Characterization of synapsin I binding to small synaptic vesicles

The binding of synapsin I, a synaptic vesicle-associated phosphoprotein, to small synaptic vesicles has been examined. For this study, synapsin I was purified under nondenaturing conditions from rat brain, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and characterized. Small synaptic vesicles were purified from rat neocortex by controlled pore glass chromatography as the last purification step, and binding was characterized at an ionic strength equivalent to 40 mM NaCl. After removal of endogenous synapsin I, exogenous dephospho-synapsin I bound with high affinity (Kd, 10 +/- 6 nM) to synaptic vesicles. The binding saturated at 76 +/- 40 micrograms synapsin I/mg of vesicle protein, which corresponded to the amount found endogenously in purified vesicles. Synapsin I binding exhibited a broad pH optimum around pH 7. Other basic proteins, specifically myelin basic protein and histone H2b, did not compete with synapsin I for binding to vesicles. Other membranes purified from rat brain and membranes derived from human erythrocytes did not show the high affinity binding site for synapsin I found in vesicles. The binding of three different forms of phosphosynapsin I to vesicles was investigated. Synapsin I, phosphorylated at sites 2 and 3 by purified calcium/calmodulin-dependent protein kinase II, bound with a 5-fold lower affinity to the vesicles than did dephospho-synapsin I. In contrast, synapsin I, phosphorylated at site 1 by purified catalytic subunit of cAMP-dependent protein kinase, bound with an affinity close to that of dephospho-synapsin I. Synapsin I phosphorylated on all three sites bound to the vesicles with an affinity comparable to that of synapsin I phosphorylated on sites 2 and 3. Under conditions of higher ionic strength (150 mM NaCl equivalent), synapsin I bound with a 5-fold lower affinity to vesicles, and no effect of phosphorylation on binding was observed under these conditions.     

Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation.

The distribution of the synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on synaptic vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to synaptic vesicles at rest. The value of this ratio was intermediate in synaptic vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with synaptic vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release.     

Heterogeneous distribution of synaptophysin and protein 65 in synaptic vesicles isolated from rat cerebral cortex

Rat brain cerebral cortex derived synaptic vesicles sedimenting on a 0.4 M sucrose solution were further fractionated according to size by column chromatography on Sephacryl-1000 and analyzed for their binding activities of antibodies directed against the vesicle-associated proteins synaptophysin, synapsin I, protein 65 and clathrin. Whereas synapsin I and particularly protein 65 and clathrin are associated with a large range of vesicle sizes, synaptophysin elutes with small vesicles only. Using monoclonal antibodies against either synaptophysin or protein 65 and polyacrylamide beads for solid matrix immunoprecipitation, significant differences could be revealed in the protein composition of the resulting vesicle populations. Whereas synapsin I is associated with both synaptophysin and protein 65 immunoprecipitated vesicle populations, synaptophysin appears to be only a minor constituent of vesicles precipitated with anti-protein 65. Vesicles precipitated with anti-synaptophysin antibodies are enriched in acetylcholine. Our results suggest that the vesicle membrane protein synaptophysin and protein 65 may not have a ubiquitous distribution among synaptic vesicles. Protein 65 containing large vesicle populations contain little synaptophysin and synaptophysin is mainly associated with synaptic vesicles of small diameter.     

Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation

Synapsin I (protein I) is a neuron-specific phosphoprotein, which is a substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases. In two accompanying studies (De Camilli, P., R. Cameron, and P. Greengard, and De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354 and 1355-1373) we have shown, by immunocytochemical techniques at the light microscopic and electron microscopic levels, that synapsin I is present in the majority of, and possibly in all, nerve terminals, where it is primarily associated with synaptic vesicles. In the present study we have prepared a highly purified synaptic vesicle fraction from rat brain by a procedure that involves permeation chromatography on controlled-pore glass as a final purification step. Using immunological methods, synapsin I concentrations were determined in various subcellular fractions obtained in the course of vesicle purification. Synapsin I was found to copurify with synaptic vesicles and to represent approximately 6% of the total protein in the highly purified synaptic vesicle fraction. The copurification of synapsin I with synaptic vesicles was dependent on the use of low ionic strength media throughout the purification. Synapsin I was released into the soluble phase by increased ionic strength at neutral pH, but not by nonionic detergents. The highly purified synaptic vesicle fraction contained a calcium-dependent protein kinase that phosphorylated endogenous synapsin I in its collagenase-sensitive tail region. The phosphorylation of this region appeared to facilitate the dissociation of synapsin I from synaptic vesicles under the experimental conditions used.     

Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins

Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.     

Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.     

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. II. Functional effects of the Rab3A-synapsin I interaction

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760-43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.     

The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse

The axoplasm at the presynaptic active zone of excitatory synapses between parallel fibers and Purkinje cell spines contains a meshwork of distinct filaments intermingled with synaptic vesicles, seen most clearly after the rapid freezing, freeze-etch technique of tissue preparation. One set of filaments extends radially from synaptic vesicles and intersects similar filaments associated with vesicles as well as larger filaments arising from the presynaptic membrane. The small, vesicle-associated filaments appear to link synaptic vesicles to one another and to enmesh them in the vicinity of the synaptic junction. The vesicle-associated filaments could be synapsin I because they have the same molecular dimensions and are distributed in the same pattern as synapsin I immunoreactivity.     

Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.     

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

Synapsin associates with cyclophilin B in an ATP- and cyclosporin A-dependent manner

Immunophilins are ubiquitous enzymes responsible for proline isomerisation during protein synthesis and for the chaperoning of several membrane proteins. These activities can be blocked by the immunosuppressants cyclosporin A, FK506 and rapamycin. It has been shown that all three immunosuppressants have neurotrophic activity and can modulate neurotransmitter release, but the molecular basis of these effects is currently unknown. Here, we show that synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin that is particularly abundant in brain. The interaction is direct and conserved in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The binding between the two proteins can be disrupted by cyclosporin A and inhibited by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes with synapsin I in rat synaptic vesicle fractions and its levels in synaptic vesicle-containing fractions are decreased in synapsin knockout mice. These results suggest that immunophilins are involved in the complex protein networks operating at the presynaptic level and implicate the interaction between cyclophilin B and synapsins in presynaptic function.     

Binding of Synapsin I to Synaptic Vesicles: Clues from the Study of its Interactions with Liposomes

Abstract

Synapsin I is a major brain phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation-dependent fashion. The binding of synapsin I to synaptic vesicles involves interactions with the phospholipid and protein components of the vesicle membrane. The highly hydrophobic NH₂-terminal head region of the protein binds with high-affinity to acidic phospholipids and penetrates the hydrophobic core of the membrane, whereas the basic COOH-terminal tail region does not significantly contribute to this binding. The interaction with phospholipids increases the amount of α-helix in the secondary structure of synapsin I, but does not markedly affect the microenvironment of tryptophan and cysteine residues present in the head region. The results suggest that synapsin I binds to synaptic vesicle phospholipids through amphiphilic and positively charged domains present in its NH2-terminal region and that such an interaction contributes to the high-affinity binding of synapsin I to synaptic vesicles.     

The synapsins and the regulation of synaptic function

Synapsin I and II are a family of synaptic vesicle-associated phosphoproteins involved in the short-term regulation of neurotransmitter release. In this review, we discuss a working model for the molecular mechanisms by which the synapsins act. We propose that synapsin I links synaptic vesicles to actin filaments in the presynaptic nerve terminal and that these interactions are modulated by the reversible phosphorylation of synapsin I through various signal transduction pathways. The high degree of homology between the synapsins suggests that some of the functional properties of synapsin I are also shared by synapsin II.     

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.     

Glycosylation Sites Flank Phosphorylation Sites on Synapsin I

Synapsin I is concentrated in nerve terminals, where it appears to anchor synaptic vesicles to the cytoskeleton and thereby ensures a steady supply of fusion-competent synaptic vesicles. Although phosphorylation-dependent binding of synapsin I to cytoskeletal elements and synaptic vesicles is well characterized, little is known about synapsin I's O-linked N-acetylglucosamine (O-GlcNAc) modifications. Here, we identified seven in vivo O-GlcNAcylation sites on synapsin I by analysis of HPLC-purified digests of rat brain synapsin I. The seven O-GlcNAcylation sites (Ser55, Thr56, Thr87, Ser516, Thr524, Thr562, and Ser576) in synapsin I are clustered around its five phosphorylation sites in domains B and D. The proximity of phosphorylation sites to O-GlcNAcylation sites in the regulatory domains of synapsin I suggests that O-GlcNAcylation may modulate phosphorylation and indirectly affect synapsin I interactions. With use of synthetic peptides, however, the presence of an O-GlcNAc at sites Thr562 and Ser576 resulted in only a 66% increase in the Km of calcium/calmodulin-dependent protein kinase II phosphorylation of site Ser566 with no effect on its Vmax. We conclude that O-GlcNAcylation likely plays a more direct role in synapsin I interactions than simply modulating the protein's phosphorylation.     

Synapsin I, a neuron-specific phosphoprotein interacting with small synaptic vesicles and F-actin

Synapsin I is a neuron-specific phosphoprotein which is a substrate for cAMP- and Ca2+/calmodulin-dependent protein kinases. It is specifically localized to the cytoplasmic side of small synaptic vesicles. The interaction of synapsin I with the synaptic vesicle membrane is complex in nature, since it is modulated by phosphorylation and involves binding of different domains of the molecule to phospholipid and protein components of synaptic vesicles. Synapsin I is also able to interact with actin filaments in a phosphorylation-dependent manner. Because of these properties, it has been hypothesized that synapsin I acts as a dynamic link between synaptic vesicles an the actin meshwork of the nerve terminal, thereby modulating the release of neurotransmitter.     

Synapsin Ia, Synapsin Ib, Protein IIIa, and Protein IIIb, Four Related Synaptic Vesicle-Associated Phosphoproteins, Share Regional and Cellular Localization in Rat Brain

The regional and cellular distribution of four synaptic vesicle-associated proteins, synapsins Ia and Ib (Mr 86,000 and 80,000, collectively referred to as synapsin I) and proteins IIIa and IIIb (Mr 74,000 and 55,000, collectively referred to as protein III), has been compared in selected rat brain regions, using both radioimmunoassays and back-phosphorylation assays. Lesions of several neuronal populations in the basal ganglia (corticostriatal fibers, intrinsic striatal neurons, striatonigral fibers, nigrostriatal fibers) induced decreases in the levels of these various proteins that were highly correlated (r = 0.96-0.97). Moreover, the synaptic vesicle-associated phosphoproteins displayed a similar and widespread distribution throughout the CNS. This evidence for colocalization indicates that the majority of, and possibly all, CNS neurons and nerve terminals may contain both forms of synapsin I and both forms of protein III.     

Synapsin I: an actin-bundling protein under phosphorylation control

Synapsin I is a neuronal phosphoprotein comprised of two closely related polypeptides with apparent molecular weights of 78,000 and 76,000. It is found in association with the small vesicles clustered at the presynaptic junction. Its precise role is unknown, although it probably participates in vesicle clustering and/or release. Synapsin I is known to associate with vesicle membranes, microtubules, and neurofilaments. We have examined the interaction of purified phosphorylated and unphosphorylated bovine and human synapsin I with tubulin and actin filaments, using cosedimentation, viscometric, electrophoretic, and morphologic assays. As purified from brain homogenates, synapsin I decreases the steady-state viscosity of solutions containing F-actin, enhances the sedimentation of actin, and bundles actin filaments. Phosphorylation by cAMP-dependent kinase has minimal effect on this interaction, while phosphorylation by brain extracts or by purified calcium- and calmodulin-dependent kinase II reduces its actin-bundling and -binding activity. Synapsin's microtubule-binding activity, conversely, is stimulated after phosphorylation by the brain extract. Two complementary peptide fragments of synapsin generated by 2-nitro-5-thiocyanobenzoic cleavage and which map to opposite ends of the molecule participate in the bundling process, either by binding directly to actin or by binding to other synapsin I molecules. 2-Nitro-5-thiocyanobenzoic peptides arising from the central portion of the molecule demonstrate neither activity. In vivo, synapsin I may link small synaptic vesicles to the actin-based cortical cytoskeleton, and coordinate their availability for release in a Ca++-dependent fashion.     

Synapsin I Is Associated with Cholinergic Nerve Terminals in the Electric Organs of Torpedo, Electrophorus, and Malapterurus and Copurifies with Torpedo Synaptic Vesicles

Using an affinity-purified monospecific polyclonal antibody against bovine brain synapsin I, the distribution of antigenically related proteins was investigated in the electric organs of the three strongly electric fish Torpedo marmorata, Electrophorus electricus, Malapterurus electricus and in the rat diaphragm. On application of indirect fluorescein isothiocyanate-immunofluorescence and using alpha-bungarotoxin for identification of synaptic sites, intense and very selective staining of nerve terminals was found in all of these tissues. Immunotransfer blots of tissue homogenates revealed specific bands whose molecular weights are similar to those of synapsin Ia and synapsin Ib. Moreover, synapsin I-like proteins are still attached to the synaptic vesicles that were isolated in isotonic glycine solution from Torpedo electric organ by density gradient centrifugation and chromatography on Sephacryl-1000. Our results suggest that synapsin I-like proteins are also associated with cholinergic synaptic vesicles of electric organs and that the electric organ may be an ideal source for studying further the functional and molecular properties of synapsin.     

Synaptophysin and synapsin I as tools for the study of the exo-endocytotic cycle

Synaptophysin, an integral protein of the synaptic vesicle membrane, and synapsin I, a phosphoprotein associated with the cytoplasmic side of synaptic vesicles, represent useful markers that allow to follow the movements of the vesicle membrane during recycling. The use of antibodies against these proteins to label nerve terminals during experimental treatments which stimulate secretion has provided evidence that during the exo-endocytotic cycle synaptic vesicles transiently fuse with the axolemma, from which they are specifically recovered. When recycling is blocked, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and to diffusion of the vesicle components in the plane of the membrane.