Both SM proteins (for Sec1/Munc18-like proteins) and SNARE soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, Munc18-1 known to bind tightly syntaxin-1, only when syntaxin-1 a closed conformation that incompatible with complex formation. We now show also binds assembled...

Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. binds to the SNARE syntaxin-1 folded into a closed conformation complexes containing open syntaxin-1. Understanding which steps in fusion depend on latter interaction whether competes with other factors such as complexins complex binding is elucidate mechanisms involved. In this study, we show that lentiviral expression of rescues abrogation release knockout mice. We describe point...

The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N -ethylmaleimide-sensitive factor attachment (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, synaptobrevin/VAMP (vesicle-associated membrane protein) constitute core fusion machinery for synaptic vesicle exocytosis. Strikingly, interacts with neuronal SNARE in two distinct modes (i.e., isolated syntaxin-1 alone a “closed” conformation assembled complexes containing an “open” conformation). However, it is unclear whether of...

α-Latrotoxin, a potent excitatory neurotoxin, binds to two receptors: G-protein-coupled receptor calledCIRL/latrophilin 1 (CL1) and cell-surface protein called neurexin Iα. We now show that CL1 belongs family of closely related receptors CL1, CL2, CL3. CLs exhibit an unusual multidomain structure with similar alternative splicing large extra- intracellular sequences. share domains other receptors, lectins, olfactomedins/myocilin. In addition, contain novel, widespread cysteine-rich domain...

Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations distinct functions have been proposed for different occasionally even the same Rab3 protein. This is exemplified by studies demonstrating deletion Rab3A knock-out mice results dysregulation final stages exocytosis, whereas overexpression neuroendocrine cells causes nearly complete inhibition Ca2+-triggered We now examined properties all assays, with...

Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo- exocytosis. Phosphorylation phosphatidylinositol at D-4 position, an essential step biosynthesis PIPs, appears be catalyzed by two biochemically distinct enzymes. However, only one these enzymes has been molecularly characterized. We now describe a novel class 4-kinases that probably corresponds missing element metabolism....

Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins function in exocytosis. Among the tested, found besides previously described Rab3 proteins, only members of Rab11 family (Rab11a, 11b, and 25) impaired Ca 2+ -induced Rab11b, which is enriched brain, had strongest effect. Consistent with role exocytosis, were colocalized other vesicle on secretory vesicles mature synaptic brain. Rab11b mutants fix GTP- or...

The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. mechanism action these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on primary structure δ-latroinsectotoxin (δ-LIT), novel insect-specific toxin from BWSV, 1214 amino acids. δ-LIT...

α-Latrotoxin induces neurotransmitter release by stimulating synaptic vesicle exocytosis via two mechanisms: (1) A Ca²⁺-dependent mechanism with neurexins as receptors, in which α-latrotoxin acts like a Ca²⁺ ionophore, and (2) Ca²⁺-independent CIRL/latrophilins directly stimulates the transmitter machinery. Here, we show that requires SNARE-proteins synaptobrevin/VAMP SNAP-25, and, at least partly, active-zone protein Munc13-1. In contrast, induced does not...

Newly synthesized phosphatidylinositol phosphates have been implicated in many membrane-trafficking reactions. They are essential for exocytosis of norepinephrine PC12 cells and chromaffin cells, suggesting a function membrane fusion. We now studied the role synaptic vesicle using synaptosomes. Under conditions where phosphorylation phosphatidylinositols is blocked, secretion was nearly abolished whereas glutamate GABA release still elicited. Thus phosphatidylinositides only some fusion...

Cleavage of amyloid-β precursor protein (APP) by α-,β-, and γ-secretases releases an extracellular fragment called APPS, small Aβ peptides, a short APP intracellular domain that may provide transcriptional signal analogous to the Notch domain. cleavage is activated ligands on cell surface, but cellular localization remains unclear. We now show in transfected cultured cells, plasma membrane SNARE syntaxin 1A, when expressed as full-length protein, disrupts Golgi apparatus blocks trans-Golgi...

Recent studies suggest that spontaneous and action potential-evoked neurotransmitter release processes are independently regulated. However, the mechanisms uncouple two forms of neurotransmission remain unclear. In cultured mouse rat neurons, we show C2 domain-containing protein copine-6 is localized to presynaptic terminals binds synaptobrevin2 as well other SNARE proteins in a Ca²⁺-dependent manner. interaction with selectively suppresses reaction requires tandem tryptophan...

The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share unique architecture and contain tandem C2 domains Von Willebrand factor type A (VWA) domain. bind calcium, phospholipids, other mediate the transient association these with biological membranes at elevated calcium levels. VWA domain also binds is involved protein–protein interactions. Here, we provide comprehensive review sequences, structures,...

Phosphatidylinositol phosphates (PIPs) perform central functions in signal transduction and membrane traffic. Synaptojanin is a PIP 5-phosphatase that expressed brain-specific ubiquitous splice variants thought to constitute the major mammalian brain (Woscholski, R., Finan, P.M., Radley, E., Totty, N.F., Sterling, A.E., Hsuan, J.J., Waterfield, M. D., Parker, P. J. (1997) Biol. Chem. 272, 9625-9628). We now describe synaptojanin 2, novel isoform of that, similar 1, contains an N-terminal...

Protein aggregates containing ubiquitin (Ub) are commonly observed in neurodegenerative disorders, implicating the involvement of proteasome system (UPS) their pathogenesis. Here, we aimed to generate a mouse model for monitoring UPS function using green fluorescent protein (GFP)-based substrate that carries "noncleavable" N-terminal moiety (Ub^G76V). We engineered transgenic mice expressing fusion protein, consisting following: (1) Ub^G76V, GFP, and synaptic vesicle...

α-Latrotoxin, a potent stimulator of exocytosis from neurons and neuroendocrine cells, has been studied intensively, but the mechanisms its actions are poorly understood. Here, we developed new method to generate active recombinant α-latrotoxin conducted structure/function analysis toxin in stimulating Ca 2+ -dependent exocytosis. α-Latrotoxin consists conserved N-terminal domain C-terminal ankyrin-like repeats. After cleavage an N-terminally fused purification tag glutathione S -transferase...

Synaptophysin and synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes regulated. show synaptosomes treated with three stimuli induce exocytosis (a depolarizing K(+) solution, the excitatory neurotoxin alpha-latrotoxin, or Ca(2+)-ionophore ionomycin), heteromultimerization...