Dipak Haldar, Ph.D.

Function of the Mitochondrial Outer Membrane

We are convinced that mitochondrial outer membrane (MOM) has an important function in cellular lipid metabolism. Our research is mainly focused on the MOM enzyme glycerophosphate acyltransferase (GAT). This enzyme catalyzes the first and committed step in the pathway of glycerolipid synthesis. GAT can switch the fate of long-chain fatty acyl-CoA molecules synthesized by acyl-CoA synthetase (ACS) from undergoing b-oxidation to synthesis of lysophosphatidic acid (LPA).

Several lines of evidence suggest that the mitochondrial GAT can contribute to the asymmetric distribution of fatty acids found in cellular phospholipids particularly phosphatidyl choline (Vancura and Haldar, 1992). The LPA synthesized by mitochondrial GAT can be converted to PA and finally transferred to the inner membrane and converted to cardiolipin (Schlame and Haldar, 1993). Alternatively, the LPA can exit the mitochondria and with the help of fatty acid binding protein be transported to the endoplasmic reticulum where the LPA is converted to PA and presumably to other phospholipids (Vancura and Haldar, 1992). The physiological function the endoplasmic reticular GAT is presently unclear.

We have used immobilized substrate, inhibitor or activator to determine the orientation of the catalytic sites of mitochondrial GAT, monoacyl-GAT and ACS. All of these lipid-metabolizing enzymes have their catalytic sites exposed to the cytosolic surface of the MOM (Hesler, Olymbios and Haldar, 1990; Chakraborty, Vancura, Balija and Haldar, 1999). The similar orientation of the catalytic sites of these enzymes raises the possibility of substrate channeling.

We have purified rat liver mitochondrial GAT to homogeneity (Vancura and Haldar, 1994) and cloned and sequenced its cDNA (Nikonov, Morimoto and Haldar, 1998). The purified protein is inactive and requires addition of exogenous phospholipids for activity. Dioleyl derivatives of some phospholipids are much more effective than the corresponding dipalmityl derivatives in reconsituting GAT activity. These results suggest that mitochondrial GAT that prefers saturated fatty acyl-CoA's as substrate, may function to maintain a balance of saturated and unsaturated fatty acids in membranes.

Computer analysis of the mitochondrial GAT cDNA indicated that the protein has two transmembrane regions, the inter-transmembrane region is exposed to the cytosol, has several protein kinase sites, and a targeting sequence near the N-terminal region. We have provided experimental evidence for the existence of the two transmembrane regions and the overall topography of the enzyme in the transverse plane of the MOM (Balija, Chakraborty, Nikonov, Morimoto and Haldar, 2000). Protein kinase C, casein kinase II and tyrosine kinase stimulate the mitochondrial GAT as do ATP and citrate -- two allosteric modulators. We have used several chimeric proteins consisting of different regions of the N-terminal end of mitochondrial GAT and green fluorescence protein to experimentally establish the targeting sequence of the protein.

We are presently working on transcriptional and posttranslational control of mitochondrial GPAT by hormones and growth factors.