RESUMEN
Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.
Asunto(s)
Linfocitos B/inmunología , Cadenas Ligeras de Clatrina/genética , Endocitosis/inmunología , Eliminación de Gen , Cambio de Clase de Inmunoglobulina , Animales , Linfocitos B/patología , Corteza Cerebral/citología , Corteza Cerebral/inmunología , Cadenas Ligeras de Clatrina/inmunología , Regulación de la Expresión Génica , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/genética , Hígado/citología , Hígado/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/citología , Miocardio/inmunología , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/inmunología , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Receptores Opioides delta/genética , Receptores Opioides delta/inmunología , Receptores de Factores de Crecimiento Transformadores beta/agonistas , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1·SNAP-25) and v-/t-SNARE (VAMP2·syntaxin 1·SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.
Asunto(s)
Fusión de Membrana/fisiología , Proteínas Munc18/metabolismo , Proteínas SNARE/química , Animales , ADN/química , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Modelos Biológicos , Péptidos/química , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Ratas , TemperaturaRESUMEN
Regulated exocytosis requires tight coupling of the membrane fusion machinery to a triggering signal and a fast response time. Complexins are part of this regulation and, together with synaptotagmins, control calcium-dependent exocytosis. Stimulatory and inhibitory functions have been reported for complexins. To test if complexins directly affect membrane fusion, we analyzed the 4 known mammalian complexin isoforms in a reconstituted fusion assay. In contrast to complexin III (CpxIII) and CpxIV, CpxI and CpxII stimulated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-pin assembly and membrane fusion. This stimulatory effect required a preincubation at low temperature and was specific for neuronal t-SNAREs. Stimulation of membrane fusion was lost when the carboxy-terminal domain of CpxI was deleted or serine 115, a putative phosphorylation site, was mutated. Transfer of the carboxy-terminal domain of CpxI to CpxIII resulted in a stimulatory CpxIII-I chimera. Thus, the carboxy-terminal domains of CpxI and CpxII promote the fusion of high-curvature liposomes.