Stinulation of Phosphatidylethanolamine Exchange by Castor Bean Cytosol

Cytosol proteins prepared from castor bean endosperm (4-day-old) s stimulate the exchange of [IHlpbosphatidylethanolamine between liposomes and mitochondria. The acceleration of the exchange depends on the quantity of cytosol proteins, the time of incubation, and the respective amounts of liposomes and mitochondria. On a per seeding basis, the active proteins are essentiafly located in the endosperm, whereas the roots and the cotyledons are less rich in these proteins. Cytosols prepared from various plants, including potato tuber (7, 8), cauliflower buds and Jerusalem artichoke tuber (6), and castor bean endosperm (5, 18, 19), contain proteins that are able to accelerate the exchange of phospholipids between organelles (microsomes and mitochondria) or between liposomes and organelles. The exchange of phosphatidylcholine, a major phospholipid in plant organelles, has been studied in castor bean endosperm (5, 17, 18). No particular study has been devoted to the exchange of phosphatidylethanolamine, another major phospholipid of castor bean mitochondria (4). This paper shows that cytosol proteins from castor bean endosperm actively stimulate the exchange of phosphatidylethanolamine between liposomes and mitochondria. A preliminary account of this work has been presented (2). MATERIALS AND METHODS Plant Material. Seeds of castor bean (Ricinus communis L., var. Sanguin Vilmorin) were grown in moist Vermiculite in darkness at 30 C. The endosperm halves were removed after 4 days of germination. Isolation of Mitochondria. Mitochondria are isolated from endosperm halves (100 g fresh weight) and purified by sucrose gradient centrifugation according to Douady et al. (5). The mitochondrial suspensions (about 10 mg ml-') were immediately used or stored at -20 C. Proteins were determined by the biuret method with BSA as standard. Isolation of Cytosol Proteins. The postmitochondrial supernatant, obtained from the endosperm halves, was treated at pH 5.1 (7). After centrifugation of the supernatant at 16,000g for 10 mi, the proteins were precipitated with ammonium sulfate (75% saturation) and dialyzed overnight against 30 volumes of buffer A (0.25 M sucrose, I mm EDTA, 50 mM Tris-HCI [pH 7.4]). Preparation of I3HjPbosphatidylethanolamine. Potato tuber slices (50 g fresh weight) were shaken for 16 h at 25 C in a solution of [1-3HJethanol-2-amine hydrochloride (Amersham/Searle) (0.1 mCi/50 ml water). The lipids were then extracted and [3H]phos1No. 5 ofthe series: "Proteins and the Intracellular Exchange ofLipids." phatidylethanolamine was purified by TLC on silica gel plates (Merck) using chloroform-acetone-methanol-acetic acid-water (40:20:10:10:5, v/v) as solvent mixture for the separation of lipids (6). The specific radioactivity of [3Hjphosphatidylethanolamine was equal to about 7 nCi nmol'. Preparation of Liposomes. For 10 exchange assays, 1.25 mg soybean phosphatidylcholine (Sigma), 106 dpm I3Hlphosphatidylethanolamine, and 0.5 x 106 dpm cholesteryl-[1-14CJoleate were introduced in a flask. After evaporation of the solvent, the residue was suspended in 5 ml buffer A. The suspension was sonicated for 30 min at 20-30 C under N2 (MSE disintegrator, 150 w). After adjustment of the volume to 5 ml, the liposomes were immediately used or stored at +4 C no longer than 2 days. Exchange Assays. Mitochondria (2.2-3 mg of protein) were mixed with labeled liposomes (0.5 ml) and eventually 544 mg cytosol proteins, in a final volume of 2.5 ml, adjusted with buffer A. After incubation at 30 C for 15-120 min, 5 ml cold buffer A were added and the mixture was centrifuged at 16,000g for 10 min. The lipids were extracted from the mitochondrial pellets and from the supernatants containing the liposomes (5). After evaporation of the solvent, the lipids were suspended in 2 ml tolueneethanol (4:1, v/v). 3H and 14C contents were determined in an Intertechnique-Kontron scintiUlation counter. Phospholipid phosphorus was determined according to Shibuya et al. (15).