Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

Quetglas, Stéphanie; Iborra, Cécile; Sasakawa, Nobuyuki; Haro, Luc de; Kumakura, Konosuke; Sato, Kazuki; Lévêque, Christian; Seagar, Michael

doi:10.1093/emboj/cdf404

Cited by 117 publications

(131 citation statements)

References 45 publications

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“…Release of hGH from toxin-treated, permeabilized PC12 cells transfected with botulinum neurotoxin type B (botB)-resistant syb2 (Q76V,F77W) was measured essentially as described previously (Quetglas et al, 2002) in the presence of recombinantly expressed endopeptidase light chain (LC) of botB (botB/ LC). BotB/LC was purified as described previously (Sutton et al, 2005), was of high homogeneity as assessed by SDS-PAGE, and it showed endopeptidase activity comparable with native botulinum neurotoxin by using an in vitro vesicle-associated membrane protein peptide cleavage assay (Sutton et al, 2005).…”

Section: Toxin Rescue Assaysmentioning

confidence: 99%

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Fdez

Jowitt

Wang

et al. 2008

MBoC

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The interactions underlying the cooperativity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during neurotransmission are not known. Here, we provide a molecular characterization of a dimer formed between the cytoplasmic portions of neuronal SNARE complexes. Dimerization generates a two-winged structure in which the C termini of cytosolic SNARE complexes are in apposition, and it involves residues from the vesicleassociated SNARE synaptobrevin 2 that lie close to the cytosol-membrane interface within the full-length protein.Mutation of these residues reduces stability of dimers formed between SNARE complexes, without affecting the stability of each individual SNARE complex. These mutations also cause a corresponding decrease in the ability of botulinum toxin-resistant synaptobrevin 2 to rescue regulated exocytosis in toxin-treated neuroendocrine cells. Moreover, such synaptobrevin 2 mutants give rise to a dominant-negative inhibition of exocytosis. These data are consistent with an important role for SNARE complex dimers in neurosecretion.

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Section: Toxin Rescue Assaysmentioning

confidence: 99%

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Fdez

Jowitt

Wang

et al. 2008

MBoC

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“…Amperometric measurement of exocytotic catecholamine release was performed as described previously (Ohyama et al, 2002;Quetglas et al, 2002) except that the chromaffin cells were stimulated with 60 mM KCl. Microinjection was performed using a 6-d-old culture of chromaffin cells on collagencoated coverslips.…”

Section: Amperometrymentioning

confidence: 99%

“…For these reasons, we used chromaffin cell exocytosis to investigate the function of the Ca 2ϩ -dependent interaction between syntaxin-1A and myosin-Va. Amperometric measurements were used to examine the physiological role of myosin-Va-syntaxin-1A binding because it is a powerful method not only for quantitative measurement of exocytosis but also for characterizing the mechanism of exocytosis (Segre et al, 2000;Fisher et al, 2001). We therefore performed an amperometric assay of catecholamine release for dense-core vesicles in chromaffin cells (Ohyama et al, 2002;Quetglas et al, 2002), which are known to possess myosin-Va (Rosé et al, 2003). We found that the syntaxin-1A specifically reduced the exocytotic frequency, whereas syntaxin-1A [191-240 L222E] fragment had no effect (Figure 4, A and B).…”

Section: Interference With Syntaxin-1a-myosin-va Complex Formation Inmentioning

confidence: 99%

“…systems such as central neurons (Burgoyne and Morgan, 2003). Moreover, it is the most suitable system for examining whether a biochemical interaction plays a physiological role in exocytosis (Fisher et al, 2001;Ohyama et al, 2002;Quetglas et al, 2002). For these reasons, we used chromaffin cell exocytosis to investigate the function of the Ca 2ϩ -dependent interaction between syntaxin-1A and myosin-Va. Amperometric measurements were used to examine the physiological role of myosin-Va-syntaxin-1A binding because it is a powerful method not only for quantitative measurement of exocytosis but also for characterizing the mechanism of exocytosis (Segre et al, 2000;Fisher et al, 2001).…”

Section: Interference With Syntaxin-1a-myosin-va Complex Formation Inmentioning

confidence: 99%

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Myosin-Va Regulates Exocytosis through the Submicromolar Ca²+-dependent Binding of Syntaxin-1A

Watanabe

Nomura

Ohyama

et al. 2005

MBoC

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Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 M Ca 2؉ . Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca 2؉ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca 2؉ . INTRODUCTIONMyosin-V, a processive molecular motor, conveys vesicles and other organelles along F-actin (Mercer et al., 1991;Espreafico et al., 1992;Cheney et al., 1993;Reck-Peterson et al., 2000;Vale, 2003). This unconventional myosin is a member of the class-V myosins, which are expressed in all eukaryotic species from yeast to mammals (Reck-Peterson et al., 2000;Matsui, 2003;Vale, 2003). Myosin-V is a dimeric protein (Cheney et al., 1993). Each monomer is composed of a head region, a long neck domain containing six tandem IQ-motifs, and a tail region (Reck-Peterson et al., 2000). The head acts as a plus-end ATPase-dependent molecular motor to move myosin-V along F-actin (Reck-Peterson et al., 2000). The long neck region is thought to act as a lever arm to regulate the motor activity of the head and to maintain the large step size of myosin-V via bound light chains. In higher eukaryotes, the light chains consist mainly of calmodulin (CaM) bound to the IQ-motifs in the neck domain (Cheney et al., 1993;Vale, 2003). In addition, the globular tail of myosin-V interacts with membrane-bound vesicles. In these ways, myosin-V plays a central role in intracellular polarized transport (Reck-Peterson et al., 2000;Matsui, 2003;Vale, 2003).Among the three isoforms of myosin-V in higher vertebrates, myosin-Va is the most abundant, and it is highly enriched in the brain (Espreafico et al., 1992), particularly in the neurons (Tilelli et al., 2003). Several lines of evidence indicate that synaptic vesicles, which undergo the Ca 2ϩ -regulated exocytosis, are one of the most important cargoes for myosin-Va (Prekeris and Terrian, 1997;Bridgman, 1999;Tilelli et al., 2003). In addition, Myo2p, a yeast h...

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“…Several proteins have been invoked in regulating synaptobrevin. For instance, calmodulin was reported to bind to the C-terminal region of synaptobrevin (residues 77-90) in a calcium-dependent manner (Quetglas et al, 2002). Calmodulin was shown to compete with membrane binding of this region, and it was proposed that calmodulin binding inhibits rather than activates synaptobrevin (de Haro et al, 2004).…”

mentioning

confidence: 99%

Determinants of Synaptobrevin Regulation in Membranes

Siddiqui

Vites

Stein³

et al. 2007

MBoC

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Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion. INTRODUCTIONCommunication between neurons is mediated by neurotransmitters that are released from presynaptic nerve endings by Ca 2ϩ -dependent exocytosis of synaptic vesicles. Exocytotic fusion of the vesicle with the synaptic plasma membrane is mediated by the proteins synaptobrevin 2/VAMP2, SNAP-25, and syntaxin 1 (Jahn and Scheller, 2006;Rizo et al., 2006). These proteins are members of the SNARE protein family that are involved in all fusion events of the secretory pathway. SNAREs are characterized by stretches of 60-70 amino acids arranged in heptad repeats, termed SNARE motifs (Weimbs et al., 1997;Fasshauer et al., 1998b;Bock et al., 2001;Day et al., 2006). Syntaxin and synaptobrevin each contain a single SNARE motif that is located adjacent to a C-terminal transmembrane domain. In contrast, SNAP-25 contains two SNARE motifs connected by a palmitoylated linker region that serves as membrane anchor.Synaptobrevin resides in synaptic vesicles, whereas SNAP-25 and syntaxin 1 reside in the plasma membrane. The SNARE motifs of syntaxin, SNAP-25, and synaptobrevin readily assemble into quarternary bundles of ␣-helices (Fasshauer et al., 1997;Sutton et al., 1998). Assembly would thus lead to a tight connection between the membranes. According to this view, assembly is nucleated at the Nterminal ends of the SNARE-motifs and proceeds toward the C-terminal membrane anchor ("zippering"), resulting in a strained "trans"-complex . During membrane merger, the trans-complex would relax into a "cis"-complex in which the transmembrane domains are aligned in parallel. To regenerate the SNAREs for another round of fusion, SNARE complexes need to be disassembled by the AAAϩ-ATPase NEM-sensitive factor (NSF) in conjunction with cofactors termed soluble NSF attachment proteins (SNAPs; Sollner et al., 1993).Although the "zippering" hypothesis of SNARE function has received a lot of experimental support, it is still unclear...

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Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

Cited by 117 publications

References 45 publications

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Myosin-Va Regulates Exocytosis through the Submicromolar Ca²+-dependent Binding of Syntaxin-1A

Determinants of Synaptobrevin Regulation in Membranes

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Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

Cited by 117 publications

References 45 publications

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A

Determinants of Synaptobrevin Regulation in Membranes

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Myosin-Va Regulates Exocytosis through the Submicromolar Ca²+-dependent Binding of Syntaxin-1A