The chemical structure of synaptic membranes

Brain Research, 62 (1973) 405--411

405

© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands

THE CHEMICAL STRUCTURE OF SYNAPTIC MEMBRANES

I. G. MORGAN, J.-P. ZANETTA, W. C. BRECKENRIDGE, G. VINCENDON ANDG. GOMBOS Centre de Neurochimie du C N R S et Institut de Chimie Biologique, Facult~ de Mddecine, 67085 Stras, bourg Cedex (France)

Based upon the now classical techniques of Whittaker 47 and De Robertis 14, we have recently reported a method for preparing synaptosomal plasma membranes (SPM) with purity over 80 ~o31. The major contaminant appears to be fragments of the outer mitochondrial membrane 31, and several parameters suggest that contamination with glial membranes is limited zg. With similar modifications, we have prepared synaptic vesicles (SV) with a purity of 90 ~o, the only contaminant being fragments of the SPM 30. SV were richer in lipid than the SPM (lipid-protein ratios of 1.5 and 1.2 respectively). In lipid class composition, the SV contained only cholesterol and phospholipids 5, whereas SPM contained cholesterol, phospholipids and gangliosides4. Lipid sialic acid is highly concentrated in SPM, and is in fact as reliable a marker for the SPM as is (Na + + K+)-ATPasel, a and neuraminidase 4z. The cholesterol-phospholipid molar ratio of the SPM was lower than that found for other plasma membranes 1s,21,37,45 but was similar to that of the SV. A low cholesterol-phospholipid molar ratio has been reported for other excitable plasma membranes7,9, 4s. The phospholipid compositions of the two membranes were very similar4, 5. Ethanolamine and choline phosphoglycerides were the major phospholipids. Serine phosphoglycerides were present in lower amounts and only low levels of inositol phosphoglycerides and sphingomyelins were detected. The latter result is interesting, since generally plasma membranes contain appreciable amounts of sphingomyelinis, 21,37,45. A low level of sphingomyelin may be a feature of other excitable membranesg, 48. The fact that only low levels of lysophosphatidyl choline were detected is important. It has been suggested that this phospholipid could mediate the presumptive vesicle membrane-plasma membrane fusion which occurs during exocytosis, at least in the adrenal medulla 1. While lysolecithin does induce membrane fusione6,35, this hypothesis, in its simple form, cannot be applied to the central nervous system, and it appears difficult to apply to other secretory systems12,24,~s,46. Two features of the fatty acid compositions of the SV and SPM phospholipids stand out. The ethanolamine and serine phosphoglycerides contain high levels of docosahexaenoic acid4,5, but this fatty acid does not appear to be specific to the functionally coupled SV and SPM, since the phospholipids of synaptosomal mito-

4O6 TABLE

I. G . M O R G A N

el a[.

1

CHARACTERISTICS OF THE LIPID COMPOSITIONS OF SYNAPTIC MEMBRANES

Synaptosomal plasma

Svnaptic vesicles

Synaptosomal mitochondria

membranes

Low cholesterol-phospholipid molar ratio (relative to non-neural plasma membranes) Low sphingomyelin content (relative to non-neural plasma membranes) High ganglioside content Sphingomyelin fatty acids, predominantly stearic acid High levels of docosahexaenoic acid in ethanolamine and serine phosphoglycerides

÷ I

÷

+

chondria were also rich in docosahexaenoic acid ~. Since non-synaptosomal mitochondria contain levels of docosahexaenoic acid around half that of the synaptosomal mitochondria 2, it may be that the phospholipids of neuronal mitochondria, and more generally of neuronal membranes, contain this fatty acid in concentrations higher than those found in the phospholipids of astrocyte and oligodendrocyte membranes. In support of this possibility docosahexaenoic acid does seem to be present at high levels in several tissues which contain excitable membranes 6,7,9,aa,a4,4s. The second feature is that both SV and SPM sphingomyelins were virtually devoid of long chain fatty acids4,L Stearic acid was the major component, in contrast to the situation with the sphingomyelins of myelin and isolated oligodendroc/tes 16, cultured astrocytes z and most other tissues 42. It may be significant that neuronal gangliosides have a fatty acid composition which is similar to that of the synaptosomal sphingomyelins 3s. From these results, which are in agreement with those of others 1°,15,~2, it is at least possible to conclude that the characteristic features of the lipid composition of the SPM are reproduced in that of the SV - - with the exception of the ganglioside content (Table I). While the reasons for this similarity are obscure, objections previously raised to the exocytosis mechanism of transmitter release based on the different compositions of the SV and SPM do not seem to be valid. Since the biochemical and morphological evidence for an exocytosis-endocytosis cycle during transmitter release is now rather convincing 8,19,2°,23,32,a6,4°,41, the problem is to explain how the gangliosides do not become involved in the process of membrane fusion. This could be explained by accepting a fluid mosaic model for membranes 39 where only the SV patches, devoid of gangliosides, would be involved in the exocytosis-endocytosis cycle, or by placing the gangliosides in the outer layer of the greater membrane 25. We have also determined the protein and glycoprotein compositions of the SV and SPM, after reduction and carboxymethylation of the membranes (Fig. 1). The

CHEMICAL STRUCTURE OF SYNAPTIC MEMBRANES

407

Fig. 1. Protein profiles of synaptosomal plasma membranes (SPM), synaptic junctions (SJ) and synaptic vesicles (SV). Membranes were dissolved in 1% SDS, 1% fl-mercaptoethanol, 2 M urea and dialysed overnight against 30 m M iodoacetamide in 0.2 ~ SDS. The carboxymethylated polypeptide chains were electrophoresed on 12 ~ acrylamide gels in the presence of 0.2 ~ SDS. Gels were stained with amido black.

SPM contain a large number of polypeptide chains, of which three form the major components, and correspond in molecular weight to the polypeptide chains of purified bovine brain (Na + -k K+)-ATPase44. This is in agreement with the high (Na + ÷ K+) ATPase activity of the SPM 31. The SPM contain three low molecular weight glycoproteins and several high molecular weight bands. By contrast, SV have simple protein and glycoprotein profiles. There are 7 major polypeptide chains, and one major low molecular weight glycoprotein. Several high molecular weight glycoproteins are also present. Neither by determining molecular weights (Table II) nor by mixing experiments have we been able to detect protein or glycoprotein bands specific to the SV such as has been reported by other workers 27. All the bands detected in the SV could be seen in the SPM. At the present time, these results can be interpreted in terms of a relatively long incorporation of the SV membrane into the SPM during the exocytosis-endocytosis cycle. Other interpretations of' these results have not been excluded however, and it will be necessary to investigate the dynamics of" the system to establish whether the SV proteins are temporarily incorporated into the SPM during transmitter release.

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I.G. MORGAN et al.

TABLE 1I MOLECULARWEIGHTSOF SYNAPTOSOMALPLASMAAND SYNAPTICVESICLEMEMBRANEPROTEINSAND GLYCOPROTEINS(determined in 12 × 2.5 acrylamide gels in 0.2 ~ SDS) Synaptosomal plasma membranes

Synaptic vesicles

Proteins

Glycoproteins

Proteins

Glycoproteins

120,000

128,000 96,000

120,000

66,000

64,000 58,000 53,000

68,000

43,000

41,500

210,000 180,000 160,000

125,000 98,000 93,000

64,000 58,000 53,000 52,000

49,000 42,000 39,000 37,500 35,700 32,500 30,300

29,200 24,800 22,500 20,800 18,100 16,100 14,000 12,000

48,000

45,000

3~500

34,000

23,000

35,800 32,500 29,600 24,500 21,100 18,200 13,800 12,000

Figures in italics give the molecular weights of the major species.

The fact that certain stimulation conditions lead to an increased synaptic surface area8,19, 36 is in accord with this idea. Methods have been reported for isolating synaptic junction-enriched fractions (SJ) from Triton X-100 extracted SPM 11,13,17. The morphological and chemical characterization of these fractions is far from complete, but preliminary results on their protein and glycoprotein composition are of some interest. Several of the SV protein bands, which are present in the SPM as only minor components, are concentrated in the putative SJ fractions (Fig. 1). However not all the SV bands are found in the SJ fractions, and there are several bands which are not found in the SV but which are present in the SJ. It is tempting to relate the latter bands to the intrinsic constituents of the synaptic j unction, but this identification is, at the moment, only tentative. However it may be possible to relate our observations to those of Streit et al. 41 who suggest that the liaison between the SV and the SPM at the point of fusion may be relatively stable. F r o m their results on the number of presumptive exocytosis sites per sq./zm of synaptic junction surface, it is possible to calculate that there should be

CHEMICAL STRUCTUREOF SYNAPTIC MEMBRANES

409

approximately 2 sq. #m of vesicle membrane per sq. #m of synaptic junction. In this case it would not be surprising to find a concentration of SV-like proteins in SJ fractions. Our studies on the chemical composition of the SV and SPM have thus shown that both these membranes have a characteristic lipid composition, of which certain features may be generally characteristic of excitable membranes. Both membranes have relatively simple protein compositions, and isolation and analysis of individual proteins now seem feasible. These studies in no way contradict the possibility of an exocytosis-endocytosis cycle for the SV in nerve-endings, and in particular the common proteins of SV, SPM and SJ may be explained by such a cycle. Further studies are in progress to investigate the cytological significance of our observations. This work has been in part supported by grants from the Fondation pour la Recherche Mddicale fran~aise and the Institut National de la Santd et de la Recherche Mddicale (contract 7111698). WCB was a Fellow of the Medical Research Council of Canada. IGM and JPZ are Attachds de Recherche and GG is Chargd de Recherche of the Centre National de la Recherche Scientifique.

1 BLASCHKO,H., FIREMARK,H., SMITH,A. D., AND WINKLER,H., Lipids of the adrenal medulla. Lysolecithin, a characteristic constituent of chromaffin granules, Biochem. J., 104 (1967) 545-549. 2 BRECKENRIDGE,W. C., Unpublished results. 3 BRECKENRIDGE,W. C., GOMBOS,G., AND MORGAN,I. G., The dososahexaenoic acid of the phospholipids of synaptic membranes, vesicles and mitochondria, Brain Research, 33 (1971) 581-583. 4 BRECKENRIDGE,W. C., GOMBOS,G., AND MORGAN,I. G., The lipid composition of adult rat brain synaptosomal plasma membranes, Biochim. biophys. Acta (Amst.), 266 (1972) 695-707. 5 BRECKENRIDGE,W. C., MORGAN,I. G., ZANETTA,J. P., AND VINCENDON,G., Adult rat brain synaptic vesicles. II. Lipid composition, Biochim. biophys. Acta (Amst.), 320 (1973) 681-686. 6 BRECKENRIOGE,W. C., ET VINCENDON,G., L'acide docosahexaenoIque de l'organe 61ectriquede la Torpille, C.R. Aead. Sci. (Paris), 273 (1971) 1337-1339. 7 CAMEJO,G., VILLEGAS,G., BARNOLA,F. V., AND VILLEGAS,R., Characterization of two different membrane fractions isolated from the first stellar nerve of the squid Dosidicus gigos, Biochim. biophys. Acta (Amst.), 193 (1969) 247-259. 8 CECCARELLI,B., HURLBUT,W. P., AND MAURO,A., Depletion of vesicules from frog neuromuscular junctions by prolonged tetanic stimulation, J. Cell Biol., 54 (1972) 30-38. 9 CHACKO,G. K., GOLDMAN,n . E., ANDPENNOCK,B. E., Composition and characterization of the lipids of garfish (Lepisosteus osseus) olfactory nerve, a tissue rich in axonal membrane, Biochim. biophys. Acta (Amst.), 280 (1972) 1-16. 10 COTMAN, C. W., BLANK, M. L., MOEHL, A., AND SNYDER, F., Lipid composition of synaptic plasma membranes isolated from rat brain by zonal centrifugation, Biochemistry, 8 (1969) 4606-4612. 11 COTMAN,C. W., AND TAYLOR,D., Isolation and structural studies on synaptic complexes from rat brain, J. Cell Biol., 55 (1972) 696-711. 12 DA PRADA,M., PLETSCHER,A., AND TRANZER,J. P., Lipid composition of membranes of aminestorage organelles, Biochem. J., 127 (1972) 681-683. 13 DAVIS, G. A., AND BLOOM, e. E., Proteins of synaptic junctional complexes, J. Cell BioL, 47 (1970) 46A. 14 DE ROBERTI$,E., AND RODRIGUEZDE LORESARNAIZ,G., Structural components of the synaptic region. In A. LmTVlA(Ed.), Handbook ofNeurochemistry, Vol. II, Plenum Press, New York, 1969, pp. 365-392. 15 EICHBERG,J., WHITTAKER,V. e., AND DAWSON, R. M. C., Distribution of lipids in subcellular particles of guinea-pig brain, Biochem. J., 92 (1964) 91-100.

410

J.G. MORGAN et al.

16 FEWSTER,M. E., AND MEAD, J. F., Fatty acid and fatty aldehyde composition of glial cell lipids isolated from bovine white matter, J. Neurochem., 15 (1968) 1303 1312. 17 FISZER,S., AND DE ROBERTIS,E., Action of Triton X-100 on ultrastructure and membrane-bound enzymes of isolated nerve-endings from rat brain, Brain Research, 5 (1967) 31-44. 18 HENNING,R., KAULEN,H. D., ANDSTOFEEL,W., Isolation and chemical composition of the lysosomal and the plasma membrane of the rat liver cell, Hoppe-Seylers Z. physio/. Chem., 351 (1970) 1191-1199. 19 HEUSER,J., AND REESE,T. S., Stimulation-induced uptake and release of peroxidase from synaptic vesicles in frog neuromuscular junctions, Anat. Ree., 172 (1972) 329-330. 20 HOLTZMANN,E., FREEMAN,A. R., AND KASHNER, L. A., Stimulation-dependent alterations in peroxidase uptake at lobster neuromuscular junctions, Science, 173 (1971) 733-736. 21 KEENAN,T. W., MORRE,D. J., OLSON,D. E., YUNGHANS,W. N., ANDPATTON,S., Biochemical and morphological comparison of plasma membrane and milk fat globule membrane from bovine mammary gland, J. Cell Biol., 44 (1970) 80-93. 22 KISHIMOTO,Y., AGRANOEF,B. W., RADIN, N. S., AND BURTON, R. M., Comparison of the fatty acids of lipids of subcellular brain fractions, J. Neurochem., 16 (1969) 397 404. 23 KORNELIUSSEN,H., Ultrastructure of normal and stimulated motor endplates with comments on the origin and fate of synaptic vesicles, Z. Zellforsch., 130 (1972) 28--57. 24 LAGERCRANTZ,H., Lipids of the sympathetic nerve trunk vesicles. Comparison with adrenomedullary vesicles, Acta physiol, scand., 82 (1971) 567-570. 25 LEHNINGER,A., The neuronal membrane, Proe. nat. Acad. Sci. (Wash.), 60 (1968) 1069-1080. 26 LucY, J. A., The fusion of biological membranes, Nature (Lond.), 227 (1970) 814-817. 27 McBRIDE, W. J., AND VAN TASSEL,J., Resolution of proteins from subfractions of nerve-endings, Brain Research, 44 (1972) 177-187. 28 MELDOLESI,J., JAMIESON,J. D., AND PALADE,G. E., Composition of cellular membranes in the pancreas of the guinea pig. II. Lipids, J. Cell Biol., 49 (1971) 130-149. 29 MORGAN,I. G., REITH,M., MARINARI,U., BRECKENRIDGE,W. C., ANDGO~iBOS, G., The isolation and characterization of synaptosomal plasma membranes. In V. ZAMBOTTI,G. TETTAMANTIAND M. ARRIGONI(Eds.), Glycolipids, Glycoproteins and Mucopolysaccharides of the Nervous System, Plenum Press, New York, 1972, pp. 209-228. 30 MORGAN,I. G., VINCENDON,G., AND GOMBOS,G., Adult rat brain synaptic vesicles. I. Isolation and characterization, Biochim. biophys. Aeta (Amst.), 320 (1973) 671-680. 31 MORGAN,I. G., WOLFE, L. S., MANDEL,P., AND GOMBOS,G., Isolation of plasma membranes from rat brain, Biochim. biophys. Aeta (Amst.), 241 (1971) 737 751. 32 MusIcK, J., AND HUBBARD, J. I., Release of protein from mouse motor nerve terminals, Nature (Lond.), 237 (1972) 279-281. 33 N~ELSEN,N. C., FLEISCHER, S., AND MACCONNELL,D. G., Lipid composition of bovine retinal outer segment fragments, Biochim. biophys. Acta (Amst.), 211 (1970) 10-19. 34 POINCELO'I-,R. P., AND ABRAHAMSON,E. W., Fatty acid composition of bovine rod outer segments and rhodopsin, Bioehim. biophys. Aeta (Amst.), 202 (1970) 382-385. 35 POOLE, A. R., HOWELL, J. I., AND LUCY, J. A., Lysolecithin and cell fusion, Nature (Lond.), 227 (1970) 810-813. 36 Pvsn, J. J., AND WILEY, R. G., Morphological alterations of synapses in electrically stimulated superior ganglia of the cat, Science, 176 (1972) 191-193. 37 RYA, T. K., SKIPSKI, V. P., BARCLAY,M., ESSNER, E., AND ARCHIBALD,F. M., Lipid composition of rat liver plasma membranes, J. biol. Chem., 244 (1969) 5528-5536. 38 ROUSER, G., AND YAMAMOTO,A., The fatty acid composition of total gangliosides in normal human whole brain at different ages, J. Neurochem., 19 (1972) 2697-2698. 39 SINGER, S. J., AND NICOLSON, G. L., The fluid mosaic model of the structure of cell membranes, Science, 175 (1972) 720-731. 40 SMITH, A. D., Summing up: some implications of the neuron as a secreting cell, Phil. Trans. B, 261 (1971) 423-437. 41 STREIT,P., AKERT, K., SANDRI,C., LIVINGSTON,R. B., AND MOOR, H., Dynamic ultrastructure of presynaptic membranes at nerve terminals in the spinal cords of rats. Anesthetized and unanesthetized preparations compared, Brain Research, 48 (1972) 11-26. 42 SVENNERHOLM,E., ST.g.LLBERG-STENHAGEN,S., AND SVENNERHOLM,L., Fatty acid composition of sphingomyelins in blood, spleen, placenta, liver, lung and kidney, Biochim. biophys. Acta (Amst.), 125 (1966) 60-69.

CHEMICAL STRUCTURE OF SYNAPTIC MEMBRANES

411

43 TETTAMANTI,G., MORGAN, I. G., GOMBO$,G., VINCENDON,G., AND MANDEL, P., Sub-synaptosomal localization of brain particulate neuraminidase, Brain Research, 47 (1972) 515-518. 44 UESUGI,S., DULAK, N. C., DIXON,J. F., HEXUM,T. D., DAHL, J. L., PERDUE,J. F., AND HOKIN, L. E., Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. VI. Large scale partial purification and properties of a Lubrol-solubilized bovine brain enzyme, J. biol. Chem., 246 (1971) 531-543. 45 WEINSTEIN,D. B., MARSH,J. B., GLICK, M. C., AND WARREN, L., Membranes of animal cells. IV. Lipids of the L cell and its surface membrane, J. biol. Chem., 244 (1969) 4103-4111. 46 WHITE, D. A., AND HAWTHORNE,J. N., Zymogen secretion and phospholipid metabolism in the pancreas, Biochem. d., 120 (1970) 533-538. 47 WHITTAKER,V. P., The synaptosome. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. 11, Plenum Press, New York, 1969, pp. 327-364. 48 ZAMBRANO, F., CELLINO, M., AND CANESSA-FISHER,M., The molecular organization of nerve membranes. IV. The lipid composition of plasma membranes from squid retinal axons, J. Membrane Biol., 6 (1971) 289-303.