Identification of a new glycerol-3-phosphate acyltransferase isoenzyme, mtGPAT2, in mitochondria

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.     

Membrane topology of murine glycerol-3-phosphate acyltransferase 2

Glycerol-3-phosphate acyltransferase (GPAT) is a rate-limiting enzyme in mammalian triacylglycerol biosynthesis. GPAT is a target for the treatment of metabolic disorders associated with high lipid accumulation. Although the molecular basis for GPAT1 activation has been investigated extensively, the activation of other isoforms, such as GPAT2, is less well understood. Here the membrane topology of the GPAT2 protein was examined using an epitope-tag-based method. Exogenously expressed GPAT2 protein was present in the membrane fraction of transformed HEK293 cells even in the presence of Na(2)CO(3) (100 mM), indicating that GPAT2 is a membrane-bound protein. Trypsin treatment of the membrane fraction degraded the N-terminal (FLAG) and C-terminal (myc-epitope) protein tags of the GPAT2 protein. Bioinformatic analysis of the GPAT2 protein sequence indicated four hydrophobic sequences as potential membrane-spanning regions (TM1-TM4). Immunoblotting of the myc-epitope tag, which was inserted between each TM region of the GPAT2 protein, showed that the amino acid sequence between TM3 and TM4 was protected from trypsin digestion. These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. These results also suggest that the enzymatically active motifs I-III of the GPAT2 protein face the cytosol, while motif IV is within the membrane. It is expected that the use of this topological model of GPAT2 will be essential in efforts to elucidate the molecular mechanisms of GPAT2 activity in mammalian cells.     

Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds

The glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.1.15) from sunflower (Helianthus annuus L.) microsomes has been characterised and partially purified. The in vitro determination of activity was optimized, and the maximum value for GPAT activity identified between 15 and 20 days after flowering. The apparent Michaelis–Menten K_m for the glycerol 3-phosphate was 354 μM. The preferred substrates were palmitoyl-CoA = linoleoyl-CoA > oleoyl-CoA with the lowest activity using stearoyl-CoA. High solubilisation was achieved using 0.75% Tween80 and the solubilised GPAT was partially purified by ion-exchange chromatography using a Hi-Trap DEAE FF column, followed by gel filtration chromatography using a Superose 12 HR column. The fraction containing the GPAT activity was analysed by SDS-PAGE and contained a major band of 60.1 kDa. Finally, evidence is provided which shows the role of GPAT in the asymmetrical distribution, between positions sn-1 and sn-3, of saturated fatty acids in highly saturated sunflower triacylglycerols. This work provides background information on the sunflower endoplasmic reticulum GPAT which may prove valuable for future modification of oil deposition in this important crop.     

甘油-3磷酸转酰酶氨基酸与植物抗冷性关系初探

甘油 - 3磷酸转酰酶 (GPAT)与植物抗冷性密切相关。南瓜 (Cucurbitamoschata)与黑子南瓜 (Cucurbitaficifolia)同属不同种 ,亲缘关系较近 ,但却存在显著的抗冷性差异。南瓜及黑子南瓜GPAT基因的克隆 ,可以使我们从二者推导的有限氨基酸的差异中对GPAT氨基酸组成及其与植物抗冷性作一定的探讨。发现在南瓜与黑子南瓜 13个不同的氨基酸残基中有 3个与抗冷性植物拟南芥菜 (Arabidopsisthaliana)、豌豆 (Pisumsativum)、红花 (Carthamustincto rius)和菠菜 (Spinaciaoleracea)等相同 ,可能与黑子南瓜比南瓜更具抗冷性的原因有关。比较南瓜、黑子南瓜、豌豆、红花、拟南芥菜和菠菜等植物中GPAT基因推导的氨基酸序列发现 ,在比较抗冷的拟南芥菜、红花、豌豆和菠菜等植物中 ,虽然它们之间的亲缘关系都比较远 ,但某些位点上的氨基酸残基却完全相同 ,而与南瓜等抗冷性较差的植物不同 ,这些位点的氨基酸残基可能也与GPAT对底物酰基的选择性有关。     

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an approximately 89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.     

Stimulation of rat liver glycerol-3-phosphate acyltransferase activity by acid- and heat-stable low-molecular-weight substances from skeletal muscle of rats treated with insulin

Experiments were conducted to examine a possible mechanism of activation of rat liver microsomal glycerol-3-phosphate acyltransferase (GPAT) by insulin. Fractions of Mr 1100 were prepared from hind-limb muscles of rats, which had been given intravenous injections of insulin or saline, by a procedure which involved acidification (pH 3.8) and heating (100 degrees C), followed by chromatography on Sephadex G-25 in 50 mM formic acid. These fractions were shown to modify the activity of microsomal GPAT from the livers of fed rats which had not been treated with insulin. The difference in GPAT activity between microsomes supplemented with the Mr 1100 material and those treated with an equal volume of 50 mM formic acid from before the void volume of the column was determined. Relative to the formic acid control, the Mr 1100 material from saline-treated rats decreased GPAT activity, whereas Mr 1100 material from insulin-treated rats increased GPAT activity and the difference of 0.64 nmol/min/mg microsomal protein was significant (P less than 0.01). Fractions of approximately 3000 Da were found to behave in a similar manner and caused a significant (P less than 0.01) increase in GPAT activity of 0.46 nmol/min/mg microsomal protein. These substances, which stimulate GPAT activity, may be related to the putative insulin mediator substance.     

Mitochondria‐type GPAT is required for mitochondrial fusion

Glycerol‐3‐phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER‐GPAT and mitochondrial (Mt)‐GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt‐GPAT is essential for mitochondrial fusion. Mutation of Mt‐GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt‐GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP‐1 and by overexpression of mitochondrial fusion protein FZO‐1/mitofusin, suggesting that the fusion/fission balance is affected by Mt‐GPAT depletion. Mitochondrial fragmentation was also observed in Mt‐GPAT‐depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt‐GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt‐GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion.     

Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase

Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis.     

Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV)

Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.     

Thematic review series: glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity

Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids. The existence of multiple enzyme isoforms with GPAT activity was predicted many years ago when GPAT activities with distinct kinetic profiles and sensitivity to inhibitors were characterized in two subcellular compartments, mitochondria and microsomes. We now know that mammals have at least four GPAT isoforms with distinct tissue distribution and function. GPAT1 is the major mitochondrial GPAT isoform and is characterized by its resistance to sulfhydryl-modifying reagents, such as N-ethylmaleimide (NEM). GPAT2 is a minor NEM-sensitive mitochondrial isoform. The activity referred to as microsomal GPAT is encoded by two closely related genes, GPAT3 and GPAT4. GPAT isoforms are important regulators of cellular triglyceride and phospholipid content, and may channel fatty acids toward particular metabolic fates. Overexpression and knock-out studies suggest that GPAT isoforms can play important roles in the development of hepatic steatosis, insulin resistance, and obesity; GPAT isoforms are also important for lactation. This review summarizes the current state of knowledge on mammalian GPAT isoforms.     

Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: Functional divergence of the dilysine ER retrieval motif in plant cells

Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N- and C-termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (–KK–COOH rather than the prototypic –KKXX–COOH or –KXKXX–COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (––X–X–K/R/D/E––; where are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C-termini of other GPAT8s from various plant species, further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.     

A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment.

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5--6-fold higher in the parenchymal cells.     

A second gene for acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase in squash, Cucurbita moschata cv. Shirogikuza(*), codes for an oleate-selective isozyme: molecular cloning and protein purification studies

A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5-40.5 kDa, pI values of 6.75-7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.     

Studies on Acetyl-Coenzyme A Synthetase of Yeast: Inhibition by Long-Chain Acyl-Coenzyme A Esters

Long-chain acyl-coenzyme A (CoA) compounds (palmityl, stearyl, and oleyl) were found to be potent inhibitors of acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae strain LK2G12 from aerobic, but not from nonaerobic, cells. The effectiveness of the inhibitors of the aerobic enzyme was in the following order: palmityl-CoA < stearyl-CoA < oleyl-CoA. Short-chain acyl-CoA compounds (propionyl, butyryl, and valeryl) and long-chain fatty acids had no effect on ACS from either source. The inhibition by oleyl-CoA was found to be dependent on enzyme concentration, whereas the inhibition by palmityl- and stearyl-CoA was independent of ACS concentration. Inhibition by palmityl-CoA was noncompetitive with respect to both acetate and CoA, and with increasing concentration of inhibitor the pattern was sigmoidal, with a Hill value of 3.24. At maximally inhibitory concentrations of palmityl-CoA, a small amount of enzyme activity remained. This noninhibitable enzyme in aerobic cells was shown not to be of nonaerobic origin.     

冷胁迫下王百合GPAT基因保守区克隆及表达分析

以王百合为试验材料,通过同源克隆和巢式PCR方法从4℃低温诱导的王百合试管苗中分离得到了王百合GPAT基因的保守区序列,采用DNAman软件和BLASTN对该序列进行分析并分别从蛋白和基因角度分析了GPAT基因在4℃冷诱导情况下的表达情况.结果显示:(1)该保守区长744 bp,推测其编码247个氨基酸,氨基酸序列存在1个高度保守的区域(WIAPSGGRDRP),经过Blast比对分析发现,该保守区序列为LPLAT基因超级家族酶类的催化活性区,此家族多为催化酰基辅酶A(acylCoAs)或者酰基载体蛋白(acylACPs)中的酰基与受体蛋白结合的酰基转移酶类.(2)冷诱导促进GPAT基因的表达,随冷诱导时间延长,基因表达量不断增大,诱导4 h有大量表达,16 h表达量达到最高,16 h之后表达量随着冷诱导时间的延长逐渐下降,72 h时的表达量与0 h处理时基本一致.研究表明,GPAT在百合抵抗冷胁迫的过程中具有重要的作用.     

Aging and acyl-CoA binding protein alter mitochondrial glycerol-3-phosphate acyltransferase activity

It is well known that cellular function declines with age. Since phosphatidic acid (PtdOH) biosynthesis is central to the generation of membrane phospholipids, the hypothesis that aging decreases PtdOH biosynthesis was tested. Glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LAT) activities were examined in isolated mitochondria and microsomes from young and old rat liver. The results show that mitochondrial GPAT preference for palmitoyl-CoA over oleoyl-CoA was only observed if albumin or acyl-CoA binding protein (ACBP) were present in the assay in the young rats. Furthermore, mitochondrial GPAT activity was significantly reduced in the presence of albumin and ACBP in aged mitochondria using palmitoyl-CoA as the substrate. These data show, for the first time, that mitochondrial GPAT acyl-CoA preference is due to the presence of a protein that binds acyl-CoAs, not the enzyme itself, and that aging significantly reduces mitochondrial GPAT activity.     

Phylogenetic utility of the glycerol-3-phosphate acyltransferase gene: evolution and implications in Paeonia (Paeoniaceae)

The nuclear-encoded chloroplast-expressed glycerol-3-phosphate acyltransferase (GPAT) gene has been found to be single-copy in a number of angiosperm families. In this study we investigated the phylogenetic utility of the GPAT gene at the interspecific level using the genus Paeonia (Paeoniaceae) as an example. An approximately 2.3- to 2.6-kb fragment of the GPAT gene, containing a large intron of more than 2 kb, was amplified, cloned, and sequenced from 19 accessions representing 13 Paeonia species. The GPAT gene phylogeny inferred by parsimony analysis supported interspecific relationships that were previously unresolved, suggesting that large introns of low-copy nuclear genes are particularly informative in the resolution of close relationships at low taxonomic levels. Whereas the GPAT phylogeny is largely congruent with the previous phylogenetic hypothesis of Paeonia, it shows a significant discordance involving the paraphyly of section Paeonia. Given evidence of an ancient duplication and the subsequent silencing of one GPAT locus in P. anomala, this discordance is most likely the result of paralogy. Two distinct genomic clones containing partial GPAT genes were isolated from P. anomala. The GPAT sequence from one clone corresponded to the functional copy of the gene, and the second genomic clone was determined to contain a GPAT pseudogene. The insertion of a retrotransposon in an intron of this pseudogene may have been responsible for the silencing of this GPAT locus in P. anomala. This study suggests that, although it is unlikely that universal nuclear gene markers free from paralogy are usually available, low-copy nuclear genes can be very useful in plant phylogenetic reconstruction, especially at low taxonomic levels, as long as the evolutionary dynamics of the genes are carefully examined.     

GPAT3 and GPAT4 are regulated by insulin-stimulated phosphorylation and play distinct roles in adipogenesis

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of glycerolipids. Mammals have at least four GPAT isoforms. Here we report the further characterization of the two recently identified microsomal GPAT3 and GPAT4. Both enzymes are highly expressed in adipose tissues. However, while GPAT3 is highly (∼60-fold) induced during adipocyte differentiation, GPAT4 induction is only modest (∼5-fold), leading to a lower abundance of GPAT4 mRNA in adipocytes. While overexpression of GPAT3 and GPAT4 in either insect or mammalian cells results in a comparable increase of GPAT activity, shRNA-mediated knockdown of GPAT3, but not GPAT4, in 3T3-L1 adipocytes led to a significant decrease in GPAT activity, a profound inhibition of lipid accumulation, and a lack of expression of several adipogenic markers during adipocyte differentiation. These data suggest that GPAT3 may encode the major GPAT isoform in adipocytes and play an important role in adipogenesis. Furthermore, we have shown that both GPAT3 and GPAT4 are phosphorylated by insulin at Ser and Thr residues, leading to increased GPAT activity that is sensitive to wortmannin. Our results reveal a link between the lipogenic effects of insulin and microsomal GPAT3 and GPAT4, implying their importance in glycerolipid biosynthesis.     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

A conserved seven amino acid stretch important for murine mitochondrial glycerol-3-phosphate acyltransferase activity. Significance of arginine 318 in catalysis

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. We previously cloned the cDNA sequence to murine mitochondrial GPAT (Yet, S-F., Lee, S., Hahm, Y. T., and Sul, H.S. (1993) Biochemistry 32, 9486-9491). We expressed the protein in insect cells which was targeted to mitochondria, purified, and reconstituted mitochondrial GPAT activity using phospholipids (Yet, S.-F., Moon, Y., and Sul, H. S. (1995) Biochemistry 34, 7303-7310). Deletion of the seven amino acids from mitochondrial GPAT, (312)IFLEGTR(318), which is highly conserved among acyltransferases in glycerolipid biosynthesis, drastically reduced mitochondrial GPAT activity. Treatment of mitochondrial GPAT with arginine-modifying agents, phenylglyoxal and cyclohexanedione, inactivated the enzyme. Two highly conserved arginine residues, Arg-318, in the seven amino stretch, and Arg-278, were identified. Substitution of Arg-318 with either alanine, histidine, or lysine reduced the mitochondrial GPAT activity by over 90%. On the other hand, although substitution of Arg-278 with alanine and histidine decreased mitochondrial GPAT activity by 90%, replacement with lysine reduced activity by only 25%. A substitution of the nonconserved Arg-279 with either alanine, histidine, or lysine did not alter mitochondrial GPAT activity. Moreover, R278K mitochondrial GPAT still showed sensitivity to arginine-modifying agents, as in the case of wild-type mitochondrial GPAT. These results suggest that Arg-318 may be critical for mitochondrial GPAT activity, whereas Arg-278 can be replaced by a basic amino acid. Examination of the other conserved residues in the seven amino acid stretch revealed that Phe-313 and Glu-315 are also important, but conservative substitutions can partially maintain activity; substitution with alanine reduced activity by 83 and 72%, respectively, whereas substituting Phe-313 with tyrosine and Glu-315 with glutamine had even lesser effect. In addition, there was no change in fatty acyl-CoA selectivity. Kinetic analysis of the R318K and R318A mitochondrial GPAT showed an 89 and 95%, respectively, decrease in catalytic efficiency but no major change in substrate binding as indicated by the K(m) values for palmitoyl-CoA and glycerol 3-phosphate. These studies indicate importance of the conserved seven amino acid stretch for mitochondrial GPAT activity and the significance of Arg-318 for catalysis.