SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors ␣-, -, and ␥-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant ␣-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in ␣-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.Neurons release their neurotransmitters by the Ca 2ϩ -dependent exocytosis of synaptic vesicles. In recent years, several membrane proteins have been identified which are required for exocytotic membrane fusion. These proteins include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) 1 and the synaptic membrane proteins syntaxin and SNAP-25, collectively referred to as SNAREs. Synaptobrevin and syntaxin both contain a single transmembrane domain at the C terminus (1, 2). SNAP-25 does not contain a transmembrane domain but carries palmitoyl side chains attached to cysteine residues in the middle of the sequence (3, 4). Homologues of these proteins have been identified in many eukaryotic cells including yeast, suggesting that fusion of trafficking vesicles with their respective target membranes is mediated by a conserved mechanism (2,(5)(6)(7)(8).While the evidence linking synaptobrevin, SNAP-25, and syntaxin to exocytosis is compelling, their precise role is unknown. In detergent extracts of brain membranes, the three proteins form a tight complex (9, 10). A ternary complex with properties similar to the native complex can be formed using recombinant proteins lacking their transmembrane anchors (11). Both native and recombinant complexes can be disassembled by the concerted action of the ATPase NSF and the protein ␣-SNAP (9, 10, 12, 13). The latter two proteins are soluble, abundant, and highly conserved through evolution. They are essential for the fusion of trafficking vesicles with their target membranes (9, 10).The assembly and disassembly of the SNARE proteins has not yet been integrated into a coherent picture of exocytosis. However, any interference with these reactions seriously inhibits membrane fusion (6 -8). To further understand these essential reac...
The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimidesensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.Synaptobrevin (also referred to as VAMP), SNAP-25, and syntaxin are crucial components of the exocytotic apparatus in neurons (1-4). Synaptobrevin is exclusively localized to synaptic vesicles whereas syntaxin and SNAP-25 are mainly localized to the neuronal plasma membrane. Any interference with the function of these proteins, e.g., proteolysis by clostridial neurotoxins (5, 6) or genetic deletion (7), inhibits exocytotic neurotransmitter release. Relatives of these proteins participate in many intracellular membrane traffic steps in all eukaryotic cells (3), suggesting that membrane fusion is mediated by a common and conserved mechanism.Despite compelling evidence linking syntaxin, SNAP-25, and synaptobrevin to exocytosis, it is not understood how these proteins operate in the sequence of events that leads to vesicle docking and membrane fusion. In detergent extracts, the three proteins form a stable complex that binds the soluble proteins ␣͞͞␥-SNAP and N-ethylmaleimide-sensitive factor (NSF), leading to their designation as v-SNAREs (synaptobrevin, vesicular SNAp-REceptors) and t-SNAREs (syntaxin and SNAP-25, target membrane SNAp REceptor) (8, 9). ATPhydrolysis by NSF causes disassembly of the complex. A complex with very similar properties also can be formed from recombinant proteins lacking their transmembrane domains (10-12). Based on these, observations it has been suggested that v-SNARE-t-SNARE interactions are responsible for docking of vesicles at their target membrane and that the conformational change caused by NSF contributes to membrane fusion (1, 9). The specificity of v-SNARE-t-SNARE pairing would ensure that trafficking vesicles only dock at an appropriate target membrane. These proposals are referred to as the SNARE hypothesis (1) and have gained wide acceptance in the field (1-3).Recently, we have observed that, unlike other residents of the presynaptic plasma membrane, significant amounts of syntaxin and SNAP-25 are also localized to synaptic vesicles (13). Using two independent and complementary approaches, we now report that a ternary complex containing syntaxin, synaptobrevin, and SNAP-25 can form in the membrane of synaptic vesicles in the absence of plasm...
Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a subpopulation present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein.Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.In eukaryotic cells, the nuclear envelope, which separates the nucleoplasm from the cytoplasm, is composed of the inner and outer nuclear membranes (INM 1 and ONM, respectively), the latter membrane being continuous with the endoplasmic reticulum. The two membranes are separated by a thin lumen and are joined at nuclear pores (1-3). Underlying the INM is a meshwork of various lamin isoforms (4) that are in close contact with the INM and its resident proteins (5). Characterized INM proteins in mammalian cells include the lamin B receptor (6), lamin-associated polypeptides 1 and 2 (Lap1 and Lap2) (7), emerin (8), and Man1 (9). These proteins possess a hydrophilic N-terminal region that protrudes into the nucleoplasm as well as one or more hydrophobic regions leading to predicted single or multispanning transmembrane domains. INM proteins are immobilized in the nuclear envelope through their interaction with lamins and/or heterochromatin (10, 11).Beside their structural role, lamina components also exert additional functions through their ability to interact with effector proteins involved in various regulatory processes. For example, the lamin B receptor directly binds to HP1 (12), a heterochromatin protein involved in transcription repression. Lap2 (13,14), emerin (15), and Man1 (16) interact with the barrier-to-autointegration factor, a 10-kDa DNA-binding protein (17). The range of regulatory functions pe...
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