Direct Interaction of the Rat unc-13 Homologue Munc13-1 with the N Terminus of Syntaxin

Abstract

unc-13 mutants in Caenorhabditis elegans are characterized by a severe deficit in neurotransmitter release. Their phenotype is similar to that of the C. elegans unc-18 mutation, which is thought to affect synaptic vesicle docking to the active zone. This suggests a crucial role for the unc-13 gene product in the mediation or regulation of synaptic vesicle exocytosis. Munc13-1 is one of three closely related rat homologues of unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. In the present study, we demonstrate by three independent methods that the C terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homologue Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin.Nerve cells store neurotransmitters in synaptic vesicles. These vesicles dock to a specialized region of the synaptic plasma membrane, the active zone, where they undergo a maturation or priming process. Upon depolarization and a consequential rise in the intracellular calcium concentration, primed vesicles release their content by exocytosis. Following release, vesicular membrane and protein components are retrieved by endocytosis and recycled through an early endosomal compartment. From there, synaptic vesicles bud off for a new round of regulated exocytosis (1, 2).During the last decade, the molecular mechanisms underlying synaptic vesicle exocytosis and recycling have been the focus of numerous studies. In particular, the components of the exocytotic synaptic core complex, the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25, 1 have been analyzed in detail. These proteins are absolutely essential for neurotransmitter release, and their complex formation was originally thought to mediate the vesicl...

Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

“…Mammalian homologues munc13-1, -2, and -3 (munc13s) were originally cloned from rat brain, and similar to Unc-13 they possess both DAG-and Ca 2ϩ -binding domains (Brose et al, 1995). Syntaxin, synaptobrevin, SNAP 25 (Betz et al, 1997), and Doc2 (Orita et al, 1997) were found to coimmunoprecipitate with munc13s, consistent with the suggestion that this new family of DAG-binding proteins is involved in vesicle trafficking and neurotransmitter release. Recently we cloned a human renal homologue, hmunc13, which is expressed in cultured renal mesangial and cortical epithelial cells (Song et al, 1998).…”

“…A number of SNARE proteins, such as yeast Sed5p (Banfield et al, 1994) and mVps45 (Tellam et al, 1997), mammalian syntaxin 6 (Bock et al, 1997), VAMP4, Syntaxin 13, and mVtib (Advani et al, 1998), have all been reported to be localized to the Golgi. Rat munc13-1 has been shown to interact with a number of proteins involved in vesicle docking and trafficking, such as syntaxin (Betz et al, 1997) and Doc2 (Orita et al, 1997). Interaction of munc13-1 and Doc2 was stimulated by DAG and has been suggested to be involved in Ca 2ϩ -dependent exocytosis (Orita et al, 1997).…”

Specific Interaction between SNAREs and Epsin N-terminal Homology (ENTH) Domains of Epsin-related Proteins in trans-Golgi Network to Endosome Transport

“…Yeast Two-hybrid Assays-The yeast two-hybrid screen was performed as described (15) in L40 cells using pLexN with the N-terminal domain of mouse vti1b (amino acids 1-128) as bait vector and a library derived from rat E18 embryonic brain RNA in pVP16 -3 as prey vector (16). Positive cells were selected on plates with minimal medium lacking uracil, lysine, tryptophan, leucine, and histidine complemented with 2.5 mM 3-aminotriazole.…”

Active zones for presynaptic plasticity in the brain

“…131 Munc13s unfold and activate the SNARE protein syntaxin and promote the formation of the SNARE complex. [131][132][133] In addition, recent studies have shown that Munc13 proteins play an important role in potentiation of neurotransmitter release in short-term synaptic plasticity. 125 Rosenmund et al 125 using knockout mice for Munc13-1 and Munc13-2, have shown that in cultured hippocampal neurons there are two functional classes of synapses depending on the differential localization of Munc13-1 and Munc13-2 in the same neuron along a single axon.…”