Identification and Characterization of the Major Proteins of Mammalian Brain Synaptic Vesicles

Highly purified rat and cow brain synaptic vesicles contain major proteins with molecular weights of approximately 74,000, 60,000, 57,000, 40,000, 38,000, and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the major proteins on synaptic vesicles was confirmed by immunoprecipitation of intact rat brain synaptic vesicles with a synaptic vesicle-specific monoclonal antibody. The 40,000-Mr protein appeared to be identical to the 38,000-Mr integral membrane glycoprotein, p38 or synaptophysin, previously identified as a major component of mammalian synaptic vesicles. The isoelectric point of the 75,000-Mr proteins from either rat or cow brain synaptic vesicles is 5.0, and the pI of the 57,000-Mr protein is approximately 5.1 in both species. The similarity in size and charge of several major proteins in rat and cow synaptic vesicles suggests a high degree of structure conservation of these proteins in diverse mammalian species and raises the possibility that a set of functions common to most or all mammalian synaptic vesicles is mediated by these proteins.     

How peptide hormone vesicles are transported to the secretion site for exocytosis

Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.     

Vesicles Bearing Toxoplasma Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption

Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.     

beta'-COP, a novel subunit of coatomer.

Several lines of evidence favour the hypothesis that intracellular biosynthetic protein transport in eukaryotes is mediated by non-clathrin-coated vesicles (for a review see Rothman and Orci, 1992). The vesicles have been isolated and a set of their surface proteins has been characterized as coat proteins (COPs). These COPs exist in the cytosol as a preformed complex, the coatomer, which was prior to this study known to contain six subunits: four (alpha-, beta-, gamma- and delta-COP) with molecular weights between 160 and 58 kDa, and two additional proteins of approximately 36 and 20 kDa, epsilon- and xi-COP. Here we describe a novel subunit of the coatomer complex, beta'-COP. This subunit occurs in amounts stoichiometric to the established COPs both in the coatomer and in nonclathrin-coated vesicles and shows homology to the beta-subunits of trimeric G proteins.     

Phospholipid diversity: correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called "fusion proteins". This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Proteomics analyses of microvesicles released by Drosophila Kc167 and S2 cells

Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions depending on their origin. One subtype designated circulating microvesicles (MVs) provides a novel form of intercellular communication and recent work suggested the release and uptake of morphogens in vesicles by Drosophila cells. In this study, we have examined cells of the hemocyte-like cell lines Kc167 and S2 and identified secreted vesicles in the culture supernatant. The vesicles were isolated and found to have characteristics comparable to exosomes and plasma membrane MVs released by mammalian cells. In wingless-transfected cells, the full-length protein was detected in the vesicle isolates. Proteomics analyses of the vesicles identified 269 proteins that include various orthologs of marker proteins and proteins with putative functions in vesicle formation and release. Analogous to their mammalian counterparts, the subcellular origin of the vesicular constituents of both cell lines is dominated by membrane-associated and cytosolic proteins with functions that are consistent with their localization in MVs. The analyses revealed a significant overlap of the Kc167 and S2 vesicle proteomes and confirmed a close correlation with non-mammalian and mammalian exosomes.     

A complete set of SNAREs in yeast

Trafficking of cargo molecules through the secretory pathway relies on packaging and delivery of membrane vesicles. These vesicles, laden with cargo, carry integral membrane proteins that can determine with which target membrane the vesicle might productively fuse. The membrane fusion process is highly conserved in all eukaryotes and the central components driving membrane fusion events involved in vesicle delivery to target membranes are a set of integral membrane proteins called SNAREs. The yeast Saccharomyces cerevisiae has served as an extremely useful model for characterizing components of membrane fusion through genetics, biochemistry and bioinformatics, and it is now likely that the complete set of SNAREs is at hand. Here, we present the details from the searches for SNAREs, summarize the domain structures of the complete set, review what is known about localization of SNAREs to discrete membranes, and highlight some of the surprises that have come from the search.     

Detection of the basic fibroblast growth factor low affinity binding site in cardiac sarcolemmal vesicles

Bovine cardiac sarcolemmal (SL) vesicles contain basic fibroblast growth factor (bFGF)-binding proteins. Binding to native SL vesicles was specific and saturable with a Kd of 6.9 nM and a Bmax of 15.2 pmoles bFGF/mg vesicle protein. Using radioiodinated bFGF as a probe, autoradiography of SL proteins subjected to SDS-PAGE and electroblotting onto nitrocellulose revealed a set of 3-4 bands, of an apparent molecular weight of 100-150 kDa. bFGF binding to these bands was reduced by pretreatment of SL vesicles with heparinase. Binding was abolished by treatment of blot strips with heparinase or high salt concentrations (greater than 0.6 NaCl) but not endoglycosidase F. bFGF-binding activity remained associated with the membrane fraction following an alkaline wash, which removed peripheral membrane proteins. These data suggest that the cardiac SL contains an integral proteoglycan(s) which may be a low affinity binding/storage site of endogenous bFGF.     

Proteomic investigations of the synaptic vesicle interactome

Synaptic vesicles are key organelles in chemical signal transmission allowing neurons to communicate with each other and neighboring cells. The numerous tasks of synaptic vesicles are governed by a unique set of proteins. Recently, proteomic studies have been performed by several laboratories employing mass spectrometry and immunoblotting in order to identify the complete proteinaceous inventory of the purified synaptic vesicle compartment. Surprisingly, several fold more proteins were assigned to the organelle than previously anticipated. Despite several novel candidates, a large variety of proteins assumed to be only transiently associated with the vesicular compartment turned out to be constitutive components of the synaptic vesicle proteome. In recent years, the focus on protein-protein interactions has led to a deeper understanding of functional aspects in cellular trafficking. Several proteins acting in concert in defined cellular processes build an interactome. This article will survey the interacting partners during the entire synaptic vesicle life cycle identified by proteomic approaches. This includes anterograde and retrograde axonal transport of the synaptic vesicle membrane compartment, transport within the presynapse to the active zone, priming, docking, exocytosis, endocytosis, recycling and neurotransmitter reuptake to replenish the pool of exocytosis-competent synaptic vesicles.     

Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.     

High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease

A unique set of high molecular weight proteins was identified in junctional sarcoplasmic reticulum (SR) vesicles isolated from both cardiac muscle and skeletal muscle. These high Mr proteins were not present in free SR vesicles isolated from either tissue, nor were they observed in purified sarcolemmal fractions. The junctional SR high Mr proteins migrated as doublets in sodium dodecyl sulfate-polyacrylamide gels and exhibited apparent Mr values between 290,000 and 350,000. The high Mr proteins bound calmodulin; they were the principal proteins labeled in the cardiac and skeletal muscle SR subfractions by azido-125I-calmodulin. The high Mr proteins were also substrates for an endogenous Ca2+-calmodulin-dependent protein kinase activity, as well as exogenously added catalytic subunit of cAMP-dependent protein kinase. In addition, the junctional SR high Mr proteins were the major SR proteins degraded by a Ca2+-activated protease purified from smooth muscle. Control experiments verified the separation of junctional SR vesicles and free SR vesicles from both muscle types. Junctional SR vesicles were enriched in calsequestrin, and they exhibited Ca2+ uptake which was stimulated up to 10-fold by either ryanodine or ruthenium red. Free SR vesicles were deficient in calsequestrin and were insensitive to these two agents. Localization of the cardiac and skeletal muscle high Mr proteins to the junctional SR, coupled with demonstration of their nearly identical biochemical properties, suggests that the proteins are homologous and are likely to have similar functions in both types of striated muscle.     

Organization of lymphocyte plasma membrane. Surface protein-membrane matrix interactions in B-cell lines of different stages of differentiation

Composition of surface proteins and their interactions with cytoskeleton or membrane matrix were compared in tumor B-cell lines of different stages of B-lymphocyte maturation. All studied B-cell lines were found to share a similar set of cell surface proteins, which are tightly associated with the cytoskeleton. The increase in amount of detergent-unextractable cell surface proteins with B-cell maturation suggested that differentiation of B lymphocytes was accompanied by development of specific interactions between surface proteins and elements of the cytoskeleton or membrane matrix. Using a recently developed procedure for lymphocyte plasma membrane fractionation we demonstrate changes in distribution of cell surface proteins in membrane matrix-rich and membrane matrix-poor plasma membrane fractions during B-lymphocyte maturation. Thus, cell surface proteins of the mature B-cell line MOPC-315 were predominantly found in the plasma membrane vesicles of a high buoyant density. These vesicles mostly contained plasma membrane proteins tightly associated with elements of the membrane matrix. In immature B cells (line 70Z3) virtually all surface proteins were detected in both low and high buoyant density membrane vesicles. The tendency to increased associations between surface proteins and cytoskeleton/membrane matrix with maturation of B cells could not be explained by increased amounts of filamentous actin, since no correlation was found between the amount of globular or filamentous actin and the degree of surface protein-cytoskeleton (membrane matrix) interactions.     

Signals involved in targeting membrane proteins to synaptic vesicles

1. Synaptic vesicles (SVs) mediate fast regulated secretion of classical neurotransmitters. In order to perform their task SVs rely on a restrict set of membrane proteins. The mechanisms responsible for targeting these proteins to the SV membrane are still poorly understood.2. Likewise, little is known about the intracellular routes taken by these proteins in their way to SV membrane. Recently, several domains and motifs necessary for correct localization of SV proteins have been identified.3. In this review we summarize the sequence motifs that have been identified in the cytoplasmic domains of SV proteins that are involved in endocytosis and targeting of SVs. We suggest that the vesicular acetylcholine transporter, a protein found predominantly in synaptic vesicles, is perhaps a model protein to understand the pathways and interactions that are used for synaptic vesicle targeting.     

Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.     

Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils

Human neutrophils release vesicles when activated in vitro and in vivo, in local and systemic inflammation. We have suggested that the presence of these vesicles is due to ectocytosis, defined as the release of rightside-out oriented vesicles expressing a select set of membrane proteins. Herein we have characterised the vesicles released by neutrophils to be ectosomes with specific properties. They contained cytosolic F-actin indicating their outside-out orientation. They bound Annexin V, suggesting that they expose phosphatidylserine, similarly to platelet microparticles. They expressed a subset of cell surface proteins (selectins and integrins, complement regulators, HLA-1, FcgammaRIII, and CD66b, but not CD14, FcgammaRII, and CD87). There was no specificity for transmembrane or glycosyl-phosphatidylinositol-linked proteins and, unexpectedly, L-selectin, known to be cleaved from the surface of activated neutrophils, was present. Ectosomes exposed active enzymes released by neutrophils upon degranulation (matrix metalloproteinase-9, myeloperoxidase, proteinase 3, and elastase). In particular, released myeloperoxidase was able to bind back to ectosomes. The purified complement protein C1q and C1q from serum bound to ectosomes as well. Another aspect of ectosomes was that they became specifically adherent to monocytic and endothelial cells. These observations suggest that neutrophil-derived ectosomes have unique characteristics that make them candidates for playing roles in inflammation and cell signaling.     

Biogenesis of synaptic vesicle-like structures in a pheochromocytoma cell line PC-12

The presence of unique proteins in synaptic vesicles of neurons suggests selective targeting during vesicle formation. Endocrine, but not other cells, also express synaptic vesicle membrane proteins and target them selectively to small intracellular vesicles. We show that the rat pheochromocytoma cell line, PC12, has a population of small vesicles with sedimentation and density properties very similar to those of rat brain synaptic vesicles. When synaptophysin is expressed in nonneuronal cells, it is found in intracellular organelles that are not the size of synaptic vesicles. The major protein in the small vesicles isolated from PC12 cells is found to be synaptophysin, which is also the major protein in rat brain vesicles. At least two of the minor proteins in the small vesicles are also known synaptic vesicle membrane proteins. Synaptic vesicle-like structures in PC12 cells can be shown to take up an exogenous bulk phase marker, HRP. Their proteins, including synaptophysin, are labeled if the cells are surface labeled and subsequently warmed. Although the PC12 vesicles can arise by endocytosis, they seem to exclude the receptor-mediated endocytosis marker, transferrin. We conclude that PC12 cells contain synaptic vesicle-like structures that resemble authentic synaptic vesicles in physical properties, protein composition and endocytotic origin.     

Phospholipid diversity: Correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called “fusion proteins”. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

真核细胞内膜泡运输的分子机制

真核细胞内一些蛋白质需靠膜泡进行定向运输,膜泡是在外衣蛋白的作用下形成的,根据外衣蛋白的不同,膜泡分为笼蛋白,COPⅠ和COPⅡ外衣膜泡,这些外衣膜泡分别在细胞内不同供膜（donor membrane)处形成,因为被运输蛋白具有分选信号可与供膜上相应的受体结合,所以能被包裹在特异的膜泡之中,在膜泡形成过程中,外衣蛋白在“芽生”膜泡的细胞质侧组装成笼状外衣,帮助“芽生”膜泡从供膜处脱落,一旦笼状外衣膜泡脱离供膜,笼状外衣蛋白便发生解聚而成为无衣膜泡,无衣膜泡在Rab蛋白的调控下可定向运输蛋白质,而解聚后的外衣蛋白可重新介导新的外衣膜泡形成。     

Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers

We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.