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.