RESUMO
Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Membrana/química , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cranio-lenticulo-sutural dysplasia (CLSD) is an autosomal recessive syndrome characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects mapped to chromosome 14q13-q21 (ref. 1). Here we show, using a positional cloning approach, that an F382L amino acid substitution in SEC23A segregates with this syndrome. SEC23A is an essential component of the COPII-coated vesicles that transport secretory proteins from the endoplasmic reticulum to the Golgi complex. Electron microscopy and immunofluorescence show that there is gross dilatation of the endoplasmic reticulum in fibroblasts from individuals affected with CLSD. These cells also exhibit cytoplasmic mislocalization of SEC31. Cell-free vesicle budding assays show that the F382L substitution results in loss of SEC23A function. A phenotype reminiscent of CLSD is observed in zebrafish embryos injected with sec23a-blocking morpholinos. Our observations suggest that disrupted endoplasmic reticulum export of the secretory proteins required for normal morphogenesis accounts for CLSD.
Assuntos
Anormalidades Múltiplas/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mutação , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Catarata/genética , Modelos Animais de Doenças , Embrião não Mamífero , Ossos Faciais/anormalidades , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Transporte Proteico/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Proteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Anormalidades Craniofaciais/genética , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , Retículo Endoplasmático/fisiologia , Fibroblastos/fisiologia , Humanos , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Osteoblastos/fisiologia , Transporte Proteico , Proteínas de Transporte Vesicular/genéticaRESUMO
A yeast plasma membrane protein, Chs3p, transits to the mother-bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)-binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPgammaS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPgammaS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.
Assuntos
Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Transporte/metabolismo , Células Cultivadas , Quitina Sintase/metabolismo , Vesículas Revestidas/fisiologia , Lipossomos/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ácidos Mirísticos/metabolismo , Transporte Proteico/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major-minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major-minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major-minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP-dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Proteínas Fúngicas/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Retículo Endoplasmático Rugoso/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/análise , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Transporte VesicularRESUMO
Vesicle transport requires four steps: vesicle formation, movement, tethering, and fusion. In yeast, two Rab GTPases, Ypt31/32, are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles. Vesicle production is coupled to transport via direct interaction between Ypt31/32 and the yeast myosin V, Myo2. Here we show that Myo2 interacts directly with Sec4 and the exocyst subunit Sec15. Disruption of these interactions results in compromised growth and the accumulation of secretory vesicles. We identified the Sec15-binding region on Myo2 and also identified residues on Sec15 required for interaction with Myo2. That Myo2 interacts with Sec15 uncovers additional roles for the exocyst as an adaptor for molecular motors and implies similar roles for structurally related tethering complexes. Moreover, these studies predict that for many pathways, molecular motors attach to vesicles prior to their formation and remain attached until fusion.
Assuntos
Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Exocitose , Fusão de Membrana , Modelos Moleculares , Proteínas Motores Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/química , Miosina Tipo V/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Secretórias/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.
Assuntos
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Proteínas de Membrana/química , Trifosfato de Adenosina/química , Animais , Células CHO , Cricetinae , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Glutationa Transferase/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Microssomos Hepáticos/metabolismo , Plasmídeos/metabolismo , Presenilina-1 , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Frações Subcelulares/metabolismoRESUMO
Secretory proteins traffic from the ER to the Golgi via COPII-coated transport vesicles. The five core COPII proteins (Sar1p, Sec23/24p, and Sec13/31p) act in concert to capture cargo proteins and sculpt the ER membrane into vesicles of defined geometry. The molecular details of how the coat proteins deform the lipid bilayer into vesicles are not known. Here we show that the small GTPase Sar1p directly initiates membrane curvature during vesicle biogenesis. Upon GTP binding by Sar1p, membrane insertion of the N-terminal amphipathic alpha helix deforms synthetic liposomes into narrow tubules. Replacement of bulky hydrophobic residues in the alpha helix with alanine yields Sar1p mutants that are unable to generate highly curved membranes and are defective in vesicle formation from native ER membranes despite normal recruitment of coat and cargo proteins. Thus, the initiation of vesicle budding by Sar1p couples the generation of membrane curvature with coat-protein assembly and cargo capture.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/fisiologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Membranas Intracelulares/ultraestrutura , Bicamadas Lipídicas/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação/fisiologia , Estrutura Secundária de Proteína/fisiologia , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte VesicularRESUMO
The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Lipossomos/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Domínio Catalítico , Exocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Transport of secretory proteins out of the endoplasmic reticulum (ER) is mediated by vesicles generated by the COPII coat complex. In order to understand how cargo molecules are selected by this cytoplasmic coat, we investigated the functional role of the Sec24p homolog, Lst1p. We show that Lst1p can function as a COPII subunit independently of Sec24p on native ER membranes and on synthetic liposomes. However, vesicles generated with Lst1p in the absence of Sec24p are deficient in a distinct subset of cargo molecules, including the SNAREs, Bet1p, Bos1p and Sec22p. Consistent with the absence of any SNAREs, these vesicles are unable to fuse with Golgi membranes. Furthermore, unlike Sec24p, Lst1p fails to bind to Bet1p in vitro, indicating a direct correlation between cargo binding and recruitment into vesicles. Our data suggest that the principle role of Sec24p is to discriminate cargo molecules for incorporation into COPII vesicles.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Membrana/fisiologia , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Lipossomos/metabolismo , Fusão de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Subunidades Proteicas , Proteínas Qc-SNARE , Proteínas SNARE , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Relação Estrutura-AtividadeRESUMO
The yeast plasma membrane H(+)-ATPase Pma1p is one of the most abundant proteins to traverse the secretory pathway. Newly synthesized Pma1p exits the endoplasmic reticulum (ER) via COPII-coated vesicles bound for the Golgi. Unlike most secreted proteins, efficient incorporation of Pma1p into COPII vesicles requires the Sec24p homolog Lst1p, suggesting a unique role for Lst1p in ER export. Vesicles formed with mixed Sec24p-Lst1p coats are larger than those with Sec24p alone. Here, we examined the relationship between Pma1p biosynthesis and the requirement for this novel coat subunit. We show that Pma1p forms a large oligomeric complex of >1 MDa in the ER, which is packaged into COPII vesicles. Furthermore, oligomerization of Pma1p is linked to membrane lipid composition; Pma1p is rendered monomeric in cells depleted of ceramide, suggesting that association with lipid rafts may influence oligomerization. Surprisingly, monomeric Pma1p present in ceramide-deficient membranes can be exported from the ER in COPII vesicles in a reaction that is stimulated by Lst1p. We suggest that Lst1p directly conveys Pma1p into a COPII vesicle and that the larger size of mixed Sec24pLst1p COPII vesicles is not essential to the packaging of large oligomeric complexes.
Assuntos
Ceramidas/biossíntese , ATPases Translocadoras de Prótons/química , Proteínas de Saccharomyces cerevisiae/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Membrana Celular/metabolismo , Ceramidas/metabolismo , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Cinética , Metabolismo dos Lipídeos , Microscopia Eletrônica , Microssomos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Transporte Proteico , Temperatura , Fatores de Tempo , Leveduras/metabolismoRESUMO
We have characterized the mechanisms of cargo selection into ER-derived vesicles by the COPII subunit Sec24p. We identified a site on Sec24p that recognizes the v-SNARE Bet1p and show that packaging of a number of cargo molecules is disrupted when mutations are introduced at this site. Surprisingly, cargo proteins affected by these mutations did not share a single common sorting signal, nor were proteins sharing a putative class of signal affected to the same degree. We show that the same site is conserved as a cargo-interaction domain on the Sec24p homolog Lst1p, which only packages a subset of the cargoes recognized by Sec24p. Finally, we identified an additional mutation that defines another cargo binding domain on Sec24p, which specifically interacts with the SNARE Sec22p. Together, our data support a model whereby Sec24p proteins contain multiple independent cargo binding domains that allow for recognition of a diverse set of sorting signals.