Identification of a New Glycerol-3-phosphate Acyltransferase Isoenzyme, mtGPAT2, in Mitochondria

Abstract

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT-/- mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT-/- liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT-/- mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT-/- mice. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT-/- mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT-/- liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT-/- mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT-/- mice. The initial and rate-limiting step of glycerolipid synthesis is the acylation of glycerol 3-phosphate with long-chain fatty acyl-CoA to form 1-acyl-glycerol 3-phosphate (LPA). 1The abbreviations used are: LPA, lysophosphatidic acid; DHAP, dihydroxyacetone phosphate; DTT, dithiothreitol; G3P, glycerol 3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; NEM, N-ethylmaleimide; TAG, triacylglycerol; nt, nucleotides. 1The abbreviations used are: LPA, lysophosphatidic acid; DHAP, dihydroxyacetone phosphate; DTT, dithiothreitol; G3P, glycerol 3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; NEM, N-ethylmaleimide; TAG, triacylglycerol; nt, nucleotides. This reaction is catalyzed by two glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) isoenzymes that are encoded by different genes (1Coleman R.A. Lewin T.M. Muoio D.M. Annu. Rev. Nutr. 2000; 20: 77-103Google Scholar). One isoform is present in the endoplasmic reticulum membrane (microsomal GPAT), and the other is located in the outer mitochondrial membrane (mtGPAT). Although the microsomal GPAT has not been cloned or purified, its activity is easily distinguished, because, unlike mtGPAT, the microsomal isoform is inhibited by sulfhydryl reagents (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar). In most tissues, the microsomal GPAT activity is 10 times higher than that found in the mitochondrial fraction, but in liver, mtGPAT contributes 30–50% of the total activity (1Coleman R.A. Lewin T.M. Muoio D.M. Annu. Rev. Nutr. 2000; 20: 77-103Google Scholar). Microsomal GPAT and mtGPAT also differ in their acyl-CoA substrate preference. The microsomal isoform esterifies both saturated and unsaturated long-chain acyl-CoAs equally well, but mtGPAT prefers C16:0-CoA (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar). In rat liver, kidney, and heart, mtGPAT activity is 3–10-fold higher with C16:0-CoA than with other long-chain saturated or unsaturated acyl-CoA substrates (3Bremer J. Bjerve K.S. Borrebaek B. Christiansen R. Mol. Cell Biochem. 1976; 12: 113-125Google Scholar, 4Haldar D. Tso W.-W. Pullman M.E. J. Biol. Chem. 1979; 254: 4502-4509Google Scholar, 5Monroy G. Rola F.H. Pullman M.E. J. Biol. Chem. 1972; 247: 6884-6894Google Scholar, 6Monroy G. Kelker H.C. Pullman M.E. J. Biol. Chem. 1973; 248: 2845-2852Google Scholar). The activity of recombinant mouse mtGPAT purified from an insect cell expression system is 2-fold higher with C16:0-CoA than with C18:1-, C18:2n6-, C18:3n3-, or C20: 4n6-CoA (7Yet S.-F. Moon Y.K. Sul H.S. Biochemistry. 1995; 34: 7303-7310Google Scholar). Because most naturally occurring glycerolipids contain saturated fatty acids at the sn-1 position and unsaturated fatty acids at the sn-2 position, it was proposed that mtGPAT establishes the initial asymmetric distribution of fatty acids (4Haldar D. Tso W.-W. Pullman M.E. J. Biol. Chem. 1979; 254: 4502-4509Google Scholar). This hypothesis was borne out by studies with GPAT-/- mice showing that, compared with the wild-type mice, their liver phosphatidylethanolamine and phosphatidylcholine have 20% less palmitate in the sn-1 position and 36–40% more arachidonate in the sn-2 position (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). These results confirm both the important role mtGPAT plays in the positioning of 16:0 at the sn-1 position of phospholipids and its indirect role in positioning 20:4 at the sn-2 position. Although the microsomal GPAT is present in the same subcellular membrane fraction as the terminal enzymes for TAG synthesis, physiological data suggest that TAG formation is regulated by the mitochondrial isoform (1Coleman R.A. Lewin T.M. Muoio D.M. Annu. Rev. Nutr. 2000; 20: 77-103Google Scholar, 8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar, 9Coleman R.A. Lee D.P. Prog. Lipid Res. 2004; 43: 134-176Google Scholar, 10Igal R.A. Wang S. Gonzales-Baro M. Coleman R.A. J. Biol. Chem. 2001; 276: 42205-42212Google Scholar). Because only the mitochondrial GPAT isoform has been cloned (11Yet S.-F. Lee S. Hahm Y.T. Sul H.S. Biochemistry. 1993; 32: 9486-9491Google Scholar, 12Bhat B.G. Wang P. Kim J.-H. Black T.M. Lewin T.M. Fiedorek T.F. Coleman R.A. Biochim. Biophys. Acta. 1999; 1439: 415-423Google Scholar), changes in activity of the two isoenzymes can be compared, but not changes in mRNA abundance or protein expression. In general, mtGPAT mRNA, protein, and activity in liver and adipose tissue increase with carbohydrate feeding and with insulin stimulation, whereas microsomal GPAT activity does not change (11Yet S.-F. Lee S. Hahm Y.T. Sul H.S. Biochemistry. 1993; 32: 9486-9491Google Scholar, 13Saggerson E.D. Carpenter C.A. Biochem. J. 1987; 243: 289-292Google Scholar, 14Shin D.-H. Paulauskis J.D. Moustaid N. Sul H.S. J. Biol. Chem. 1991; 266: 23834-23839Google Scholar, 15Lewin T.M. Granger D.A. Kim J.H. Coleman R.A. Arch. Biochem. Biophys. 2001; 396: 119-127Google Scholar). In addition, mtGPAT mRNA is upregulated by sterol regulatory element-binding protein-1c, a potent activator of lipogenesis (16Ericsson J. Jackson S.M. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1997; 272: 7298-7305Google Scholar). The role for mtGPAT in up-regulating TAG synthesis is also supported by studies of overexpressed rat mtGPAT in Chinese hamster ovary cells. Expression of recombinant rat mtGPAT in Chinese hamster ovary cells results in a 3.8-fold increase in NEM-resistant GPAT activity, a 4-fold increase in [14C]oleate incorporation into TAG, and a 30% decrease in [14C]oleate incorporation into phospholipids (10Igal R.A. Wang S. Gonzales-Baro M. Coleman R.A. J. Biol. Chem. 2001; 276: 42205-42212Google Scholar). To confirm the critical role of mtGPAT in the regulation of TAG synthesis, mtGPAT null mice were generated (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). The knockout mice weigh less than controls and have reduced gonadal fat pad weights, lower hepatic TAG content, a lower plasma TAG and very low density lipoprotein TAG, and decreased secretion of TAG from liver (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). These data suggest that mtGPAT is required for normal synthesis of TAG in both fat cells and hepatocytes but show that the microsomal GPAT also contributes. We were surprised to find that mitochondria isolated from liver of mtGPAT-/- retained considerable GPAT activity but that this activity, unlike that of mtGPAT, was inactivated by NEM. Further, an anti-GPAT immunoblot analysis of GPAT-/- liver mitochondria detected an immunoreactive protein with approximately the same molecular mass as mtGPAT. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT-/- liver mitochondria had different isoelectric points. These results suggested the presence of a second mtGPAT isoform. Further characterization showed that, unlike the mtGPAT activity in wild type samples, the NEM-sensitive GPAT activity in mitochondria from liver of knockout animals did not prefer C16:0-CoA, was inhibited by DHAP and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that liver mitochondria from mtGPAT-/- mice express a novel GPAT activity. Chemicals—All chemicals were purchased from Sigma if not otherwise indicated. [2-3H]Glycerol was from PerkinElmer Life Sciences. Lipid were from were by the of at and mtGPAT-/- and wild type mice were by PCR as (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). were a with to and of and Microsomal mtGPAT mice and wild type mice were with of and by was and in 10 was with 10 and in a and at for to and were by the at for 10 The microsomal fraction was by the for at mitochondrial and microsomal fractions were in at were by the using as the of the and Microsomal of the liver subcellular fractions was by the activity of G. P. G. J. Cell Biol. Scholar), and for endoplasmic reticulum and mitochondria, were an gel to a membrane and with recombinant rat mtGPAT in and from the immunoreactive were by the membrane with and reagents two-dimensional mitochondria of were to of and and The was and in a for The was for an at temperature and at to the gel were at with the protein were a protein isoelectric cell for The gel was for with and the was to isoelectric the gel were for 10 with in 20% and in for an 10 with The gel were an gel to proteins in the second was as and GPAT 3-phosphate was from purified, and as J. Lipid Res. Scholar). GPAT activity was in a fatty DTT, and R.A. J. Biol. Chem. Scholar). The reaction was by of mitochondrial or microsomal protein to the were for 10 at temperature indicated. are in the were at Lipid from the GPAT were and in The were a with for LPA, and The were in The were to and the to LPA, and were into for GPAT-/- an NEM-sensitive GPAT GPAT knockout mice were generated by a of the with and by PCR genotyping (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). To that we had mtGPAT in the knockout we from mtGPAT and mice with two for rat mtGPAT, and The of mtGPAT is of and T.M. Wang P. Coleman R.A. Biochemistry. 1999; Scholar). are The a of that and B, of the identified an in liver from mtGPAT-/- mice (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). These Northern that mtGPAT-/- mice expression of the mtGPAT we were surprised to find that the mitochondria of mtGPAT-/- mice a GPAT activity that was sensitive to we a of the novel GPAT activity in mitochondrial fractions from livers of wild type and mtGPAT null mice. the GPAT activity in mitochondria has been as NEM-resistant glycerol-3-phosphate acyltransferase activity, because sulfhydryl reagents only the GPAT activity present in (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar). In initial characterization of the mtGPAT null mice, we liver total membrane fractions in the presence and of NEM. In total membrane GPAT activity in the presence of was only of the NEM-resistant activity in wild type mice (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar), with the of microsomal GPAT activity that is D.M. R.M. J. Biol. Chem. 1976; Scholar, D.M. R.M. Arch. Biochem. Biophys. Scholar). The NEM-sensitive GPAT were similar in wild type and mtGPAT knockout mice. purified mitochondria from mtGPAT-/- mice, however, GPAT activity that was of the activity present in mitochondria from wild type mice the glycerolipid in the for GPAT activity in mitochondria were by a similar distribution of LPA, and was in from wild type and mtGPAT null mice We that the GPAT activity present in mitochondria from the knockout be an NEM-sensitive To confirm this liver mitochondria from wild type and mtGPAT null mice were for GPAT activity with of GPAT activity in wild type mitochondria was in the presence of as (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google however, as as inhibited of activity in mitochondria from mtGPAT-/- mice. These data GPAT activity in wild type and knockout animals and show that the GPAT activity in liver mitochondria from mtGPAT null mice is similar to the microsomal GPAT of and mitochondrial activity in of total activity in activity in of total activity in activity in of total activity in fractions in a activity in mtGPAT-/- liver mitochondria is activity was in liver mitochondria from wild type and mtGPAT knockout mice. were at the for The GPAT was at temperature with of the of two at were for wild type and for mtGPAT knockout liver are from a GPAT activity from wild type and mtGPAT-/- mice to NEM, polymixin B, and activity was in liver mitochondria from wild type and mtGPAT knockout mice and in from wild type mice. the of from a at in the different from for for wild type mitochondria, and for knockout of and microsomal fractions were purified and microsomal fractions were purified the NEM-sensitive and NEM-resistant the NEM-sensitive and NEM-resistant the NEM-sensitive and NEM-resistant the NEM-sensitive and NEM-resistant not and microsomal fractions were purified The the NEM-sensitive and NEM-resistant not in a from mtGPAT-/- a GPAT mitochondria from the mtGPAT-/- mice an anti-mtGPAT immunoreactive protein with a molecular mass similar to that of mtGPAT The mitochondria were not with endoplasmic reticulum and immunoreactive protein was detected in the fraction with that the microsomal and mitochondrial GPAT isoforms are distinct (1Coleman R.A. Lewin T.M. Muoio D.M. Annu. Rev. Nutr. 2000; 20: 77-103Google Scholar, R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar, S.-F. Lee S. Hahm Y.T. Sul H.S. Biochemistry. 1993; 32: 9486-9491Google and that their are very two-dimensional analysis was in to the anti-GPAT immunoreactive proteins in wild type and mtGPAT-/- liver The immunoreactive protein detected in mtGPAT knockout mouse liver had a different isoelectric than the protein detected in wild type mice In the two and and were detected in a of liver mitochondria from wild type and mtGPAT null mice. In liver mitochondria from mtGPAT-/- mice is whereas the used was recombinant rat mtGPAT protein, we that the novel immunoreactive protein was a novel GPAT with considerable to mtGPAT. Characterization of GPAT in GPAT-/- sensitivity of GPAT activity in knockout liver mitochondria the presence of a GPAT To this we the for the two glycerol 3-phosphate and The for in wild type mitochondria was and in the same as studies of purified recombinant mouse mtGPAT (7Yet S.-F. Moon Y.K. Sul H.S. Biochemistry. 1995; 34: 7303-7310Google Scholar). The for in mitochondria from the mtGPAT null mice was similar Microsomal GPAT had an for of is the substrate for mtGPAT (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google unsaturated fatty acyl-CoA a that is lower (3Bremer J. Bjerve K.S. Borrebaek B. Christiansen R. Mol. Cell Biochem. 1976; 12: 113-125Google Scholar, 4Haldar D. Tso W.-W. Pullman M.E. J. Biol. Chem. 1979; 254: 4502-4509Google Scholar, 5Monroy G. Rola F.H. Pullman M.E. J. Biol. Chem. 1972; 247: 6884-6894Google Scholar, 6Monroy G. Kelker H.C. Pullman M.E. J. Biol. Chem. 1973; 248: 2845-2852Google Scholar). The only of mouse mtGPAT showed that recombinant purified from insect cells had a 2-fold for C16:0-CoA compared with at (7Yet S.-F. Moon Y.K. Sul H.S. Biochemistry. 1995; 34: 7303-7310Google Scholar). In mtGPAT-/- liver mitochondria, in was and C16:0-CoA but wild type liver mitochondria showed a 30% for C16:0-CoA as compared with at In wild type and mtGPAT-/- mice, the for and not be because of inhibition at higher than The of a for C16:0-CoA for activity from mtGPAT null mitochondria that of the microsomal GPAT (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar). Because the microsomal GPAT from rat fat cells and liver also DHAP, as a R.A. J. Biol. Chem. Scholar, D.M. R.M. J. Biol. Chem. 1976; Scholar), we the ability of DHAP to GPAT activity. of DHAP inhibited acylation in mitochondria from mtGPAT-/- liver but not in wild type The for DHAP was to be similar to for rat microsomal GPAT R.A. J. Biol. Chem. Scholar, R.A. R.M. J. Biol. Chem. 1980; Scholar). liver microsomal GPAT activity was not inhibited by DHAP at was present in the but at both mouse and rat liver microsomal GPAT were inhibited 30% not similar to that used lower R.A. J. Biol. Chem. Scholar). we conclude that DHAP is a of the novel GPAT activity present in knockout liver The to inhibition by DHAP is similar to microsomal GPAT, whereas in wild type mitochondria, DHAP does not mtGPAT activity. GPAT in mtGPAT-/- by B, and to and mtGPAT are by polymixin B, and (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar), we the of mitochondrial GPAT from wild type and GPAT-/- liver mitochondria GPAT in wild type mitochondria, and knockout mitochondria were similar and The of and did not GPAT a potent of did not or GPAT activity in wild type or knockout LPA, the of the GPAT GPAT activity equally in mitochondria from wild type and mtGPAT-/- mice, similar to R.A. Biochim. Biophys. Acta. Scholar). microsomal GPAT activity is inhibited for at R.A. J. Biol. Chem. Scholar), we the temperature sensitivity of mtGPAT. In to GPAT activity in wild type mitochondria, GPAT activity from mtGPAT-/- liver mitochondria was sensitive to with of the glycerol-3-phosphate acyltransferase activity for at B, an that with microsomal GPAT and mtGPAT D. 1987; 22: Scholar, D. Arch. Biochem. Biophys. Scholar). In liver mitochondria from GPAT-/- mice, inhibited GPAT activity similar to microsomal GPAT, whereas mtGPAT from wild type mice was only inhibited 20% In to we did not of wild type mtGPAT, because lower of polymixin D. 1987; 22: Scholar, D. Arch. Biochem. Biophys. Scholar). Because both temperature and polymixin the K.S. Annu. Rev. Biochem. Scholar), we out the that the membrane of wild type and knockout liver mitochondria differ to sensitivity to temperature and polymixin (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). GPAT activity in wild type and knockout liver mitochondria were with the of or to the acyltransferase studies have that in the rat mtGPAT activity whereas the microsomal activity is inhibited or (2Bell R.M. Coleman R.A. Annu. Rev. Biochem. 1980; 49: 459-487Google Scholar). GPAT activity in wild type mitochondria, but change was in mitochondria from mtGPAT-/- mice The of inhibited GPAT activity in mitochondria from wild type and mtGPAT null mice, and These data hypothesis that a novel GPAT is present in liver mitochondria from mtGPAT null mice. GPAT is a critical in glycerolipid synthesis because it catalyzes the initial and step required for the formation of triacylglycerol and of the two distinct GPAT isoenzymes have been an NEM-sensitive isoform present in the endoplasmic reticulum membrane and an NEM-resistant present in the outer mitochondrial that an mitochondrial GPAT isoform the total to We the for the GPAT for the microsomal for the cloned mitochondrial GPAT, and for the activity in this and Northern showed of the mtGPAT in the mtGPAT-/- mice, and the of animals in liver TAG and in the of the (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). we liver mitochondria and from mice, we that unlike mitochondria from wild type liver, the knockout liver mitochondria an NEM-sensitive GPAT activity (mtGPAT2) The of an NEM-sensitive GPAT in knockout liver mitochondria with the presence of an anti-mtGPAT immunoreactive protein (mtGPAT2) with a similar molecular mass but a different isoelectric than GPAT from wild type liver mitochondria is not that the rat mtGPAT glycerol-3-phosphate the was recombinant rat liver mtGPAT, and GPAT and 1-acyl-glycerol acyltransferase isoforms a of the low molecular of the 1-acyl-glycerol of isoforms R.A. Lee D.P. Prog. Lipid Res. 2004; 43: 134-176Google Scholar). In a similar rat long-chain acyl-CoA acyl-CoA and T.M. Kim J.-H. Granger D.A. Coleman R.A. J. Biol. Chem. 2001; 276: Scholar). The mtGPAT did not protein in liver that the from the mitochondrial We that is in null mice, because the of a immunoreactive protein with the of a mitochondrial NEM-sensitive GPAT activity are distinct from those of for data be that mtGPAT has two that only of the and that the GPAT activity in liver mitochondria is to a increase in the of the We not this is the because Northern (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar), with that of the two critical that have been for acyltransferase activity T.M. Wang P. Coleman R.A. Biochemistry. 1999; Scholar), to in mice. In addition, the of GPAT in knockout liver mitochondria differ from wild Although contain or more epitopes by its differ from those of activity in liver mitochondria from is inhibited by NEM, this activity from the NEM-resistant In addition, data the that is a mitochondrial membrane GPAT, because this GPAT activity was NEM-resistant E.D. J. Biochem. Scholar). Further also from and other saturated and unsaturated acyl-CoAs equally and is inhibited by DHAP, by but not unlike is temperature-sensitive, inhibited by polymixin B, and not by acetone. Although the of are of the activity the microsomal because mitochondrial fractions less than of the endoplasmic reticulum and the immunoblot analysis showed anti-mtGPAT immunoreactive protein in microsomal fractions In addition, activity is not to be a mitochondrial DHAP acyltransferase activity, because this activity is NEM-resistant P. J. Biol. Chem. Scholar). the of a anti-mtGPAT immunoreactive protein in mtGPAT-/- liver mitochondria with a distinct GPAT activity, similar in its to those of but not detectable in wild type of a second mtGPAT isoform in GPAT protein expression activity. studies in show that, compared with liver and adipose mtGPAT protein expression is in and but that NEM-resistant GPAT activity is low T.M. Wang P. Coleman R.A. Biochemistry. 1999; Scholar). is that is in and by the NEM-sensitive activity in mitochondria not have been because activity was in a total membrane and the GPAT activity was in the presence of (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). To the and proteins are of NEM-sensitive and GPAT activity in purified mitochondrial and microsomal fractions from Although the of is not at this it is distinct from because mice weigh less and have reduced gonadal fat pad weights, lower hepatic TAG content, lower plasma TAG, low density lipoprotein TAG, and decreased hepatic TAG secretion (8Hammond L.E. Gallagher P.A. Wang S. Hiller S. Kluckman K.D. Posey-Marcos E.L. Maeda N. Coleman R.A. Mol. Cell Biol. 2002; 22: 8204-8214Google Scholar). the novel for the ability of to TAG in fat cells and we that in the initial step in the synthesis of mitochondrial phospholipids This hypothesis is with the presence of activity in wild type and heart, M. a tissue in of the of sensitivity, temperature sensitivity, of stimulation, and DHAP were in wild type liver mitochondria, it that be in mouse liver, to a critical of mitochondrial is to the GPAT R.A. Lee D.P. Prog. Lipid Res. 2004; 43: 134-176Google Scholar, T.M. Wang P. Coleman R.A. Biochemistry. 1999; Scholar). from the immunoblot data is also to be similar to the synthesis of the phospholipids in mitochondria and mitochondrial proteins have a and and are both from contributes to the synthesis of TAG, a from most be important for synthesizing mitochondrial similar to protein is to and mtGPAT contain the required for substrate and R.A. Lee D.P. Prog. Lipid Res. 2004; 43: 134-176Google Scholar), is to D.-H. Paulauskis J.D. Moustaid N. Sul H.S. J. Biol. Chem. 1991; 266: 23834-23839Google Scholar). of the mouse data using acyltransferase T.M. Wang P. Coleman R.A. Biochemistry. 1999; the presence of of be glycerolipid and enzymes and 1-acyl-glycerol and and acyltransferase phosphatidylethanolamine and and and have two or more isoforms that have distinct R.A. Lee D.P. Prog. Lipid Res. 2004; 43: 134-176Google Scholar, Biochem. Scholar, J. Biol. Chem. 1993; Scholar, S. S. J. Biol. Chem. Scholar, J. N. B. S. Coleman J. Cell Biol. 1997; Scholar, S. N. 2002; Scholar, Biochim. Biophys. Acta. Scholar, S. J. Biol. Chem. Scholar, S. S. S. Scholar, S. P. B. K.D. J. Biol. Chem. 2001; 276: Scholar, D. J. J. R. R. M. J. Biol. Chem. Scholar, S. P. S. 2002; Scholar, J. Biol. Chem. Scholar, R. S. 1997; Scholar, M. J. Biol. Chem. 1991; 266: Scholar, M. J. Biol. Chem. 1997; 272: Scholar). Although characterization of the of molecular it is to that endoplasmic reticulum synthesis, mitochondrial synthesis, and TAG synthesis be by distinct GPAT We for