Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular , Animales , Membrana Celular/metabolismo , Endocitosis/fisiología , Exocitosis/fisiología , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Estructura Terciaria de Proteína , Proteínas SNARE , Sinapsis/fisiologíaRESUMEN
SNAP receptor (SNARE) complexes bridge opposing membranes to promote membrane fusion within the secretory and endosomal pathways. Because only the exocytic SNARE complexes have been characterized in detail, the structural features shared by SNARE complexes from different fusion steps are not known. We now describe the subunit structure, assembly, and regulation of a quaternary SNARE complex, which appears to mediate an early step in endoplasmic reticulum (ER) to Golgi transport. Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity binding site for sec22b, an R-SNARE. The syntaxin 5 amino-terminal domain potently inhibits SNARE complex assembly. The ER/Golgi quaternary complex is remarkably similar to the synaptic complex, suggesting that a common pattern is followed at all transport steps, where three Q-helices assemble to form a high affinity binding site for a fourth R-helix on an opposing membrane. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others assemble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1. Finally, we demonstrate that the quaternary complex containing sec22b is not an in vitro entity only, but is a bona fide species in living cells.
Asunto(s)
Retículo Endoplásmico/química , Proteínas de la Membrana/química , Proteínas de Transporte Vesicular , Animales , Sitios de Unión , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Cromatografía , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Aparato de Golgi/química , Humanos , Cinética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , TransfecciónRESUMEN
The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Células Cultivadas , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Microscopía Fluorescente , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARERESUMEN
ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Transporte Vesicular , Animales , Células COS , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Células PC12 , Pruebas de Precipitina , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Conejos , Ratas , Proteínas SNARE , Fracciones Subcelulares , Temperatura , Células Tumorales CultivadasRESUMEN
A major current issue in vesicle trafficking is whether NSF (N-ethylmaleimide-sensitive factor) and alpha-SNAP (alpha-soluble NSF attachment protein) are required prior to SNARE (SNAP receptor) complex formation to allow vesicle docking, or after docking at a step close to membrane fusion. Recent studies of yeast vacuolar fusion indicated that the requirement for ATP, NSF and alpha-SNAP could be completely satisfied prior to SNARE docking complex assembly; however, the universality of a predocking role for these factors remains to be established. The vacuolar fusion system has also been used to directly demonstrate a requirement for SNARE proteins on both fusing membranes, verifying a central postulate of current fusion models.
Asunto(s)
Proteínas Portadoras/fisiología , Endosomas/fisiología , Membranas Intracelulares/fisiología , Lisosomas/fisiología , Fusión de Membrana , Proteínas de la Membrana/fisiología , Proteínas de Transporte Vesicular , Animales , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Homeostasis , Modelos Biológicos , Proteínas SNARE , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Vacuolas/fisiologíaRESUMEN
The proposed cis-Golgi vesicle receptor syntaxin 5 was found in a complex with Golgi-associated SNARE of 28 kDa (GOS-28), rbet1, rsly1, and two novel proteins characterized herein: rat sec22b and membrin, both cytoplasmically oriented integral membrane proteins. The complex appears to recapitulate vesicle docking interactions of proteins originating from distinct compartments, since syntaxin 5, rbet1, and GOS-28 localize to Golgi membranes, whereas mouse sec22b and membrin accumulate in the endoplasmic reticulum. Protein interactions in the complex are dramatically rearranged by N-ethylmaleimide-sensitive factor. The complex consists of two or more subcomplexes with some members (rat sec22b and syntaxin 5) in common and others (rbet1 and GOS-28) mutually exclusively associated. We propose that these protein interactions determine vesicle docking/fusion fidelity between the endoplasmic reticulum and Golgi.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico/fisiología , Células COS/fisiología , Proteínas Portadoras/fisiología , Detergentes , Retículo Endoplásmico/química , Epítopos/análisis , Expresión Génica/fisiología , Aparato de Golgi/química , Mamíferos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/aislamiento & purificación , Neuronas/citología , Neuronas/metabolismo , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Conejos , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Fracciones Subcelulares/química , Vesículas Sinápticas/químicaRESUMEN
Vesicle traffic propagates and maintains distinct subcellular compartments and routes secretory products from their site of synthesis to their final destinations. As a basis for the specificity of vesicular transport reactions, each step in the secretory pathway appears to be handled by a distinct set of evolutionarily conserved proteins. Mammalian proteins responsible for vesicle trafficking at early steps in the secretory pathway are not well understood. In this report, we describe rat sec22 (rsec22) and rat bet1 (rbet1), mammalian sequence homologs of yeast proteins identified as mediators of endoplasmic reticulum-to-Golgi protein transport. rsec22 and rbet1 were expressed widely in mammalian tissues, as anticipated for proteins involved in fundamental membrane trafficking reactions. Recombinant rsec22 and rbet1 proteins behaved as integral membrane components of 28 and 18 kDa, respectively, consistent with their primary structures, which contain a predicted transmembrane domain at or near the carboxyl terminus. Recombinant rsec22 and rbet1 had distinct subcellular localizations, with rsec22 residing on endoplasmic reticulum membranes and rbet1 found on Golgi membranes. Studies with brefeldin A and nocodazole indicated that rbet1 function might involve interaction with or retention in the intermediate compartment. The distinct localizations of rsec22 and rbet1 may reflect their participation in opposite directions of membrane flow between the endoplasmic reticulum and Golgi apparatus.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Línea Celular , Chlorocebus aethiops , Clonación Molecular , Biblioteca de Genes , Humanos , Membranas Intracelulares , Mamíferos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa , Proteínas Qc-SNARE , Proteínas R-SNARE , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Regulated fusion of secretory granules with the plasma membrane in secretory cells requires ATP, Ca2+ and cytosolic as well as membrane proteins. ATP-dependent steps in Ca(2+)-activated secretion from PC12 cells require three cytosolic PEP proteins (priming in exocytosis proteins, PEP1-3), the identity of which will provide insights into the required ATP-using reactions. PEP3 was recently identified as phosphatidylinositol transfer protein (PtdInsTP), and here we report that PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K). The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequential phosphoinositide recruitment and phosphorylation explains their synergistic activity in ATP-dependent priming. Moreover, inhibition of Ca(2+)-activated secretion by PtdIns(4,5)P2-specific antibodies and phospholipase C implies that 5-phosphorylated inositides play a novel, necessary role in the regulated secretory pathway. The results indicate that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Exocitosis , Proteínas de la Membrana , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Bovinos , Citosol/metabolismo , Norepinefrina/metabolismo , Células PC12 , Proteínas de Transferencia de Fosfolípidos , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ratas , Fosfolipasas de Tipo C/metabolismoAsunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Exocitosis/fisiología , Proteínas de Transporte Vesicular , Actinas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Células PC12 , Fosfolípidos/metabolismo , Fosforilación , Ratas , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Vesículas Sinápticas/metabolismoRESUMEN
Elucidation of the reactions responsible for the calcium-regulated fusion of secretory granules with the plasma membrane in secretory cells would be facilitated by the identification of participant proteins having known biochemical activities. The successful characterization of cytosolic and vesicle proteins that may function in calcium-regulated secretion has not yet revealed the molecular events underlying this process. Regulated secretion consists of sequential priming and triggering steps which depend on ATP and Ca2+, respectively, and require distinct cytosolic proteins. Characterization of priming-specific factors (PEP proteins) should enable the ATP-requiring reactions to be identified. Here we show that one of the mammalian priming factors (PEP3) is identical to phosphatidylinositol transfer protein (PITP). The physiological role of PITP was previously unknown. We also find that SEC14p, the yeast phosphatidylinositol transfer protein which is essential for constitutive secretion, can substitute for PEP3/PITP in priming. Our results indicate that a role for phospholipid transfer proteins is conserved in the constitutive and regulated secretory pathways.
Asunto(s)
Adenosina Trifosfato/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Hígado/metabolismo , Proteínas de la Membrana , Fosfatidilinositoles/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Permeabilidad de la Membrana Celular , Cromatografía en Gel , Citosol/metabolismo , Ácido Egtácico/farmacología , Cinética , Sustancias Macromoleculares , Microsomas/metabolismo , Datos de Secuencia Molecular , Norepinefrina/metabolismo , Células PC12 , Proteínas de Transferencia de Fosfolípidos , RatasRESUMEN
The biochemical events and components responsible for ATP-dependent Ca(2+)-activated secretion remain to be identified. To simplify the molecular dissection of regulated secretion, we have resolved norepinephrine (NE) secretion from semi-intact PC12 cells into two kinetically distinct stages, each of which was studied separately to discern its molecular requirements. The first stage consisted of MgATP-dependent priming of the secretory apparatus in the absence of Ca2+. MgATP-dependent priming was readily reversible and inhibited by a broad range of protein kinase inhibitors. The second stage consisted of Ca(2+)-triggered exocytosis which, in contrast to priming, occurred in the absence of MgATP. Both priming and triggering were found to be dependent upon or stimulated by cytosolic proteins. The priming and triggering activities of cytosol were functionally distinct as indicated by differing thermolability. Furthermore, active components in cytosol resolved by gel filtration were found to support either priming or triggering, but not both. For both priming and triggering reactions, several peaks of activity were detected; one of each type of factor was partially purified from rat brain cytosol, and found to be enriched for stage-specific activity. Two partially purified factors exhibiting stage-specific activity, a approximately 20-kD priming factor and approximately 300-kD triggering factor, were able to support regulated secretion as effectively as crude cytosol when used sequentially in the partial reactions. Further characterization of stage-specific cytosolic factors should clarify the nature of MgATP- and Ca(2+)-dependent events in the regulated secretory pathway.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Norepinefrina/metabolismo , Proteínas/metabolismo , Animales , Química Encefálica , Cromatografía en Gel , Células PC12 , RatasAsunto(s)
Adaptación Psicológica , Aprendizaje/fisiología , Percepción Visual/fisiología , Adolescente , Adulto , Femenino , Humanos , Masculino , Movimiento (Física) , PsicofísicaAsunto(s)
Aprendizaje , Actividad Motora , Adaptación Ocular , Conducta , Computadores , Electrooculografía , Movimientos Oculares , Retroalimentación , Humanos , Memoria , Modelos Teóricos , Oscilometría , Proyectos Piloto , Propiocepción , Tiempo de Reacción , Percepción Espacial , Percepción VisualAsunto(s)
Cabeza , Ilusiones , Movimiento , Orientación , Percepción Visual , Conversión Analogo-Digital , Análisis de Varianza , Procesamiento Automatizado de Datos , Retroalimentación , Humanos , Teoría de la Información , Memoria , Percepción de Movimiento , Destreza Motora , Tiempo de Reacción , Factores de Tiempo , Transferencia de Experiencia en PsicologíaAsunto(s)
Adaptación Psicológica , Actividad Motora , Percepción Espacial , Percepción Visual , Femenino , Mano , Humanos , Matemática , Distorsión de la PercepciónAsunto(s)
Adaptación Ocular , Pruebas de Visión , Campos Visuales , Análisis de Varianza , Movimientos Oculares , Femenino , Fijación Ocular , Humanos , MasculinoRESUMEN
A visual target was mloved left and right in exact synchrony with vertical movements of the head. A few minutes' exposure to this novel head-movement feedback led to a change in the visual discrimlination of head movement from object movement. The critical factor in the adaptation is the novel correlation of eye and head movement elicited during the period of exposure.