Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals

Nerve endings of the posterior pituitary are densely populated by dense- core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.     

ISOLATED NERVE ENDINGS (NEUROSECRETOSOMES) FROM THE POSTERIOR PITUITARY : Partial Separation of Vasopressin and Oxytocin and the Isolation of Microvesicles

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40–80 mµ diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.     

The content and composition of gangliosides in brain subcellular fractions of the frog Rana temporaria

Ganglioside distribution in various frog brain subcellular fractions (myelin, microsomes, mitochondria, synaptosomes, plasma membranes of nerve endings and synaptic vesicles) was investigated. The synaptosomes and plasma membranes of nerve endings were found to be the main places of ganglioside localization, ganglioside concentration being 2.42 and 1.79 times higher than that in homogenates. Gangliosides were shown to be present in synaptic vesicles. The characteristic features of gangliosides from frog brain and its subcellular fractions are the predominance of polysialogangliosides with 3-5 sialic acid residues (up to 57.4%), low content of monosialogangliosides (not more than 7%) and the presence of disialogangliosides with short carbohydrate chain. The increase of ganglioside content per one nerve cell during phylogenetic development of vertebrates is discussed.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5'-nucleotidase, Na+, K+-activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000--33000 are found. A gel-electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr 160000, 147000, 34000 and 25000) was made. Comigration of major bands was detected in one-dimensional gel electrophoresis with the 42000-Mr, 35000--33000-Mr and 34000-Mr components. Upon two-dimensional gel electrophoresis the 42000-Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin-like polypeptides is unlikely. Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.     

ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND : Electron Microscopy of Fractions Obtained by Centrifugation

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.     

Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.     

Purification of bovine neurosecretory granule membranes by density gradient centrifugation

A procedure for the subfractionation of neurosecretory granules into membrane and content components is described. The procedure involves the hypotonic lysis of the secretory granule fraction and further purification of the membranes by centrifugation through a discontinuous sucrose gradient. The neurosecretory granule membranes represented 5.2% of the total proteins of the neurosecretory granule fraction and were highly enriched in cytochrome b561. Electron microscopic analysis of the purified membranes showed vesicles devoid of electrodense content.     

The fine structure of nerve endings on rat thyroid follicular cells

Summary Axon profiles in thyroid glands obtained from adult male Wistar rats were studied electron-microscopically, using common and serial thin sections.Bouton profiles of nerve fibers, resembling the terminal or en passant type, often appeared closely associated with vascular smooth muscle cells via basement membranes. These structures are probably adrenergic, since they contained mainly small-core vesicles (mean diameter: 41.2 nm), in addition to a few large-core (mean diameter: 88.4 nm) and flattened vesicles.Nerve fibers containing microtubules and sometimes mitochondria and vesicles were seen lying between basement membranes and follicular cells. The incidence of nerve fiber contacts on profiles of follicular cells was 0.0177±0.0092 (S.D.). Using serial sections, follicles were seen to have up to two nerve endings, separated from the plasma membranes of the follicular cells by a gap of 22 nm. They contained mainly flattened vesicles and several large-core vesicles (mean diameter: 95.1 nm). Small-core vesicles were rarely seen in these nerve endings. Furthermore, subsurface cistern-like rough endoplasmic reticulum was found immediately under the plasma membranes of follicular cells facing membranes of nerve endings. These results suggest that the nerve fibers in contact with follicular cells are different from the adrenergic type.     

The same 15 kDa proteolipid subunit is a constituent of two different proteins in torpedo, the acetylcholine releasing protein mediatophore and the vacuolar HATPase

Using the monoclonal antibody 15KI, we have studied, at the cellular and subcellular levels, the distribution of a 15 kDa proteolipid, identified as the subunit of mediatophore, a presynaptic membrane protein able to release acetylcholine when activated by calcium. Aside from the electric lobe, the antigen distribution in the brain of Torpedo paralleled that of the synaptic vesicle antigen SV2 and did not appear to be related to that of acetylcholine and choline acetyltransferase. The 15 kDa proteolipid antigen was therefore present in all nerve endings and not restricted to cholinergic ones. At the ultrastructural level, on cholinergic nerve endings, the antigen was detected associated to synaptic vesicles and, to a lesser extent, to the presynaptic plasma membrane. Indeed, considering the high sequence homology between the mediatophore subunit (Birman et al., 1990) and the proteolipid subunit of the vacuolar type H⁺ATPase, a major enzyme constituent of synaptic vesicles, this distribution was not surprising.

To determine whether antibody 15KI recognizes the vacuolar type H⁺ATPase, we chose a non neuronal cell type which possesses a high content of this enzyme, the kidney proton secreting epithelial cells. Indeed, antibody 15KI intensely labelled the apical plasma membrane of mitochondria rich epithelial cells in kidney tubules. A high density of the antigen was also found associated to intracellular membrane structures such as lysosomal multivesicular bodies, both in kidney epithelial cells and in electromotoneurons. The 15 kDa proteolipid antigen was associated with other vacuolar H⁺ATPase subunits in kidney membranes which was not the case in presynaptic plasma membranes. This illustrates that the 15 kDa proteolipid antigen is a constituent of two different protein complexes, which exhibit very different functional properties.     

The zinc iodide-osmium tetroxide (ZIO) reaction on nerve endings in the median eminence of the rat under normal and experimental conditions

Summary The reaction of nerve endings in the median eminence of the rat to zinc iodide-osmium tetroxide (ZIO) staining was examined electron microscopically under normal and experimental conditions. The experimental condition of catecholamine exhaustion in the nerve endings was induced by the administration of H44/68 and reserpine. Vesicles in the terminals of catecholaminergic nerves reacted similarly to ZIO staining in both normal and experimental material. The majority of synaptic vesicles in various terminals gave a positive ZIO reaction. The neurosecretory elementary granules, however, failed to react with ZIO. On the other hand, some nerve terminals in the external layer of the median eminence showed a strong positive reaction in the cytoplasmic matrix, in mitochondria as well as in synaptic vesicles. These findings strongly suggest that the ZIO-positive substance in nerve terminals is not the transmitter itself, i.e. the monoamine, but rather represents a range of substances commonly found in various kinds of synaptic vesicles and is probably proteinaceous in nature. A brief discussion is also given on the difference in ZIO reactivity between neurosecretory elementary granules and small vesicles in the hypothalamo-hypophyseal tract.This work was supported in part by a research grant from the Ministry of Education, Japan     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

Axon terminals with unusual vesicles in the brain of the polychaete,Ophryotrocha puerilis

Summary In the brain of Ophryotrocha puerilis swollen nerve endings filled with electron-lucent vesicles and aggregates of vesicles were observed. The vesicles do not resemble elementary neurosecretory granules. Tests for biogenic amines were negative; no dense-core vesicles were found. The vesicle type described here cannot be related to any of the types thus far found in nerve cells.Supported by Deutsche Forschungsgemeinschaft Pf116/3     

Phospholipid Transverse Diffusion in Synaptosomes: Evidence for the Involvement of the Aminophospholipid Translocase

We have studied in Torpedo marmorata electric organ synaptosomes the equilibration kinetics of spin-labeled phospholipid analogues initially incorporated into the outer plasma membrane monolayer. As assayed by evoked releases of both ATP and acetylcholine, the nerve endings were closed vesicles containing an energy source. The aminophospholipids (phosphatidylethanolamine and phosphatidylserine) were translocated toward the inner membrane leaflet faster and to a higher extent than their choline-containing counterparts (phosphatidylcholine and sphingomyelin). This difference was abolished by incubation of synaptosomal membranes with N-ethylmaleimide, suggesting that the accumulation of aminophospholipids in the inner layer was driven by a protein. This phenomenon is comparable with what was described in plasma membranes of other eucaryotic cells (erythrocyte, lymphocyte, platelet, fibroblast), and thus we would suggest that an aminophospholipid translocase, capable of moving the aminophospholipids from the outer to the inner layer at the expense of ATP, is also present in the synaptosomal plasma membrane.     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Ultrastructural and morphometric studies of nonsecretory neurons of the rat supraoptic nucleus

Ultrastructural and morphometric studies were made on nonsecretory and complementarily neurosecretory neurons of the rat supraoptic nucleus. 6% of perimeter of nonsecretory neuronal perikarya was covered by presynaptic endings. The value was well in agreement with that of interneurons elsewhere. The covering ratio of neurosecretory neurons was 12%. The perikarya of nonsecretory and neurosecretory neurons bear an average of 14 and 49 axon terminals, respectively. Nonsecretory neurons should be interneurons, receiving much less information than neurosecretory neurons.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

Summary The general ultrastructural features of the hypothalamo-neurohypophysial system in rats with hereditary hypothalamic diabetes insipidus (DI-rats, Brattleboro strain) are described. There is no decisively distinguishing difference between the neurons of the supraoptic and paraventricular nuclei. The neurons of both nuclei show signs of active protein synthesis. The perikarya of the neurons are markedly hypertrophic, the nuclei are large and the nucleoli prominent. In the cytoplasm there are numerous ribosomes, abundant rough-surfaced endoplasmic reticulum and extensive Golgi complexes. However, very few neurosecretory granules are to be seen. The axons of the hypothalamo-neurohypophysial tract are likewise enlarged and the paucity of neurosecretory granules is a striking feature also in the area of the tract. The majority of nerve endings in the posterior pituitary of DI-rats are devoid of neurosecretory granules. Microvesicles are abundant in the nerve endings and there are findings which suggest that microvesicles are involved either in endoor exocytosis. The signs of active protein synthesis and the concomitant paucity of neurosecretory granules are interpreted to imply transportation of the secretory proteins in an extragranular phase. The possible mode of release of the secretory proteins from the nerve endings and the role of microvesicles therein are discussed.This study has been supported by grants from the Finnish Cultural Foundation and the Sigrid Jusélius Foundation. The collaboration of Professors Antti Arstila and Tapani Vanha-Perttula is gratefully acknowledged.The Brattleboro-rats were kindly provided by Dr. Heinz Valtin, to whom we express our thanks.     

Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.     

Partial characterization of a growth-inhibiting protein in 3T3 cell plasma membranes

A plasma membrane-enriched fraction from 3T3 cells has been detergent-solubilized, and the supernatant of this solubilization was reconstituted into liposomes using soybean lecithin. When these vesicles were added to actively growing cells, cell growth rates were inhibited to levels that were comparable to those observed with the original plasma membranes (at least 50% of maximum growth rates). Liposomes without proteins, or liposomes containing proteins from SV3T3 plasma membranes did not significantly inhibit growth of 3T3 cells. Treatment of the reconstituted vesicles with urea or high concentrations of salt did not eliminate the growth-inhibiting properties of these reconstituted membranes. These results indicate that the specific growth-inhibiting factor in 3T3 cell plasma membranes is a membrane protein that has significant non-polar interactions with the membrane bilayer.