Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/ synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calciumindependent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/ SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.Cell-cell communication relies on the regulated release of transmitter molecules from secretory vesicles. These vesicles fuse with the plasma membrane in a calcium-dependent manner to release the transmitter molecules (1, 2). Despite identification of the major players involved in intracellular membrane fusion (3, 4), the molecular steps leading to vesicle fusion are still not fully understood. Synaptotagmin I, a calciumphospholipid binding protein, is essential for synchronous synaptic vesicle exocytosis, whereas membrane fusion itself relies on the three SNARE 1 proteins: synaptobrevin, also known as vesicle-associated membrane protein or VAMP, on the vesicular membrane, and syntaxin and SNAP-25, on the target plasma membrane (5-8). The three SNARE proteins form a four-helical bundle that likely drives membrane fusion (9), with the syntaxin/SNAP-25 dimer (t-SNARE dimer) being an important intermediate in this process (10, 11).Current models of calcium-triggered exocytosis depict the calcium sensor, synaptotagmin, being physically linked to the SNARE fusion machinery in anticipation of the calcium entry (12-15). Indeed, many independent studies have demonstrated that synaptotagmin I, the major brain isoform, can interact specifically with the neuronal SNAREs in the absence of calcium, as evidenced by pull-downs and affinity chromatography approaches followed by Coomassie staining (16 -20). Using the brain-purified SNAREs, syntaxin1 and SNAP-25, we were recently able to show that synaptotagmin I binds specifically and with high affinity the t-SNARE dimer (but not the monomeric SNARE proteins) in a calcium-independent manner (18). Furthermore, we found that the majority of syntaxin and SNAP-25 in neuroendocrine cells likely exist as stable t-SNARE dimers on the plasma membrane (11), suggesting that interaction of vesicular synaptotagmin with this entity may take place during the SNARE-mediated fusion of the two membranes.Because it is well established that synaptotagmin I plays a critical role in SNARE-mediated membrane fusion (3, 6, 7), the molecular basis for the observed calcium-independent physic...

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Membrane fusion during exocytosis and throughout the cell is believed to involve members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) family of proteins. The assembly of these proteins into a four-helix bundle may be part of the driving force for bilayer fusion. Regulated exocytosis in neurons and related cell types is specialized to be fast and Ca 2؉ -dependent suggesting the involvement of other regulatory proteins specific for regulated exocytosis. Among these are the complexins, two closely related proteins that bind only to the assembled SNARE complex. We have investigated the function of complexin by analysis of single vesicle release events in adrenal chromaffin cells using carbon fiber amperometry. These cells express complexin II, and overexpression of this protein modified the kinetics of vesicle release events so that their time course was shortened. This effect depended on complexin interaction with the SNARE complex as introduction of a mutation of Arg-59, a residue that interacts with synaptobrevin in the SNARE complex, abolished its effects. The data are consistent with a function for complexin in stabilizing an intermediate of the SNARE complex to allow kiss-andrun recycling of the exocytosed vesicle.The SNARE 1 (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) proteins associate to form a SNARE complex (1) that has a crucial role in membrane fusion events throughout the secretory pathway as the core of a highly conserved fusion machinery (2). The SNARE proteins syntaxin, SNAP-25, and vesicle-associated membrane protein (synaptobrevin), involved in regulated exocytosis in neurons and neuroendocrine cells, have been studied in most detail (3,4). From in vitro studies, the neuronal SNAREs are sufficient for membrane fusion when reconstituted in liposomes (5), but this occurs in a Ca 2ϩ -independent manner and with kinetics that are many orders of magnitude slower than exocytosis at the synapse suggesting an essential requirement for other proteins. A prime candidate for a protein that influences membrane fusion during regulated exocytosis is complexin (6, 7) as it binds specifically to the assembled SNARE complex (6,8,9).Regulated exocytosis differs significantly from other membrane fusion events. First, it is dependent upon an intracellular signal such as an elevated Ca 2ϩ concentration for its initiation (10). Second, in synapses it is specialized to be able to occur within tens of microseconds of Ca 2ϩ elevation (11) through the rapid formation of a transient fusion pore (12). Third, rapid retrieval of the fused membrane is essential to maintain the releasable vesicle pool (13). It is likely that proteins in addition to SNAREs are crucially important for the control and kinetics of fast, regulated exocytosis. Numerous proteins have been discovered that interact with the neuronal SNARE proteins, and some of these are not expressed in organisms such as yeast that lack regulated exocytosis. It is likely that these prote...

The regulated release of hormones and neurotransmitters is a fundamental process throughout the animal kingdom. The short time scale for the calcium triggering of vesicle fusion in regulated secretion suggests that the calcium sensor synaptotagmin and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) membrane fusion machinery are well ordered before the calcium signal. To gain insight into the organization of the prefusion protein assembly in regulated exocytosis, we undertook a structural/functional study of the vesicular synaptotagmin1 and the plasma membrane SNARE proteins, which copurify from the brain in the absence of calcium. Based on an evolutionary analysis, mutagenesis screens, and a computational protein docking approach, we now provide the first testable description of the supramolecular prefusion assembly. Perturbing the determined synaptotagmin/SNARE-interacting interface in several models of regulated exocytosis altered the secretion of hormones and neurotransmitters. These mutations also disrupted the constitutive synaptotagmin/SNARE link in full agreement with our model. We conclude that the interaction of synaptotagmin with preassembled plasma membrane SNARE proteins, before the action of calcium, can provide a precisely organized "tethering" scaffold that underlies regulated secretion throughout evolution. INTRODUCTIONIn regulated secretion, fusion of docked secretory vesicles with the plasma membrane is triggered by the rapid elevation of the intracellular calcium concentration (Katz and Miledi, 1967). The membrane fusion itself is brought about by the action of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, whereas the vesicular protein synaptotagmin (SYT) acts as the calcium sensor (Sollner et al., 1993;Sudhof and Scheller, 2001;Bonifacino and Glick, 2004). In neuroendocrine cells, the principal SNAREs are syntaxin1 and synaptosome-associated protein of 25 kDa (SNAP-25) on the plasma membrane (so-called target SNAREs or t-SNAREs), and vesicular synaptobrevin, also known as VAMP. Interaction between synaptobrevin and the two t-SNAREs leads to the formation of the ternary SNARE complex, a twisted parallel fourhelical bundle that probably drives membrane fusion (Sutton et al., 1998). With all the major players involved in vesicle fusion identified, it is becoming possible to tackle a central problem of this cellular process-how does calcium trigger vesicle fusion? Arguably, to understand the action of calcium it is essential to know 1) the extent of the assembly of the SNARE fusion proteins and 2) their organization in relation to the calcium sensor, SYT, before calcium-triggered events.It would be reasonable to assume that the SNAREs are at least partially preassembled, and the plasma membrane syntaxin and SNAP-25 are the first obvious candidates for being in such a ready state (An and Almers, 2004;Rickman et al., 2004b). Importantly, synaptobrevin binds syntaxin and SNAP-25 with high-affinity only when the two...

Abstract:The assembly of gap junction intercellular communication channels was studied by analysis of the molecular basis of the dysfunction of connexin 32 mutations associated with the X-linked form of CharcotMarie-Tooth disease in which peripheral nervous transmission is impaired. A cell-free translation system showed that six recombinant connexin 32 mutated proteins-four point mutations at the cytoplasmic amino terminus, one at the membrane aspect of the cytoplasmic carboxyl terminus, and a deletion in the intracellular loop-were inserted into microsomal membranes and oligomerised into connexon hemichannels with varying efficiencies. The functionality of the connexons was determined by the ability of HeLa cells expressing the respective connexin cDNAs to transfer Lucifer yellow. The intracellular trafficking properties of the mutated connexins were determined by immunocytochemistry. The results show a relationship between intracellular interruption of connexin trafficking, the efficiency of intercellular communication, and the severity of the disease phenotype. Intracellular retention was explained either by deficiencies in the ability of connexins to oligomerise or by mutational changes at two targeting motifs. The results point to dominance of two specific targeting motifs: one at the amino terminus and one at the membrane aspect of the cytoplasmically located carboxyl tail. An intracellular loop deletion of six amino acids, associated with a mild phenotype, showed partial oligomerisation and low intercellular dye transfer compared with wild-type connexin 32. The results show that modifications in trafficking and assembly of gap junction channels emerge as a major feature of Charcot-Marie -Tooth X-linked disease.