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

Abstract

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including Nethylmaleimide (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 88.8 kDa 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 μM; 0.25μCi) [1-14 C]oleate for 6 h 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 ∼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.The synthesis of triacylglycerol and all glycerophospholipids begins with the acylation of glycerol-3-phosphate with long-chain fatty acyl-CoA to produce 1-acylglycerol-3-phosphate. This reaction is catalyzed by glycerol-3-phosphate acyltransferase (GPAT 1 ; EC 3.1.3.9) which exhibits the lowest specific activity of all enzymes in the glycerol-3-phosphate pathway, suggesting that it is the rate limiting step [1].Three mammalian GPAT activities have been differentiated based on their subcellular location and biochemical properties [2,3]. GPAT activity is present in microsomal and mitochondrial Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. fractions. In most tissues, 90% of the activity is attributed to a microsomal GPAT which is sensitive to inactivation by sulfhydryl reagents such as NEM. Microsomal GPAT3, prominent in adipose tissue, has been cloned [4]. Microsomal GPAT activity does not appear to be regulated in liver. In contrast, the mitochondrial isoform (GPAT1) 2 that has been purified [5] and cloned [6,7], is highly regulated by i...

Creatine maintains intestinal homeostasis and protects against colitis

“…For example, delta(4)-desaturase, sphingolipid 2 (Degs2) regulates biosynthesis of the lipid ceramide, which is a regulator of whole organism metabolism (26)(27)(28). In addition, mitochondrial translation initiation factor 3 (Mtif3), glycine N-acyltransferase (Glyat), and glycerol-3-phosphate acyltransferase-2 (Gpat2) are localized to the mitochondria and presumably exert their functions there (29)(30)(31).…”

Thematic Review Series: Glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity

“…GPAT2 mRNA is most abundant in testis (?50-fold higher compared with other tissues), but is also detectable in most tissues, with highest expression in liver and brown adipose tissue. Unlike GPAT1, GPAT2 mRNA in the liver is not regulated by fasting (68). Little is known about the function of GPAT2.…”

“…This activity, termed GPAT2, showed a substrate and inhibitor profile similar to microsomal GPAT, but could be distinguished from microsomal GPAT by its ability to cross-react with antibodies raised against mtGPAT1 (49). The gene encoding this activity was cloned based on its relatively high sequence similarity to mtGPAT1 (27% identity; 70% similarity), and was termed GPAT2 (68,69). When overexpressed in mammalian cells, GPAT2 localizes to mitochondria and confers an ?30% increase in NEM-sensitive GPAT activity (68,69).…”

“…The gene encoding this activity was cloned based on its relatively high sequence similarity to mtGPAT1 (27% identity; 70% similarity), and was termed GPAT2 (68,69). When overexpressed in mammalian cells, GPAT2 localizes to mitochondria and confers an ?30% increase in NEM-sensitive GPAT activity (68,69). GPAT2 mRNA is most abundant in testis (?50-fold higher compared with other tissues), but is also detectable in most tissues, with highest expression in liver and brown adipose tissue.…”

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells

“…GS cells turned out to be quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. Moreover, using the GS cells, we showed by coimmunoprecipitation and mass analysis that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein that has a catalytic domain for the synthesis of lysophosphatidic acid from glycerol-3-phosphate and long-chain acyl-CoA (Wang et al 2007), is one of MILI-binding proteins. We further showed in gene knockdown experiments that GPAT2 plays a critical role in piRNA production.…”