RESUMO
The Copines are a family of evolutionary conserved calcium-binding proteins found in most eukaryotic organisms from protists to humans. They share a unique architecture and contain tandem C2 domains and a Von Willebrand factor type A (VWA) domain. C2 domains in Copines bind calcium, phospholipids, and other proteins and mediate the transient association of these proteins with biological membranes at elevated calcium levels. The VWA domain also binds calcium and is involved in protein-protein interactions. Here, we provide a comprehensive review of the sequences, structures, expression, targeting, and function of the entire family of known Copine proteins (Copine 1-9 in mammals) with a particular emphasis on their functional roles in the mammalian brain. Neuronal Copines are implicated in a wide array of processes from cell differentiation to synaptic transmission and plasticity and are also linked to several pathological conditions from cancers to brain diseases. This review provides the most up-to-date insights into the structure and function of Copines, with an emphasis on their role in brain function.
Assuntos
Proteínas de Ligação ao Cálcio , Cálcio , Proteínas de Transporte , Animais , Humanos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Encéfalo/metabolismo , Mamíferos/metabolismoRESUMO
Neural systems encode information in the frequency of action potentials, which is then decoded by synaptic transmission. However, the rapid, synchronous release of neurotransmitters depletes synaptic vesicles (SVs), limiting release at high firing rates. How then do synapses convey information about frequency? Here, we show in mouse hippocampal neurons and slices that the adaptor protein AP-3 makes a subset of SVs that respond specifically to high-frequency stimulation. Neurotransmitter transporters slot onto these SVs in different proportions, contributing to the distinct properties of release observed at different excitatory synapses. Proteomics reveals that AP-3 targets the phospholipid flippase ATP8A1 to SVs; loss of ATP8A1 recapitulates the defect in SV mobilization at high frequency observed with loss of AP-3. The mechanism involves recruitment of synapsin by the cytoplasmically oriented phosphatidylserine translocated by ATP8A1. Thus, ATP8A1 enables the subset of SVs made by AP-3 to release at high frequency.
Assuntos
Complexo 3 de Proteínas Adaptadoras , Adenosina Trifosfatases , Fosfolipídeos , Transmissão Sináptica , Vesículas Sinápticas , Animais , Camundongos , Fosfolipídeos/metabolismo , Sinapses/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Complexo 3 de Proteínas Adaptadoras/metabolismo , Adenosina Trifosfatases/metabolismoRESUMO
The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.
Assuntos
Fusão de Membrana , Proteínas SNARE , Proteínas SNARE/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Ligação Proteica , Peptídeos/químicaRESUMO
Recent studies suggest that spontaneous and action potential-evoked neurotransmitter release processes are independently regulated. However, the mechanisms that uncouple the two forms of neurotransmission remain unclear. In cultured mouse and rat neurons, we show that the two C2 domain-containing protein copine-6 is localized to presynaptic terminals and binds to synaptobrevin2 as well as other SNARE proteins in a Ca2+-dependent manner. Ca2+-dependent interaction of copine-6 with synaptobrevin2 selectively suppresses spontaneous neurotransmission in a reaction that requires the tandem tryptophan residues at the C-terminal region of synaptobrevin2. Accordingly, copine-6 loss of function augmented presynaptic Ca2+ elevation-mediated neurotransmitter release. Intracellular Ca2+ chelation, on the other hand, occluded copine-6-mediated suppression of release. We also evaluated the molecular specificity of the copine-6-dependent regulation of spontaneous release and found that overexpression of copine-6 did not suppress spontaneous release in synaptobrevin2-deficient neurons. Together, these results suggest that copine-6 acts as a specific Ca2+-dependent suppressor of spontaneous neurotransmission.SIGNIFICANCE STATEMENT Synaptic transmission occurs both in response to presynaptic action potentials and spontaneously, in the absence of stimulation. Currently, much more is understood about the mechanisms underlying action potential-evoked neurotransmission compared with spontaneous release. However, recent studies have shown selective modulation of spontaneous neurotransmission process by several neuromodulators, suggesting specific molecular regulation of spontaneous release. In this study, we identify copine-6 as a specific regulator of spontaneous neurotransmission. By both gain-of-function and loss-of-function experiments, we show that copine-6 functions as a Ca2+-dependent suppressor of spontaneous release. These results further elucidate the mechanisms underlying differential regulation of evoked and spontaneous neurotransmitter release.
Assuntos
Proteínas de Transporte/metabolismo , Neurônios/fisiologia , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Humanos , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Protein aggregates containing ubiquitin (Ub) are commonly observed in neurodegenerative disorders, implicating the involvement of the ubiquitin proteasome system (UPS) in their pathogenesis. Here, we aimed to generate a mouse model for monitoring UPS function using a green fluorescent protein (GFP)-based substrate that carries a "noncleavable" N-terminal ubiquitin moiety (Ub(G76V)). We engineered transgenic mice expressing a fusion protein, consisting of the following: (1) Ub(G76V), GFP, and a synaptic vesicle protein synaptobrevin-2 (Ub(G76V)-GFP-Syb2); (2) GFP-Syb2; or (3) Ub(G76V)-GFP-Syntaxin1, all under the control of a neuron-specific Thy-1 promoter. As expected, Ub(G76V)-GFP-Syb2, GFP-Syb2, and Ub(G76V)-GFP-Sytaxin1 were highly expressed in neurons, such as motoneurons and motor nerve terminals of the neuromuscular junction (NMJ). Surprisingly, Ub(G76V)-GFP-Syb2 mice developed progressive adult-onset degeneration of motor nerve terminals, whereas GFP-Syb2 and Ub(G76V)-GFP-Syntaxin1 mice were normal. The degeneration of nerve terminals in Ub(G76V)-GFP-Syb2 mice was preceded by a progressive impairment of synaptic transmission at the NMJs. Biochemical analyses demonstrated that Ub(G76V)-GFP-Syb2 interacted with SNAP-25 and Syntaxin1, the SNARE partners of synaptobrevin. Ultrastructural analyses revealed a marked reduction in synaptic vesicle density, accompanying an accumulation of tubulovesicular structures at presynaptic nerve terminals. These morphological defects were largely restricted to motor nerve terminals, as the ultrastructure of motoneuron somata appeared to be normal at the stages when synaptic nerve terminals degenerated. Furthermore, synaptic vesicle endocytosis and membrane trafficking were impaired in Ub(G76V)-GFP-Syb2 mice. These findings indicate that Ub(G76V)-GFP-Syb2 may compete with endogenous synaptobrevin, acting as a gain-of-function mutation that impedes SNARE function, resulting in the depletion of synaptic vesicles and degeneration of the nerve terminals. SIGNIFICANCE STATEMENT: Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.
Assuntos
Doença dos Neurônios Motores/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Ubiquitina/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Doença dos Neurônios Motores/patologia , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ubiquitina/genéticaRESUMO
Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca(2+)-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca(2+)-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle-associated SNAREs.
Assuntos
Neurônios/citologia , Proteínas R-SNARE/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Cloreto de Amônio/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Células Cultivadas , Colecistocinina/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Humanos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas R-SNARE/genética , Interferência de RNA/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas SNARE/metabolismo , Sinapses/genética , Sinapses/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Proteína 25 Associada a Sinaptossoma/deficiência , Sintaxina 1/metabolismo , Transfecção , Valina/análogos & derivados , Valina/farmacologia , Proteína 2 Associada à Membrana da Vesícula/deficiênciaRESUMO
Recent studies suggest that synaptic vesicles (SVs) giving rise to spontaneous neurotransmission are distinct from those that carry out evoked release. However, the molecular basis of this dichotomy remains unclear. Here, we focused on two noncanonical SNARE molecules, Vps10p-tail-interactor-1a (vti1a) and VAMP7, previously shown to reside on SVs. Using simultaneous multicolor imaging at individual synapses, we could show that compared to the more abundant vesicular SNARE synaptobrevin2, both vti1a and VAMP7 were reluctantly mobilized during activity. Vti1a, but not VAMP7, showed robust trafficking under resting conditions that could be partly matched by synaptobrevin2. Furthermore, loss of vti1a function selectively reduced high-frequency spontaneous neurotransmitter release detected postsynaptically. Expression of a truncated version of vti1a augmented spontaneous release more than full-length vti1a, suggesting that an autoinhibitory process regulates vti1a function. Taken together, these results support the premise that in its native form vti1a selectively maintains spontaneous neurotransmitter release.
Assuntos
Neurônios/fisiologia , Proteínas Qb-SNARE/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Potenciais de Ação/genética , Anestésicos Locais/farmacologia , Animais , Animais Recém-Nascidos , Transporte Biológico/genética , Biofísica , Cálcio/metabolismo , Células Cultivadas , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Concentração de Íons de Hidrogênio , Macrolídeos/farmacologia , Microscopia Imunoeletrônica/métodos , Mutação/genética , Neurônios/citologia , Técnicas de Patch-Clamp , Cloreto de Potássio/farmacologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Qb-SNARE/genética , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/genética , Vesículas Sinápticas/ultraestrutura , Sinaptotagmina I/metabolismo , Tetrodotoxina/farmacologia , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Recent findings suggest that spontaneous neurotransmission is a bona fide pathway for interneuronal signaling that operates independent of evoked transmission via distinct presynaptic as well as postsynaptic substrates. This article will examine the role of spontaneous release events in neuronal signaling by focusing on aspects that distinguish this process from evoked neurotransmission, and evaluate the mechanisms that may underlie this segregation.
Assuntos
Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Animais , Transdução de SinaisRESUMO
Alpha-latrotoxin induces neurotransmitter release by stimulating synaptic vesicle exocytosis via two mechanisms: (1) A Ca(2+)-dependent mechanism with neurexins as receptors, in which alpha-latrotoxin acts like a Ca(2+) ionophore, and (2) a Ca(2+)-independent mechanism with CIRL/latrophilins as receptors, in which alpha-latrotoxin directly stimulates the transmitter release machinery. Here, we show that the Ca(2+)-independent release mechanism by alpha-latrotoxin requires the synaptic SNARE-proteins synaptobrevin/VAMP and SNAP-25, and, at least partly, the synaptic active-zone protein Munc13-1. In contrast, the Ca(2+)-dependent release mechanism induced by alpha-latrotoxin does not require any of these components of the classical synaptic release machinery. Nevertheless, this type of exocytotic neurotransmitter release appears to fully operate at synapses, and to stimulate exocytosis of the same synaptic vesicles that participate in physiological action potential-triggered release. Thus, synapses contain two parallel and independent pathways of Ca(2+)-triggered exocytosis, a classical, physiological pathway that operates at the active zone, and a novel reserve pathway that is recruited only when Ca(2+) floods the synaptic terminal.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Exocitose/fisiologia , Venenos de Aranha/farmacologia , Transmissão Sináptica/fisiologia , Animais , Viúva Negra/química , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Exocitose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Camundongos , Camundongos Knockout , Venenos de Aranha/isolamento & purificação , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Sinapses/ultraestruturaRESUMO
Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca(2+)-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1-SNARE-complexin macromolecular assembly that is poised for Ca(2+) triggering of fusion.
Assuntos
Encéfalo/metabolismo , Proteínas Munc18/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas SNARE/metabolismo , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Encéfalo/ultraestrutura , Células Cultivadas , Vetores Genéticos/genética , Substâncias Macromoleculares/metabolismo , Fusão de Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas Munc18/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Mutação Puntual/genética , Terminações Pré-Sinápticas/ultraestrutura , Ligação Proteica/genética , Ratos , Proteínas SNARE/ultraestrutura , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/ultraestrutura , Sintaxina 1/genética , Sintaxina 1/metabolismo , TransfecçãoRESUMO
Neurotransmission in the nervous system is initiated at presynaptic terminals by fusion of synaptic vesicles with the plasma membrane and subsequent exocytic release of chemical transmitters. Currently, there are multiple methods to detect neurotransmitter release from nerve terminals, each with their own particular advantages and disadvantages. For instance, most commonly employed methods monitor actions of released chemical substances on postsynaptic receptors or artificial substrates such as carbon fibers. These methods are closest to the physiological setting because they have a rapid time resolution and they measure the action of the endogenous neurotransmitters rather than the signals emitted by exogenous probes. However, postsynaptic receptors only indirectly report neurotransmitter release in a form modified by the properties of receptors themselves, which are often nonlinear detectors of released substances. Alternatively, released chemical substances can be detected biochemically, albeit on a time scale slower than electrophysiological methods. In addition, in certain preparations, where presynaptic terminals are accessible to whole cell recording electrodes, fusion of vesicles with the plasma membrane can be monitored using capacitance measurements. In the last decade, in addition to electrophysiological and biochemical methods, several fluorescence imaging modalities have been introduced which report synaptic vesicle fusion, endocytosis, and recycling. These methods either take advantage of styryl dyes that can be loaded into recycling vesicles or exogenous expression of synaptic vesicle proteins tagged with a pH-sensitive GFP variant at regions facing the vesicle lumen. In this chapter, we will provide an overview of these methods with particular emphasis on their relative strengths and weaknesses and discuss the types of information one can obtain from them.
Assuntos
Exocitose/efeitos dos fármacos , Neurotransmissores/metabolismo , Animais , Eletrofisiologia , Endocitose/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Neurotransmissores/análise , Receptores de Neurotransmissores/efeitos dos fármacos , Receptores de Neurotransmissores/metabolismo , Receptores Pré-Sinápticos/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismoRESUMO
The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal syntaxin/Munc18 interaction is essential. Introduction of an N-terminal syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.
Assuntos
Proteínas Munc18/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas SNARE/metabolismo , Sintaxina 1/metabolismo , Animais , Exocitose/fisiologia , Células HeLa , Humanos , Proteínas Munc18/química , Células PC12 , Fragmentos de Peptídeos/química , Ligação Proteica/fisiologia , Ratos , Proteínas SNARE/química , Sintaxina 1/químicaRESUMO
Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for 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 SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.
Assuntos
Proteínas Munc18/metabolismo , Neurônios/metabolismo , Proteínas SNARE/metabolismo , Animais , Cromatografia em Gel , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Proteínas Munc18/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
Alpha-latrotoxin, a potent stimulator of exocytosis from neurons and neuroendocrine cells, has been studied intensively, but the mechanisms of its actions are poorly understood. Here, we developed a new method to generate active recombinant alpha-latrotoxin and conducted a structure/function analysis of the toxin in stimulating Ca2+-dependent exocytosis. alpha-Latrotoxin consists of a conserved N-terminal domain and C-terminal ankyrin-like repeats. After cleavage of an N-terminally fused purification tag of glutathione S-transferase (GST), the recombinant toxin strongly stimulated exocytosis, whereas the GST-fused toxin was much less potent. The GST-fused toxin bound to the receptors [neurexin 1alpha; CL1 (CIRL/latrophilin 1)] as efficiently as did the GST-cleaved toxin but was much less effective in inserting into the plasma membrane and inducing cation conductance. The toxin with deletion of the last two ankyrin-like repeats still bound the receptors but could neither stimulate exocytosis nor induce cation conductance efficiently. The abilities of the mutated toxins to stimulate exocytosis correlated well with their abilities to induce cation conductance, but not their binding to the receptors. Our results indicate that (1) C-terminal ankyrin-like repeats and a free (unfused) N terminus are both required for the toxin to form pores, which is essential for Ca2+-dependent exocytosis, and (2) receptor binding alone is not sufficient to stimulate Ca2+-dependent exocytosis.
Assuntos
Anquirinas/genética , Anquirinas/metabolismo , Cálcio/metabolismo , Exocitose/fisiologia , Sequências Repetitivas de Aminoácidos , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Células PC12 , Ligação Proteica/fisiologia , Ratos , Relação Estrutura-AtividadeRESUMO
Synaptophysin and synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- and heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals that are capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes are regulated. We show that in synaptosomes treated with three stimuli that induce exocytosis (a depolarizing K(+) solution, the excitatory neurotoxin alpha-latrotoxin, or the Ca(2+)-ionophore ionomycin), the homo- and heteromultimerization of synaptophysin and synaptobrevin is increased up to 6-fold. Whereas at rest less than 10% of the total synaptobrevin and synaptophysin could be chemically cross-linked into homo- and heteromeric complexes, after stimulation up to 25% of synaptobrevin and synaptophysin are present in homo- and heteromultimers, suggesting that a large fraction of these synaptic vesicle proteins physiologically participate in such complexes. The increase in multimerization of synaptophysin and synaptobrevin was only observed in intact but not in lysed synaptosomes and could not be inhibited by general kinase or phosphatase inhibitors. The stimulus dependence of synaptophysin and synaptobrevin multimers indicates that the complexes are not composed of a fixed multisubunit structure, for example, as an ion channel, but represent distinct functional states of synaptobrevin and synaptophysin that are modulated in parallel with synaptic vesicle exo- and endocytosis.
Assuntos
Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinaptofisina/análise , Proteínas de Transporte Vesicular/análise , Animais , Química Encefálica , Reagentes de Ligações Cruzadas/farmacologia , Eletroforese em Gel de Poliacrilamida , Exocitose , Immunoblotting , Ionomicina/farmacologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Camundongos , Potássio/farmacologia , Proteínas R-SNARE , Ratos , Venenos de Aranha/farmacologia , Sinaptofisina/efeitos dos fármacos , Sinaptofisina/metabolismo , Sinaptofisina/fisiologia , Sinaptossomos , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/fisiologiaRESUMO
Cleavage of amyloid-beta precursor protein (APP) by alpha-,beta-, and gamma-secretases releases an extracellular fragment called APPS, small Abeta peptides, and a short APP intracellular domain that may provide a transcriptional signal analogous to the Notch intracellular domain. Notch cleavage is activated by extracellular ligands on the cell surface, but the cellular localization of APP cleavage remains unclear. We now show that in transfected cultured cells, the plasma membrane SNARE protein syntaxin 1A, when expressed as a full-length protein, disrupts the Golgi apparatus and blocks trans-Golgi traffic and exocytosis. In contrast, truncated syntaxin 1A1-243 selectively abolishes exocytosis but has no apparent effect on trans-Golgi traffic or Golgi structure, whereas further truncated syntaxins 1A1-236 and 1A1-230 have no effect on either exocytosis or Golgi traffic. Using these syntaxin 1A fragments, we demonstrated that blocking trans-Golgi traffic greatly impairs APP cleavage and AICD-dependent nuclear signaling, whereas blocking exocytosis alone does not affect either process, even though secretion of APPS and Abeta40 peptide is abolished. Our data suggest that, different from Notch, cleavage of APP is independent of cell surface regulation by extracellular ligands but may be controlled by intracellular signaling.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Antígenos de Superfície/química , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Precursor de Proteína beta-Amiloide/química , Animais , Células COS , Linhagem Celular , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Exocitose , Complexo de Golgi/metabolismo , Humanos , Immunoblotting , Ligantes , Peptídeos/química , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores Notch , Transdução de Sinais , Sintaxina 1 , Ativação Transcricional , TransfecçãoRESUMO
Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins that function in exocytosis. Among the Rab proteins tested, we found that besides the previously described Rab3 proteins, only members of the Rab11 family (Rab11a, 11b, and 25) impaired Ca2+-induced exocytosis. Rab11b, which is enriched in brain, had the strongest effect. Consistent with a role in exocytosis, Rab11 and Rab3 proteins were colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on mature synaptic vesicles in brain. Rab11b mutants that fix Rab11b in the GTP- or GDP-bound state both effectively inhibited Ca2+-induced exocytosis but seemed to act by distinct mechanisms: whereas GDP-bound Rab11b greatly stimulated constitutive secretion of hGH and depleted hGH stores in secretory vesicles, GTP-bound Rab11b only had a moderate effect on constitutive secretion and no effect on vesicular hGH stores. These results suggest that, consistent with a GTP-dependent regulation of Rab function, GDP-bound Rab11b indirectly inhibits Ca2+-triggered exocytosis by causing the loss of hGH from the PC12 cells, whereas GTP-bound Rab11b directly impairs Ca2+-triggered exocytosis. In contrast to neuroendocrine PC12 cells in which GTP- and GDP-bound Rab11b inhibited Ca2+-induced, but not constitutive, exocytosis, in non-neuronal cells GTP- and GDP-bound Rab11b inhibited constitutive exocytosis and caused an accumulation of cellular hGH. Viewed together, our data suggest that, in addition to other functions, Rab11 has a specific role in neuronal and neuroendocrine but not in non-neuronal cells as a GTP-dependent switch between regulated and constitutive secretory pathways.
Assuntos
Cálcio/fisiologia , Exocitose/fisiologia , Neurônios/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Cálcio/farmacologia , Fracionamento Celular , Linhagem Celular , Exocitose/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hormônio do Crescimento Humano/biossíntese , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Proteínas Luminescentes/genética , Mutação , Neurônios/citologia , Células PC12 , Potássio/farmacologia , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Sinápticas/metabolismo , Transfecção , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/farmacologiaRESUMO
Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca(2+)-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca(2+)-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lüthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca(2+)-independent exocytosis that correlated with the inhibition of regulated Ca(2+)-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knock-out studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.