Neurotransmitter release is triggered by Ca2؉ binding to a low affinity Ca 2؉ sensor, mostly synaptotagmin-1, which catalyzes SNARE-mediated synaptic vesicle fusion. Tomosyn negatively regulates Ca 2؉ -dependent neurotransmitter release by sequestering target SNAREs through the C-terminal VAMPlike domain. In addition to the C terminus, the N-terminal WD40 repeats of tomosyn also have potent inhibitory activity toward Ca 2؉ -dependent neurotransmitter release, although the molecular mechanism underlying this effect remains elusive. Here, we show that through its N-terminal WD40 repeats tomosyn directly binds to synaptotagmin-1 in a Ca 2؉ -dependent manner. The N-terminal WD40 repeats impaired the activities of synaptotagmin-1 to promote SNARE complex-mediated membrane fusion and to bend the lipid bilayers. Decreased acetylcholine release from N-terminal WD40 repeat-microinjected superior cervical ganglion neurons was relieved by microinjection of the cytoplasmic domain of synaptotagmin-1. These results indicate that, upon direct binding, the N-terminal WD40 repeats negatively regulate the synaptotagmin-1-mediated step of Ca 2؉ -dependent neurotransmitter release. Furthermore, we show that synaptotagmin-1 binding enhances the target SNARE-sequestering activity of tomosyn. These results suggest that the interplay between tomosyn and synaptotagmin-1 underlies inhibitory control of Ca 2؉ -dependent neurotransmitter release.Neurotransmitter release arises from Ca 2ϩ -dependent exocytosis of synaptic vesicles (1). At the presynaptic active zone, synaptic vesicles are docked beside Ca 2ϩ channels and primed to become fusion-competent. Upon reaching presynaptic nerve terminals, action potentials open Ca 2ϩ channels. The resulting Ca 2ϩ influx triggers fusion of primed synaptic vesicles to the presynaptic plasma membrane. Exocytotic efficacy of synaptic vesicles is adequately controlled (1-3). The number of synaptic vesicles in the readily releasable pool (RRP) 2 is extremely small (1-2% of the total number of vesicles in a presynaptic terminal) (4 -6), and the vast majority of synaptic vesicles reside in the presynaptic nerve terminal despite regular Ca 2ϩ influx (2, 3). A subset of the residing vesicles (10 -20% of the total vesicles) constitutes the recycling pool (7, 8), which undergoes exo-and endocytosis under stimulation at physiological frequency (2, 3). The synaptic vesicles in the recycling pool are accordingly mobilized to prevent depletion of the RRP (2, 3, 9), thereby enabling neurons to respond to an appropriate frequency of action potentials.The vesicle fusion process is catalyzed by three soluble Nethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors (SNAREs), syntaxin-1, SNAP-25, and VAMP2 (10, 11). Syntaxin-1 and SNAP-25 are localized within the presynaptic plasma membrane as target SNAREs (t-SNAREs), and VAMP2 resides in synaptic vesicles as a vesicular SNARE (v-SNARE). The three SNARE proteins are thought to assemble in a zipper-like fashion and form a stable SNARE complex. As a c...

Neurotransmitter release from presynaptic nerve terminals is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn, a negative regulator of neurotransmitter release, which is composed of N-terminal WD40 repeats, a tail domain, and a C-terminal VAMP-like domain, is known to inhibit SNARE complex formation by sequestering target SNAREs (t-SNAREs) upon interaction of its C-terminal VAMP-like domain with t-SNAREs. However, it remains unclear how the inhibitory activity of tomosyn is regulated. Here we show that the tail domain functions as a regulator of the inhibitory activity of tomosyn through intramolecular interactions. The binding of the tail domain to the C-terminal VAMP-like domain interfered with the interaction of the C-terminal VAMP-like domain with t-SNAREs, and thereby repressed the inhibitory activity of tomosyn on the SNARE complex formation. The repressed inhibitory activity of tomosyn was restored by the binding of the tail domain to the N-terminal WD40 repeats. These results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain controls its inhibitory activity on the SNARE complex formation, leading to a regulated inhibition of neurotransmitter release.Synaptic vesicles are transported to the presynaptic plasma membrane where Ca 2ϩ channels are located. Depolarization induces Ca 2ϩ influx into the cytosol of nerve terminals through the Ca 2ϩ channels, and this Ca 2ϩ influx initiates the fusion of the vesicles with the plasma membrane, finally leading to exocytosis of neurotransmitters (1). Soluble N-ethylmaleimidesensitive fusion protein attachment protein (SNAP) 2 receptors (SNAREs) are essential for synaptic vesicle exocytosis (2-5). Synaptic vesicles are endowed with vesicle-associated membrane protein 2 (VAMP-2) as a vesicular SNARE, whereas the presynaptic plasma membrane is endowed with syntaxin-1 and SNAP-25 as target SNAREs. VAMP-2 interacts with SNAP-25 and syntaxin-1 to form a stable SNARE complex (6 -9). The formation of the SNARE complex then brings synaptic vesicles and the plasma membrane into close apposition, and provides the energy that drives the mixing of the two lipid bilayers (3-5, 9).Tomosyn is a syntaxin-1-binding protein that we originally identified (10). Tomosyn contains N-terminal WD40 repeats, a tail domain, and a C-terminal domain homologous to VAMP-2. The C-terminal VAMP-like domain (VLD) of tomosyn acts as a SNARE domain that competes with VAMP-2. Indeed, a structural study of the VLD revealed that the VLD, syntaxin-1, and SNAP-25 assemble into a SNARE complex-like structure (referred to as tomosyn complex hereafter) (11). Tomosyn inhibits SNARE complex formation by sequestering t-SNAREs through the tomosyn complex formation, and thereby inhibits SNARE-dependent neurotransmitter release. The large N-terminal region of tomosyn shares similarity to the Drosophila tumor suppressor lethal giant larvae (Lgl), the mammalian homologues M-Lgl1 and M-Lgl2, and yeast proteins Sro7p and Sro77p (...

Background: Endoplasmic reticulum (ER) export of transmembrane proteins depends on the interaction between cargo signals and Sec24 isoforms. Results: The ⌽X⌽X⌽ sequence facilitates the ER export of model proteins and selectively binds to Sec24C. Conclusion: ⌽X⌽X⌽ is a novel ER export signal that is specifically recognized by Sec24C. Significance: This novel cargo-Sec24 interaction provides mechanistic insights into vesicular transport.

During neurite outgrowth, Rho small G protein activity is spatiotemporally regulated to organize the neurite sprouting, extension, and branching. We have previously identified a potent Rho GTPase-activating protein (GAP), RA-RhoGAP, as a direct downstream target of Rap1 small G protein in the neurite outgrowth. In addition to the Ras-associating (RA) domain for Rap1 binding, RA-RhoGAP has the pleckstrin homology (PH) domain for lipid binding. Here, we showed that phosphatidic acid (PA) bound to the PH domain and enhanced GAP activity for Rho. RA-RhoGAP induced extension of neurite in a diacylglycerol kinase-mediated synthesis of the PA-dependent manner. Knockdown of RA-RhoGAP reduced the diacylglycerol kinase-induced neurite extension. In contrast to the effect of the RA domain, the PH domain was specifically involved in the neurite extension, not in the sprouting and branching. These results indicate that PA and Rap1 cooperatively regulate RA-RhoGAP activity for promoting neurite outgrowth.Formation and extension of axons and dendrites, the socalled neurite outgrowth, are crucial events in neuronal differentiation and maturation during development of the nervous system (1, 2). As neurites extend further and acquire their final axonal and dendritic identities, neurons establish synaptic contacts and reach full maturation (3-5). These morphological changes require remodeling of the actin cytoskeleton (6). Rho small G protein is a key regulator of the actin cytoskeleton organization in neurons and has been shown to play important roles in several aspects of neurite outgrowth, such as sprouting, extension, and branching (7,8). An increase in Rho activity results in reduction of neurite sprouting, extension, and branching, whereas a decrease in Rho activity enhances neurite sprouting, extension, and branching (9 -12). The Rho activity is positively regulated by guanine nucleotide exchange factors (GEFs) 2 and negatively regulated

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