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 mtG-PAT 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 acylCoA to form 1-acyl-glycerol 3-phosphate (LPA). 1 This reaction is catalyzed by two glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) isoenzymes that are encoded by different genes (1). 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 (2). 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 (1).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 (2). 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 (3...

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

“…We previously characterized GPAT2 in mitochondria from liver obtained from GPAT1 null mice [3]. Unlike GPAT1, GPAT2 was sensitive to NEM.…”

“…1A). GPAT2 also differs from GPAT1 because it exhibits no preference for 16:0-CoA compared to 18:1-CoA [3]. Interestingly, in light of the similarities between GPAT2 and chloroplast GPATs, chloroplast GPAT is believed to mediate chilling sensitivity in plants by altering the membrane content of unsaturated fatty acids in phosphatidylglycerol [25].…”

“…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.…”

“…This isoform, GPAT2, was first recognized because a polyclonal antibody raised to full-length GPAT1 identified a protein of similar molecular mass in liver from GPAT1 null mice. Characterization of GPAT2 activity in liver mitochondria from Gpat1 −/− mice showed that, unlike GPAT1, GPAT2 is sensitive to NEM inactivation, has no preference for palmitoyl-CoA compared with oleoyl-CoA, is inhibited by dihydroxyacetone-phosphate and polymixin B, is unaffected by incubation with acetone, and is more sensitive to thermal inactivation [3]. …”

“…The 38 amino acid gap found between motifs 1 and 2 in GPAT1 and the AGPAT isoforms is 12 residues shorter, the usual 35 amino acid gap between motifs 2 and 3 is 53 residues, and the amino acid sequences of both motif 2 and motif 3 are more similar to those of plant chloroplast GPATs than to E. coli plsB or to GPAT1. Firm identification of the hypothetical GPAT2 motifs as critical for catalysis will require mutational studies.We previously characterized GPAT2 in mitochondria from liver obtained from GPAT1 null mice [3]. Unlike GPAT1, GPAT2 was sensitive to NEM.…”