Solubilization of the plastidial lysophosphatidylcholine acyltransferase from Allium porrum leaves: towards plants devoid of eukaryotic plastid lipids?

To analyse the involvement of the plastidial lysophosphatidylcholine (lyso-PC) acyltransferase in the import of the extraplastidial lipid precursors required for eukaryotic plastid lipid synthesis, we plan to obtain transgenic plants. Since no sequence of lyso-PC acyltransferase is known, the purification of this enzyme has been undertaken to establish its sequence. First we determined the conditions allowing the solubilization of this membrane-bound enzyme. It is shown that by using CHAPS as a detergent, a lyso-PC acyltransferase activity is associated with the solubilized proteins.

Transfer and subsequent metabolism of lysolipids studied by immobilizing subcellular compartments in alginate beads.

The transfer and subsequent metabolism of lysophosphatidylcholine between subcellular compartments were studied in vitro by embedding membranes in alginate beads. After several experiments to validate the process, it was demonstrated that lysophosphatidylcholine was transferred from microsomes embedded in alginate beads to immobilized chloroplasts, that this transfer involved the partition of this molecule, and that the imported lysophosphatidylcholine was further used as substrate for phosphatidylcholine biosynthesis. More generally, the technique used makes it possible to avoid any cross-contamination between compartments, to evidence a transfer of molecules, and to study the metabolism of the imported molecules in the acceptor compartment.

Occurrence of an acyl-CoA:1-acylglycerophosphorylcholine acyltransferase in plant mitochondria.

In the presence of oleoyl-CoA, purified and intact mitochondria from potato tuber formed phosphatidylcholine from labeled lysophosphatidylcholine. The labeled oleoyl moiety of the acyl-CoA was also incorporated in the absence of exogenous lysolipids, such incorporation being largely increased by the addition of exogenous lysophosphatidylcholine. In the presence of various other lysophospholipids, no synthesis of the corresponding phospholipids was observed, suggesting a high specificity of the acyltransferase towards the acyl acceptor. This enzyme was chiefly located in the outer membrane of mitochondria. These results indicate that any acylglycerophosphorylcholine transferred from the endoplasmic reticulum to mitochondria may be acylated to phosphatidylcholine.

Acylation of endogenous acyl acceptors by mouse sciatic nerve microsomes.

Phospholipid (chiefly phosphatidylcholine) labeling from radioactive acyl-CoAs by mouse sciatic nerve microsomes is observed in the absence of added acyl acceptors. The maximal acylation (ca 10% of administered) for 10 micrograms microsomal proteins is observed at relatively low amounts of oleoyl-CoA (0.2-0.3 nmol) and decreases as the acyl-CoA amount increases. Labeled lysophosphatidylcholine (almost exclusively esterified at position 2) is also observed, particularly when the [1-14C]oleoyl-CoA concentration is higher than 0.2-0.3 nmol/50 microliters. The labeled acyl group is mainly inserted in position 2] of the glycerophosphorylcholine. With 0.15 nmol labeled oleoyl-CoA, phosphatidylcholine acylation increases as a function of the protein amount and reaches 25% of the added label at 40 microgram proteins. It is evaluated that, in the presence of 10 microgram proteins, 2% of the microsomal phosphatidylcholine molecules are acylated from 0.1 nmol acyl-CoA. The acylation mechanism seems to involve an acyl exchange between acyl-CoA and phosphatidylcholine.

Synthesis of phosphatidylcholine in the chloroplast envelope after import of lysophosphatidylcholine from endoplasmic reticulum membranes.

Purified, intact chloroplasts from Allium porrum seedlings are able to synthesize phosphatidylcholine by acylating lysophosphatidylcholine (but not glycerophosphocholine) with acyl-CoAs. The acyltransferase activity is located in the envelope of chloroplasts. It is specific for lysophosphatidylcholine and the neosynthesized lipids have a C18 fatty acid esterified to the sn-2 position of the glycerol backbone ('eukaryotic lipids'). By preincubating endoplasmic reticulum membranes with labeled lysophosphatidylcholine, it was shown that this molecule could be transferred by a partition process from the endoplasmic reticulum to chloroplasts where they are acylated to yield phosphatidylcholine.