Proc. Nati. Acad. Sci. USA Vol. 75, No. 5, pp. 2348-2352, May 1978 Cell Biology Enzymatic synthesis and rapid translocation of phosphatidylcholine by two methyltransferases in erythrocyte membranes (phosphatidylethanolamine/S-adenosyl-L-methionine/phospholipases/membrane asymmetry/flip-flop) FUSAO HIRATA* AND JULIUS AXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20014 Contributed by Julius Axelrod, March 9, 1978 ABSTRACT The synthesis of phosphatidylcholine from taining 1 mM MgCl2 (8, 9). The ghosts were washed with 10 phosphatidylethanolamine is carried out by two methyltrans- volumes of 0.5 mM Tris phosphate buffer, pH 8.0, containing ferases in erythrocyte membranes. The first enzyme uses 1 mM MgCl2. Inside-out ghosts were prepared by the same phosphatidylethanoramine as a substrate, requires Mg2+, and has a high affinity for the methyl donor, S-adenosyl-L-methio- procedure except that MgCl2 was excluded from the solutions. nine. The second enzyme methylates phosphatidyl-N-mono- To complete the vesiculation, inside-out ghosts were passed methylethanolamine to phosphatidyicholine and has a low af- through a 27-gauge needle in 1-ml syringe and then purified finity for S-adenosyl-L-methionine. The first enzyme is localized on a dextran density gradient (8, 9). Both inside-out and on the cytoplasmic side of the membrane and the second en- right-side-out ghosts were washed with 10 volumes of 50 mM zyme faces the external surface. This asymmetric arrangement pH containing 5 mM MgCl2 and of the two enzymes across the membrane makes possible the Tris/glycylglycine buffer, 8.0, stepwide methylation of phosphatidylethanolamine localized resuspended in the same buffer. Sidedness and membrane on the side and facilitates the rapid transmembrane impermeability were measured by the activity of acetylcholine transfercytoplasmico the final product, phosphatidylcholine, to the ex- esterase, a marker enzyme on the exterior surface of the ternal surface of the membrane. A mechanism for an enzyme- membrane (9). In seven different preparations of inside-out mediated flip-flop of phospholipids from the cytoplasmic to the ghosts, the mean (±SD) esterase activity without Triton X-100 outer surface of erythrocyte membranes is described. was 11.5 ± 4.5% of that with Triton X-100; the value for seven different preparations of right-side-out ghosts was 90.8 + 6.5%. Phospholipids are a major structural constituent of cell mem- To measure the methyltransferase activities (7), leaky ghosts branes. These lipids are asymmetrically distributed in biological were prepared by the addition of 1% Nonidet P40 or by brief membranes with amino phosphatides largely facing the cyto- sonication in hypotonic solutions as indicated in the legends. plasmic side and choline phosphatides mainly on the outside Introduction of S-Adenosylmethionine into the Inside of (1-4). How lipid asymmetry originates and is maintained in Erythrocyte Ghosts. Three milliliters of packed cells was sus- biological membranes is important to understanding cell pended in 7 ml of 50 mM Tris/glycylglycine buffer, pH 8.0, membrane structure and function. Spontaneous diffusion across containing 5 mM Mg9l2 and 0.2 mM S-adenosyl-L-[methyl- a lipid bilayer (flip-flop) is a possible process by which lipids 3H]methionine (880 dpm/pmol) and incubated overnight could achieve asymmetry. However, the spontaneous rate of (approximately 16 hr) at 40. Before use, the ghosts were washed flip-flop has been found to be slow, taking place over several with 10 volumes of 0.9% NaCl and then resuspended in 7 ml hours (5, 6). of 100 mM Tris/glycylglycine buffer, pH 8.0, containing 5 mM Recently, we identified and described the properties of two MgCl2. methyltransferases that catalyze the stepwise methylation of The enzymatic incorporation of the [3H]methyl group into phosphatidylethanolamine to form phosphatidylcholine in the membrane lipids was started by warming the test tube con- bovine adrenal medulla (7). The observation that the substrate, taining 50 ,ul of the ghosts suspension from 40 to 370. Methyl- phosphatidylethanolamine, resides on the cytoplasmic side ation of phospholipids did not proceed to any significant degree whereas the product, phosphatidylcholine, is mainly located at 4°. The activities of the two methyltransferases were deter- on the exterior side of the membrane (1-4) has led us to study mined as described (7) except that the reactions were carried the localization of the two methyltransferases and translocation at pH 8.0 in the presence of 1% Nonidet P40. of phospholipids across erythrocyte membranes. A rapid, en- Determination of Asymmetry of Transmethylases and zyme-facilitated mechanism to achieve the asymmetry of Phospholipids. The sidedness of the two transmethylases was phospholipids is described in this communication. determined by comparing their destruction by proteolytic di- METHODS AND MATERIALS gestion of resealed inside-out and right-side-out ghosts. The reaction mixture contained 100 mM Tris/glycylglycine buffer Preparation of Inside-Out and Right-Side Out Erythrocyte (pH 8.0), 5 mM MgCl2, 1 mg of trypsin (Worthington Bio- Ghosts. Fresh samples of blood were obtained from ether- chemicals), and ghosts equivalent to 2 mg of protein in a final anesthetized male Sprague-Dawley rats (200 g) by cardiac volume of 1 ml. At various time intervals, 100-,ul aliquots were puncture with heparin as anticoagulant. All procedures were removed and mixed with 100 ,ul of a pancreatic trypsin inhibitor performed at 40 unless otherwise specified. The erythrocytes (Worthington Biochemicals) solution (2 mg/ml). Fifty micro- were washed three times with 10 volumes of 0.9% NaCl. liters of the mixture was assayed for each methyltransmethylase Right-side-out ghosts were prepared by hemolysis of the cells (7). The control was run with trypsin inhibitor added prior to with 40 volumes of 5 mM Trisphosphate buffer, pH 8.0, con- the start of the reaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked * Visiting Scientist of the National Institute of Mental Health, on leave "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate from Department of Medical Chemistry, Kyoto University Faculty this fact. of Medicine, Kyoto, 606, Japan. 2348 Downloaded by guest on September 26, 2021 Cell Biology: Hirata and Axelrod Proc. Nati. Acad. Sci. USA 75 (1978) 2349 Table 1. Properties of phospholipid methyltransferase system in Table 2. Sidedness of enzymatically methylated phospholipids in rat erythrocytes erythrocyte ghosts [3H]Methyl incorporation, [3H]Methyl incorporation in lipids, SAM, MgCl2, pmol/mg protein/hr S-Adenosyl-L- pmol/mg protein/hr methionine, MM Right-side-out Inside-out MM mM PME PDE PC Inside Medium ghosts ghosts 4 0 0.9 0.15 0.3 4 5 3.9 0.4 0.6 200 0 26.5 2.9 200 0 3.1 9.6 11.1 0 200 3.6 29.4 200 5 9.9 11.3 14.2 S-Adenosyl-L-[methyl-3H]methionine was introduced inside the Erythrocytes were lysed and centrifuged. The pellets were sonicated ghosts or was added to the medium just prior to the start of the re- for 10 sec by the use of Biosonik, N. (Brownwill Co. Ltd., Rochester, action. The incubation was carried out at 370 for 1 hr. NY). The reaction medium contained 25 mM Tris/glycylglycine, pH 9.5, containing 0.5 mM Na2 EDTA, sonicated ghosts equivalent to 0.5 mg of protein, and additions as indicated in a total volume of 50,ul. the absence of Mg2 , much less of the N-methylated com- The mixture was incubated for 60 min at 37°. The reaction products pounds was detected. However, when a high concentration of were measured after separation by thin-layer chromatography with S-adenosyl-L-methionine (0.2 mM) was incubated with soni- the solvent system n-propyl alcohol/propionic acid/chloroform/water, 3:2:2:1 (vol/vol) (7). SAM, S-adenosyl-L-methionine; PME, phos- cated ghosts in the presence of Mg2+, all three N-methylated phatidyl-N-monomethylethanolamine; PDE, phosphatidyl-NN- compounds were synthesized. After the addition of the N- dimethylethanolamine; PC, phosphatidylcholine. monomethylated derivative of phosphatidylethanolamine to the sonicated vesicles, the synthesis of di- and trimethylated lipids proceeded to almost the same extent, even in the absence When asymmetry of phospholipids in membranes was de- of Mg2+. These results indicate that the conversion of phos- termined, the reaction mixture was centrifuged at 27,000 X g phatidyl-N-monomethylethanolanine to phosphatidylcholine for 45 min at 40 and then the ghosts were resuspended in 25 ,l does not require Mg2+. Both enzymes had a pH optimum of of 100 mM Tris/glycylglycine buffer, pH 8.0, containing 10 10.5. Because S-adenosyl-L-methionine is unstable above pH mM CaC12. The reaction was started by the addition of 10 p1 9, the reaction was carried out at pH 8.0 unless otherwise of phospholipase C from Bacillus cereus (Boehringer specified. From the results described above, we have concluded Mannheim, 2 mg/ml) or from Clostridium welchii (Sigma, 10 that phosphatidylcholine is synthesized in erythrocyte mem- mg/ml) at 370. After incubation for 15 min, the reaction was branes as follows: stopped by the addition of 3 ml of chloroform/methanol/ phosphatidylethanolamine hydrochloric acid, 2:1:0.02 (vol/vol). Under these conditions, 40-56% were TFase I of the total phospholipids of the ghosts hydrolyzed o- phosphatidyl-N-monomethylethanolamine when the lipid phosphate contents were measured in mem- Mg2+, SAM branes, indicating that phospholipase C acted only on the lipid TFase II layer facing the outside medium. '- phosphatidyl-N,N-dimethylethanolamine SAM RESULTS TFase II - * phosphatidylcholine Properties of Phosphatidylethanolamine Methyltrans- SAM ferases in Erythrocytes. When the ghosts, sonicated in 25 mM in which SAM is S-adenosyl-L-methionine and TFase is phos- Tris/glycylglycine buffer, pH 8.0, were incubated with 0.2 mM pholipid methyltransferase.