EXPRESSION AND IDENTIFICATION OF P90 AS THE MURINE MITOCHONDRIAL GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. Mitochondrial GPAT, unlik e the microsomal isozyme, prefers saturated fatty acids as a substrate . We have recently reported cloning of a cDNA to an unidentified 6.8-k b mRNA by a differential hybridization. The mRNA contains an open read ing frame of 827 amino acids (p90) with 30% sequence homology in a 300 amino acid stretch to Escherichia coli GPAT. The 6.8-kb mRNA was indu ced dramatically when fasted mice were refed a high-carbohydrate diet. Here, we have expressed the open reading frame as trpE fusion protein s and used them to generate antibodies. The antibodies recognized a po lypeptide of 90 kDa (p90) when the 6.8-kb cDNA sequence was used for i n vitro transcription and translation. By Western blot analysis using these antibodies, we detected p90 in mitochondrial fractions of liver, and the p90 level was increased by refeeding. The increase in the p90 level correlated with the increase in mitochondrial GPAT activity. Mo reover, p90 was not detectable in 3T3-L1 preadipocytes but markedly in creased during adipose conversion. This increase was consistent with t he 11-fold increase we observed in N-ethylmaleimide (NEM)-resistant mi tochondrial GPAT activity during adipocyte differentiation. In additio n, we have expressed p90 in CHO cells by stable transfection. The tran sfected genes in both correct and reverse orientations produced distin ct 3.9-kb transcripts owing to the truncation of a part of the noncodi ng regions of the endogenous 6.8-kb mRNA before insertion into the pMS XND vector. The transfected CHO cells were treated with 2-aminopurine, an agent that increases expression of exogenous genes. There was a 6- fold increase in the p90 level in mitochondria of the CHO cells transf ected with the p90 sequence in the correct orientation, and the activi ty of the NEM-resistant mitochondrial GPAT also increased accordingly. Moreover, the mitochondrial GPAT of the transfected cells preferred p almitoyl CoA as a substrate over oleoyl CoA. The above correlation bet ween the level of p90 expression and GPAT activity in mitochondria and the substrate specificity of the expressed enzyme show evidence that p90 is the murine mitochondrial GPAT. We have also demonstrated here t hat the changes in mitochondrial GPAT activity during different nutrit ional and developmental conditions are due primarily to the changes in enzyme concentration, and not to modulation of the catalytic activity of the existing enzyme.