Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in glycerolipid biosynthesis. We recently cloned a cDNA to a 6.8-kb mRNA, a message that can be induced dramatically by feeding a high-carbohydrate diet [Paulauskis & Sul (1988) J. Biol. Chem. 263, 7049-7054; Shin et al. (1991) J. Biol. Chem. 266, 23834-23839], and identified the open reading frame, p90, as mitochondrial GPAT [Yet et al. (1993) Biochemistry 32, 9486-9491]. To initiate characterization of mitochondrial GPAT, we purified and reconstituted the GPAT activity using phospholipids after expressing functional enzyme in Sf9 insect cells. Infection with recombinant virus containing p90 sequence resulted in high levels of GPAT expression in mitochondria, compared to noninfected cells or cells infected with the reverse orientation insertion baculovirus. There was a dramatic increase in N-ethylmaleimide-resistant mitochondrial GPAT activity. The GPAT protein was not detectable by Western blot in noninfected Sf9 cells or in cells infected with the GPAT sequence in the reverse orientation. However, in cells infected with GPAT in the correct orientation, there was a dramatic increase in the GPAT protein that was readily detectable by Coomassie staining both in total extracts and in the mitochondrial fraction. To ease the purification, we next expressed GPAT as a polyhistidine fusion protein in insect cells. The polyhistidine tag did not interfere with targeting to mitochondria or with the catalytic activity of GPAT.(ABSTRACT TRUNCATED AT 250 WORDS)