PURIFICATION AND RECONSTITUTION OF MURINE MITOCHONDRIAL GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE - FUNCTIONAL EXPRESSION IN BACULOVIRUS-INFECTED INSECT CELLS

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the intial step in glycerolipid biosynthesis. We recently cloned a cDNA to a 6.8-kb mR NA, a message that can be induced dramatically by feeding a high-carbo hydrate diet [Paulauskis & Sul (1988) J. Biol. Chem. 263, 7049-7054; S hin et al. (1991) J. Biol. Chem. 266, 23834-23839], and identified the open reading frame, p90, as mitochondrial GPAT [Yet et al. (1993) Bio chemistry 32, 9486-9491]. To initiate characterization of mitochondria l GPAT, we purified and reconstituted the GPAT activity using phosphol ipids after expressing functional enzyme in Sf9 insect cells. infectio n with recombinant virus containing p90 sequence resulted in high leve ls of GPAT expression in mitochondria, compared to noninfected cells o r cells infected with the reverse orientation insertion baculovirus. T here was a dramatic increase in N-ethylmaleimide-resistant mitochondri al GPAT activity. The GPAT protein was not detectable by Western blot in noninfected Sf9 cells or in cells infected with the GPAT sequence i n the reverse orientation. However, in cells infected with GPAT in the correct orientation, there was a dramatic increase in the GPAT protei n that was readily detectable by Coomassie staining both in total extr acts and in the mitochondrial fraction. To ease the purification, we n ext 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. After solubilization of the m itochondrial fraction with the nonionic detergent C(12)E(8), We purifi ed the GPAT fusion protein using a Ni2+ matrix column. The purified p9 0 protein was not enzymatically active, but the GPAT activity could be reconstituted by adding crude soybean phosphatidylcholine. Other phos pholipids in decreasing order of effectiveness in reconstituting GPAT activity were phosphatidylserine, phosphatidylinositol, and phosphatid ylethanolamine. Cardiolipin, a major mitochondrial membrane phospholip id, was least effective. Using GPAT expressed in mitochondria of the S f9 insect cells, we determined the apparent K-m value for glycerol 3-p hosphate to be 0.67 mM. When various fatty acyl-CoAs were compared as acyl donors, GPAT showed preference for saturated fatty acyl-CoAs from carbon lengths of 8 to 16 as substrate, and unsaturated fatty acyl-Co As tested were only 20% as effective. We also demonstrated that the ca talytic activity of GPAT was lost when a stretch of 78 amino acids (aa 250-327), a region that has sequence homology to Escherichia coli GPA T, was deleted. GPAT, expressed at a high level in mitochondria employ ing a baculovirus system, can be purified in a single step and will be useful in the future for the structure-function studies.