title: 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.
authors.affiliation: Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Tal M
authors.initials: TM
authors.lastname: Lewin
authors.forename: Nicole M J
authors.initials: NM
authors.lastname: Schwerbrock
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Lee
authors.forename: Rosalind A
authors.initials: RA
authors.lastname: Coleman
mesh_terms: D000096:Acetone / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000906:Antibodies; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008930:Mitochondria, Liver / Q000201:enzymology*; D011112:Polymyxin B / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000900:Anti-Bacterial Agents; D000906:Antibodies; D007527:Isoenzymes; D000096:Acetone; D005992:Glycerol-3-Phosphate O-Acyltransferase; D011112:Polymyxin B