Phosphatidic Acid Synthesis in Mitochondria

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80–100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A2. The amount of phospholipase A2-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.

Several lines of evidence suggest that the mitochondrial GAT regulates the selective positioning of saturated fatty acids at the sn-1 position of glycerophospholipids. The selective positioning of the fatty acids is considered to be important in the structural and functional role of biological membranes (13,14). Unlike the microsomal GAT, the mitochondrial enzyme has strong preference for saturated fatty acyl-CoAs as substrate (4,5). In Ehrlich ascites tumor cells (15) and in primary tissue culture cells (16), the mitochondrial acyltransferase activity is inversely proportional to the randomization of fatty acids at position 1. Finally, lysophosphatidic acid (LPA), synthesized in mitochondria, can exit the organelle, be transported to the endoplasmic reticulum in the presence of liver fatty acid binding protein (L-FABP), and be converted to PA (17). Recent experiments from different laboratories also suggest that the activity of mitochondrial GAT can change under different physiological (18,19) and nutritional conditions (20,21) and in the presence of some metabolic modulators (22). These observations suggest that mitochondrial GAT can be controlled at the level of transcription as well as by modulators of activity.
Biochemical investigations using proteases suggest that the rat mitochondrial GAT is a transmembrane protein (23). Cloning and sequencing of the cDNA (GenBank TM accession number U36771) revealed the presence of two transmembrane regions (12), supporting the biochemical observation.
Our previous work suggests that acyl-CoA synthetase, which activates fatty acids, spans the MOM (24). By using desulfo-CoA-agarose, an immobilized competitive inhibitor, we established that the catalytic site of the enzyme is located in the outer aspect of the MOM. The acyl-CoA formed can be used either catabolically for ␤-oxidation of the fatty acids in the mitochondrial matrix or can be used anabolically to acylate glycerophosphate. The synthesis of acyl-CoA on the outer surface of the MOM leads to the question: which leaflet of the MOM is involved in the synthesis of LPA and PA?
This paper deals with the location of synthesis of LPA and PA in the transverse plane of the MOM and the extent of translocation of PA necessary for cardiolipin synthesis. Our result suggests that both LPA and PA are synthesized on the outer aspect of the MOM, and subsequently, PA moves to the inner membrane as a precursor of cardiolipin.
Preparation of Mitochondria and Microsome-Liver mitochondria and microsome were prepared from 175-200-g male Harlan Sprague-Dawley rats as described previously (4). The purity of both preparations was evaluated by performing GAT assay in the presence and absence of 2 mM N-ethylmaleimide, which is an inhibitor of the microsomal GAT (15). Cross-contamination between mitochondrial and microsomal fractions was Ͻ3%.
Analytical Methods-GAT activity was measured by following the incorporation of sn-[2-3 H]glycerol 3-phosphate into butanol-extractable phospholipids (15). Asolectin was omitted from the system. The concen-tration of the subcellular protein in the incubation medium was maintained between 0.2 and 0.4 mg/ml. For sedimenting mitochondria or microsomes, the incubated mixture was cooled to 0°C and spun at 10,000 ϫ g for 15 min or at 105,000 ϫ g for 60 min, respectively. The pellet was resuspended in 0.5 ml of water. The supernatant, the resuspended pellet, or whole GAT reaction mixture was treated with 1-butanol to extract the radioactive acylation products, LPA and PA, which were separated by thin layer (4) or high performance thin layer chromatography. Protein concentration was assayed as per the Bradford method (25) using bovine serum albumin as a standard.

Activity of Mitochondrial and Microsomal GAT Using
Palmitoyl-CoA-Agarose as Acyl Donor-Palmitoyl-CoA is commercially available cross-linked to 4% beaded agarose. As the linkage of agarose is with the amino group of CoA with a 7-carbon spacer, it is not possible that the activated acyl group can cross the MOM phospholipid bilayer and reach the inner aspect of the membrane. Microsomal GAT has its catalytic site on the outer surface of the membrane (7) and was used as a positive control. Fig. 1 documents the activity of mitochondrial and microsomal GAT at different concentrations of palmitoyl-CoA and palmitoyl-CoA-agarose. For both mitochondria and microsomes, their activities of GAT is over 90% in the presence of immobilized substrate when compared with the activities in presence of free palmitoyl-CoA. Since the concentration of palmitoyl-CoA in the immobilized sample cannot be accurately measured, comparison can be made between the activities in the presence of free and bound palmitoyl-CoA at their optimal level of activity.
The palmitoyl-CoA-agarose was washed four times before use. There was virtually no difference in the GAT activity using washed or unwashed samples suggesting that the beads contained no free palmitoyl-CoA. To determine whether free palmitoyl-CoA was released during the assay, we measured the incorporation of glycerophosphate into phospholipids at 1, 2, and 3 min of incubation with optimal concentrations of free and immobilized substrate. In both instances, the rate of incorporation was linear with time (results not shown). Therefore, the acyl-CoA was indeed immobilized and was not available to the inner side of the MOM. The activity of GAT in the presence of palmitoyl-CoA-agarose was not a reflection of disruption of mitochondria. The latency of cytochrome oxidase of this and similarly prepared samples revealed only 10 -15% disruption of mitochondria.
The Effect of Immobilized Polymyxin B-Agarose on Mitochon- drial and Microsomal Glycerophosphate Acyltransferase-It is known that polymyxin B stimulates the mitochondrial GAT and markedly inhibits the microsomal enzyme (Refs. 26 -28, Fig. 2A). In the presence of polymyxin B-agarose, mitochondria and microsomes showed activation and inhibition in the range of 80 -90% of the values obtained in the presence of free polymyxin B (Fig. 2C). The immobilized polymyxin B is cross-linked to 4% beaded agarose through an amino group with a spacer of 1 carbon. It is, therefore, improbable that the antibiotic can penetrate the MOM. Fig. 2 also includes the results using palmitoyl-CoA-agarose and polymyxin B (Fig. 2B) and palmitoyl-CoA-agarose and polymyxin B-agarose (Fig. 2D). Both the free and immobilized polymyxin B stimulated the mitochondrial and inhibited the microsomal GAT. For reason presently unknown, polymyxin B-agarose, at higher concentrations, is less effective in both stimulating and inhibiting mitochondrial and microsomal GAT, respectively (Fig. 2, C and D).
Acylation Products Formed in the Presence of Immobilized Substrate-Amounts of LPA and PA synthesized in mitochondria and microsomes using palmitoyl-CoA and palmitoyl-CoAagarose as acyl donor are documented in Fig. 3. Both the phospholipids were formed in equal quantities in presence of free palmitoyl-CoA in mitochondria (15). In microsomes, the amount of LPA was about 30%. A similar profile was seen with the use of palmitoyl-CoA-agarose. In the absence of bovine serum albumin, mainly PA was formed in both microsomes and mitochondria.
Role of L-FABP in the Export of Mitochondrial LPA and PA-As reported earlier (17), L-FABP stimulates the export of LPA from mitochondria. The presence of L-FABP stimulates LPA synthesis 6-fold but reduced PA synthesis by 50%. In the absence of L-FABP, mitochondria can synthesize significant  , q), or PA-loaded mitochondria were incubated for 30 min at 25°C prior to addition of these proteins (छ, ‚). The mitochondria were pelleted and processed as described in the legend to Fig. 5. Results are presented as the amount released from one aliquot (0.44 mg of mitochondrial protein). amount of PA, which remains in the mitochondria. However, this PA was marginally exported when mitochondria were exposed to L-FABP (Fig. 4). When mitochondria, loaded with PA, were immediately exposed to 35 M L-FABP, up to 21% of PA left the mitochondria. The amount of PA available for export decreased with time, suggesting that the PA became inaccessible for binding to L-FABP.
Preferential export of LPA from mitochondria due to L-FABP is documented in Fig. 5. Mitochondria, loaded with PA, were treated with liver cytosol, which resulted in partial conversion of PA to LPA and glycerides. Subsequent exposure of these mitochondria to L-FABP resulted in 94% release of LPA but only 22% release of PA and 6% release of glycerides. Data shown in Fig. 4 suggest that, with time, PA within mitochondria is becoming less available for export. This observation was confirmed by another approach. Mitochondria, loaded with PA, were exposed either to phospholipase A 2 alone or to a mixture of phospholipase A 2 and L-FABP. LPA, generated by phospholipase A 2 from PA, was exported 2.5-fold more efficiently in the presence of L-FABP and phospholipase A 2 than in the presence of phospholipase A 2 only (Fig. 6). When PA-loaded mitochondria were incubated for 30 min in buffer A prior to phospholipase A 2 and L-FABP, the amount of exported LPA was reduced to 30%. However, mitochondria after 30-min incubation contained 85-95% of originally present PA. Thus, breakdown of PA was not responsible for reduction in PA release. DISCUSSION Two main points emerge from this investigation. First, both LPA and PA are synthesized on the cytosolic side of the MOM. Second, the PA synthesized rapidly moves from the outer surface, presumably to the inner membrane where it is converted to cardiolipin (29). In the presence of the immobilized substrate palmitoyl-CoA-agarose, the GAT activity of isolated mitochondria was about the same as that in the presence of the free acyl-CoA (Fig. 1), suggesting that the outer surface of the MOM is the site of LPA formation. This conclusion is in keeping with the results obtained by the use of the immobilized stimulator of the mitochondrial GAT, polymyxin B-agarose. Since mitochondrial GAT could be stimulated approximately 2-fold in the presence of either free or immobilized polymyxin B (Fig. 2), it is suggestive that the catalytic site of mitochondrial GAT is exposed to the cytosolic side of the MOM.
If LPA is formed on the outer surface of the MOM, which side of the membrane is PA formed? Analysis of the acylation products (Fig. 3) revealed that both LPA and PA were formed in the presence of the free or agarose-bound palmitoyl-CoA. Absence of bovine serum albumin in the incubation medium similarly affected the LPA:PA ratio with the two forms of the acyl-CoA. As expected (15), in the absence of bovine serum albumin, PA was the main reaction product. These results strongly suggest that both LPA and PA are made on the outer side of the MOM. Formation of both the phospholipids is stimulated in the presence of free or agarose-bound polymyxin B (results not shown), further confirming the site of formation of the phospholipids in the transverse plane of the MOM.
The results obtained here, together with those reported earlier on the formation acyl-CoA on the cytosolic side of the MOM (24), indicate that the three enzymes, acyl-CoA synthetase, GAT, and MGAT, all can draw on the cytosolic pool of substrates. This situation raises the possibility that these three enzymes are closely located and that there could be an efficient substrate "channeling" between these enzymes.
The PA synthesized on the outer surface of the MOM leaves the organelle to a very limited extent (Figs. 4 and 6). It can be converted to diacylglycerol by phosphatidate phosphohydrolase (30) or can be converted back to LPA under certain conditions by phospholipase A 2 , which is also located in the MOM (31,32). The fate of the mitochondrially synthesized PA is its conversion to cardiolipin, the final step of which takes place in the inner membrane (33,34). There is precursor-product relationship between PA and cardiolipin when PA-loaded mitochondria are incubated in a cardiolipin-synthesizing medium. Furthermore, the final step of cardiolipin synthesis takes place on the inner side of the inner membrane (29). Thus, there is a complex topological movement of PA from the outer surface of the outer membrane to the inner surface of the inner membrane. Transport of PA from the outer to inner membrane may occur either by simple diffusion (35) or by some other mechanism, for example, involving some transport protein. However, the movement of PA from the outer to inner membrane fits in with our observation that incubation of PA-loaded mitochondria renders the phospholipid inaccessible to externally added phospholipase A 2 (Fig. 6).
It appears that each of the products of the three enzymes, acyl-CoA synthetase, GAT, and MGAT, has at least two possible fates (Fig. 7). The acyl-CoA synthesized in the mitochondria can either be transported to the mitochondrial matrix for ␤-oxidation or be acted upon by GAT to form LPA. It is known that fatty acid biosynthesis is regulated by metabolic modulators (36). Similarly, as GAT is influenced by ATP and citrate (22), they can very well be involved in this regulation. Similar to acyl-CoA, the LPA formed in the MOM has also two fates. It can either be acylated to form PA (6), or it can combine with L-FABP and be exported to the endoplasmic reticulum for conversion to PA (17,37) and presumably to other phospholipids. LPA-FABP does not act as a substrate for mitochondrial MGAT (17). A small amount of PA can leave the mitochondria. Whether this PA is transported to the endoplasmic reticulum for conversion to complex phospholipids is unknown. However, the majority of mitochondrially synthesized PA appears to be converted to cardiolipin. The dual fate of acyl-CoA, LPA, and PA suggest the existence of multiple control sites in the metabolism of these compounds.