Mitochondrial glycerol phosphate acyltransferase contains two transmembrane domains with the active site in the N-terminal domain facing the cytosol

The topography of mitochondrial glycerol-3-phosphate acyltransferase (GPAT) was determined using rat liver mitochondria and mutagenized recombinant ra t GPAT (828 aa (amino acids)) expressed in CHO cells. Hydrophobicity analys is of GPAT predicts two transmembrane domains (TMDs), residues 472-493 and 576-592. Residues 224-323 correspond to the active site of the enzyme, whic h is believed to lie on the cytosolic face of the outer mitochondrial membr ane. Protease treatment of rat liver mitochondria revealed that GPAT has a membrane-protected segment of 14 kDa that could correspond to the mass of t he two predicted TMDs plus a loop between aa 494 and 575. Recombinant GPAT constructs containing tagged epitopes were transiently expressed in Chinese hamster ovary cells and immunolocalized. Both the C and N termini epitope tags could be detected after selective permeabilization of only the plasma membrane, indicating that both termini face the cytosol. A 6-8-fold increas e in GPAT-specific activity in the transfected cells confirmed correct prot ein folding and orientation. When the C terminus and loop-tagged GPAT const ruct was immunoassayed, the epitope at the C terminus could be detected whe n the plasma membrane was permeabilized, but loop-epitope accessibility req uired disruption of the outer mitochondrial membrane. Similar results were observed when GPAT was truncated before the second TMD, again consistent wi th an orientation in which the loop faces the mitochondrial intermembrane s pace. Although protease digestion of the HA-tagged loop resulted in preserv ation of a 14-kDa fragment, consistent with a membrane protected loop domai n, neither the truncated nor loop-tagged enzymes conferred GPAT activity wh en overexpressed, suggesting that the loop plays a critical structural or r egulatory role for GPAT function. Based on these data, we propose a GPAT to pography model with two transmembrane domains in which both the N (aa 1-471 ) and C (aa 593-end) termini face the cytosol and a single loop (aa 494-575 ) faces the intermembrane space.