RESUMO
The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271-277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Catálise , Células Cultivadas , Cricetinae , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Ratos , Proteínas de Transporte Vesicular/metabolismoRESUMO
The elongated and polarized characteristics of neurons render targeting of receptors to the plasma membrane of distal axonal projections and dendritic branches a major sorting task. Although the majority of biosynthetic cargo synthesis, transport, and sorting are believed to occur in the soma, local membrane protein translation and sorting has been reported recently to take place in dendrites and axons. We investigated where endoplasmic reticulum (ER) export occurs in dendrites using an in vitro permeabilized neuron system that enables us to specifically control the assembly of ER export sites. We show that ER export sites are assembled regularly throughout the entire dendritic tree by the regulated sequential recruitment of Sar1 and COPII (coat protein complex II). Moreover, activation of metabotropic glutamate receptors leads to the recruitment of the NMDA receptor subunit NR1 to remodeled ER export sites. We propose that regulation of receptor assembly and export from the ER in dendrites plays an important role in modulating receptor surface expression and neuronal function.
Assuntos
Dendritos/fisiologia , Retículo Endoplasmático/metabolismo , Glicina/análogos & derivados , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Glicina/farmacologia , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Resorcinóis/farmacologia , Proteínas de Saccharomyces cerevisiaeRESUMO
The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat.