participate in a large number of cellular reactions that involve lipid synthesis, energy metabolism, and regulation, but how acyl-CoAs are partitioned or directed toward these diverse synthetic, degradative, and signaling pathways is not understood. Currently, five different rat ACS cDNAs have been cloned, each the product of a different gene (4 -8). Rat ACS1-5 share a common structural architecture and are further classified into two subfamilies based on amino acid identity and fatty ...

Recent studies suggest that the long-chain acyl-CoA synthetases (ACS) may play a role in channeling fatty acids either toward complex lipid synthesis and storage or toward oxidation. Each of the five members of the ACS family that has been cloned has a distinct tissue distribution and subcellular location, and is regulated independently during cellular differentiation and by diverse hormones and nuclear transcription factors including adrenocorticotropic hormone (ACTH), peroxisomal proliferator-activated receptor-alpha (PPARalpha) and sterol regulatory element binding protein. Taken as a whole, these features suggest that in liver, ACS1 and ACS5 may provide acyl-CoA destined primarily for triacylglycerol synthesis or for mitochondrial oxidation, respectively. ACS4 may provide acyl-CoA for both synthesis and peroxisomal oxidation, depending on whether the enzyme is associated with the mitochondrial-associated membrane or with peroxisomes. It should be emphasized that although the data for acyl-CoA channeling are strong, they are indirect. Rigorous testing of these predictions will be required.