RESUMO
Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Aminoácidos , Sítios de Ligação , Linhagem Celular , Imunofluorescência , Células HeLa , Humanos , Imuno-Histoquímica , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte ProteicoAssuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas do Olho/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , Membrana Celular/metabolismo , Cromatografia de Afinidade/métodos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Imunofluorescência , Células HeLa , Humanos , Ligação Proteica , Subunidades Proteicas , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Segmento Externo da Célula Bastonete/enzimologia , Saccharomyces cerevisiae , Solubilidade , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/isolamento & purificaçãoRESUMO
We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.
Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/fisiologia , Junções Íntimas/fisiologia , Transativadores , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Proteínas do Citoesqueleto/análise , Desenvolvimento Embrionário e Fetal , Feminino , Humanos , Proteínas de Membrana/análise , Camundongos , Proteínas dos Microfilamentos , Fosfoproteínas/análise , Gravidez , Junções Íntimas/química , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1 , alfa Catenina , beta Catenina , Proteínas rab de Ligação ao GTP/genéticaRESUMO
How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Neuritos , Neurônios/citologia , Proteínas de Transporte Vesicular , Animais , Transporte Biológico/efeitos dos fármacos , Toxinas Botulínicas/farmacologia , Diferenciação Celular , Exocitose/efeitos dos fármacos , Metaloendopeptidases/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Ligação Proteica , Proteínas R-SNARE , Ratos , Proteínas SNARE , Estaurosporina/farmacologia , Proteína 25 Associada a Sinaptossoma , Toxina Tetânica/farmacologiaRESUMO
The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Organelas/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Encéfalo/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Proteínas de Membrana/análise , Terminações Nervosas/metabolismo , Terminações Nervosas/ultraestrutura , Neurônios/ultraestrutura , Especificidade de Órgãos , Organelas/ultraestrutura , Células PC12 , Proteínas R-SNARE , Ratos , Vesículas Sinápticas/ultraestrutura , Toxina Tetânica/farmacologiaRESUMO
Small Rab GTPases are involved in the regulation of membrane trafficking. They cycle between cytosolic and membrane-bound forms. These membrane association/dissociation are tightly controlled by regulatory proteins. To search for proteins interacting with Rab13, a small GTPase associated with vesicles in fibroblasts and predominantly with tight junctions in epithelial cells, we screened a HeLa two-hybrid cDNA library and isolated a clone encoding a protein of 17.4 kDa. This protein, almost identical to the bovine rod cGMP phosphodiesterase delta subunit, was named human delta-PDE. The delta-PDE binds specifically to Rab13. It exhibits two putative C-terminal sequences necessary for the interaction with PDZ (PSD95, Dlg, ZO-1) domains contained in many proteins localized to specific plasma membrane microdomains. Immunofluorescence microscopic studies revealed that the vesicular stomatitis virus (VSV)-tagged delta-PDE is localized in vesicular structures accumulated near the plasma membrane in epithelial cells. Deletion of the PDZ binding motifs impair VSV-delta-PDE subcellular distribution. Purified recombinant delta-PDE had the capacity to dissociate Rab13 from cellular membranes. Our data support the proposal that delta-PDE, but not GDP dissociation inhibitor, may serve to control the dynamic of the association of Rab13 with cellular membranes.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glicoproteínas de Membrana , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Proteínas rab de Ligação ao GTP , Sequência de Aminoácidos , Animais , Bovinos , Membrana Celular/enzimologia , Células HeLa , Humanos , Células LLC-PK1 , Microscopia de Fluorescência , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologia , Suínos , Proteínas do Envelope Viral/metabolismoRESUMO
The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/farmacologia , Toxina Tetânica/farmacologia , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Antígenos de Superfície/metabolismo , Sequência de Bases , Toxinas Botulínicas/farmacologia , Células CACO-2 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , DNA Complementar , Ditiotreitol/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Etilmaleimida/farmacologia , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Coelhos , Ratos , Proteínas SNARE , Homologia de Sequência de Aminoácidos , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Proteína 3 Associada à Membrana da VesículaRESUMO
We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulin-secreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in beta-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunofluorescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decreased by the overexpression of wild-type Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human beta-cells, a different Rab3 isoform but with homologous function may replace Rab3A.
Assuntos
Proteínas de Ligação ao GTP/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Linhagem Celular , Cricetinae , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Imuno-Histoquímica , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Peso Molecular , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Especificidade da Espécie , Frações Subcelulares/metabolismo , Transfecção , Proteínas rab3 de Ligação ao GTPRESUMO
During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP-binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras-related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras-related proteins could be distinguished from their counterparts by iso-electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite-related protein was found associated with the membranes of early endosome-like structures near the parasite plasma membrane. Rab6-related protein was associated with the Golgi/trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras-related protein was cytoplasmic and plasma membrane-associated. These results are in accordance with their mammalian counterparts and support the implication of Rab-related proteins in vesicular trafficking in Plasmodium.
Assuntos
Plasmodium falciparum/metabolismo , Proteínas ras/análise , Animais , Autorradiografia , Western Blotting , Proteínas de Ligação ao GTP/análise , Imuno-Histoquímica , Plasmodium falciparum/ultraestruturaRESUMO
Rab3A is a protein associated with the membrane of synaptic vesicles and is involved in the control of the targeting or docking of these vesicles at the presynaptic membrane for the release of neurotransmitters. Here, we have examined the expression and localization of this protein during the development of the rat brain. Relative to total protein, the concentration of rab3A greatly increased during brain development. Both the intracellular localization of the protein and its cerebral distribution showed an age-dependent shift. In contrast to other synaptic vesicle proteins, rab3A was heavily concentrated in cell bodies when immature neurons were migrating and during early differentiation. Later, the protein disappeared from perikarya and had a diffuse distribution in the neuropil, indicating a redistribution to nerve terminals, its exclusive localization in the adult. In the developing somatosensory cortex, rab3A delimited the modular organization of the barrels well after the afferents have arrived but just around the time that mature synaptic activity has been observed. In the hippocampus, rab3A defined a novel "blob-like" organization of the mossy fibre terminals and its appearance in terminal fields closely preceded the known onset of long-term potentiation. The appearance of rab3A in specific terminal fields during the period of increased physiological activity suggests that this small GTP-binding protein may be an important late element in the establishment of the mature characteristics of the presynaptic terminal.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular , Córtex Cerebral/metabolismo , Embrião de Mamíferos , Proteínas de Ligação ao GTP/análise , Hipocampo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Terminações Nervosas/metabolismo , Neurônios/citologia , Especificidade de Órgãos , Coelhos/imunologia , Ratos , Ratos Sprague-Dawley , Proteínas rab3 de Ligação ao GTPRESUMO
Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. Rabphilin-3A is expressed mainly in brain, but its subcellular localization remains to be clarified. Immunohistochemical analysis has revealed that Rabphilin-3A is most abundant in the synaptic area of the rat cerebellum, retina, and neuromuscular junction. Ultrastructural analysis of the neuromuscular junction using the immunogold method indicates that Rabphilin-3A is localized on the synaptic vesicle. Subcellular fractionation analysis of rat brain has shown that Rabphilin-3A is most highly concentrated in the purified synaptic vesicle fraction. These results indicate that Rabphilin-3A is localized on the synaptic vesicle in the presynapse.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fracionamento Celular , Cerebelo/metabolismo , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Proteínas de Transporte Vesicular , Rabfilina-3ARESUMO
Rab4, a low-molecular-mass GTP-binding protein, is associated with vesicles containing Glut 4 in adipocytes. Following insulin stimulation, the translocation of Glut 4 to the plasma membrane is associated with the movement of Rab4 to the cytosol. The same modifications are induced by the phosphatase inhibitor, okadaic acid. To establish a possible role for phosphorylation in Rab4 cycling, we searched for insulin-stimulated cytosolic kinase(s) which could phosphorylate Rab4. In 3T3-L1 adipocytes, insulin induced a rapid and transient activation of cytosolic kinase(s), which phosphorylated Rab4 in vitro. At least part of the Rab4 phosphorylation can be accounted for by ERK (extracellular-signal-regulated kinases) since immunopurified ERK1 from insulin-stimulated cells phosphorylated Rab4 with a comparable time-course. Both with cytosolic extracts and immunopurified ERK1, only serine residues were phosphorylated on Rab4. The phosphorylation site was localized in the C-terminus of the molecule, and occurred very probably on Ser196. These results indicate that Rab4 is an in vitro substrate for ERK, and suggest that the insulin-induced movement of Rab4 from the Glut-4-containing vesicles to the cytosol could result from phosphorylation of Rab4 by ERK.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Éteres Cíclicos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Insulina/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Células 3T3 , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Citosol/enzimologia , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Peso Molecular , Ácido Okadáico , Fosforilação , Serina/metabolismo , Proteínas rab4 de Ligação ao GTPRESUMO
Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.
Assuntos
Compartimento Celular , Polaridade Celular , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Caderinas/metabolismo , Cálcio/metabolismo , Adesão Celular , Clonagem Molecular , DNA Complementar , Imunofluorescência , Expressão Gênica , Humanos , Junções Intercelulares/fisiologia , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína da Zônula de Oclusão-1RESUMO
Rab proteins are small GTPases highly related to the yeast Ypt1 and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Prenilação de Proteína , Proteínas rab de Ligação ao GTP , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , GTP Fosfo-Hidrolases/química , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Rab proteins, which have been implicated in the regulation of vesicular traffic, were studied in adipocytes. Rab3B, Rab3C, Rab4, and Rab8 were detected, but Rab3A was not. In the absence of insulin, Rab3B and Rab3C were cytosolic, while Rab4 and Rab8 were associated with membranes. Only Rab4 distribution was modified by insulin. In unstimulated adipocytes, most of Rab4 was found in a low density microsomal fraction, which also contained the majority of Glut 4. After insulin treatment, a 50% decrease in Rab4 content was observed, concomitantly with a departure of transporters to the plasma membrane. The dose responses for the departure of Glut 4 and Rab4 from the microsomal fractions were superimposable, half-maximal effects being obtained with 0.1 nM insulin. Rab4 was redistributed to the cytosol and its movement was reversed by insulin withdrawal. When Glut 4-containing vesicles were immunopurified with antibodies to Glut 4, Rab4 was found in the immune pellets, suggesting that Rab4 was tightly associated with the vesicles. Okadaic acid, an inhibitor of phosphatases 1 and 2A that is known to stimulate Glut 4 translocation, caused the same movement of Rab4 from low density microsomal fraction to the cytosol, while the phorbol ester 12-O-tetradecanoylphorbol-13-acetate had no effect. We suggest that insulin and okadaic acid induce a cycling of Rab4 from a vesicular fraction containing the Glut 4 transporter to the cytosol and that this cycling may participate in the insulin stimulatory action on glucose transporter translocation.
Assuntos
Tecido Adiposo/metabolismo , Desoxiglucose/metabolismo , Éteres Cíclicos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Insulina/farmacologia , Tecido Adiposo/efeitos dos fármacos , Animais , Transporte Biológico , Encéfalo/metabolismo , Células CHO , Fracionamento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Cricetinae , Citosol/metabolismo , Relação Dose-Resposta a Droga , Epididimo , Proteínas de Ligação ao GTP/isolamento & purificação , Masculino , Modelos Biológicos , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Proteínas de Transporte de Monossacarídeos/metabolismo , Ácido Okadáico , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab4 de Ligação ao GTPRESUMO
The activation of platelets by specific agonists is a tightly regulated mechanism that leads to the secretion of the dense- and alpha-granule contents. Platelets have been shown to possess small GTP-binding proteins thought to be involved in central biological processes; however, no rab proteins, which may regulate the exocytic process at different stages, have been reported. This study has shown that rab1, rab3B, rab4, rab6, and rab8 proteins, but not rab3A protein, were present in platelets and in endothelial cells. To probe their functional significance in platelets, rab3B, rab6, and rab8 proteins were further characterized with regard to their intracellular localization and their phosphorylation properties. Whereas rab3B protein was found to be mainly cytosolic, rab6 and rab8 proteins were preferentially targeted to the plasma membrane and to the alpha granules. The activation of platelets by thrombin, a potent inducer of secretion, resulted in the phosphorylation of rab3B, rab6, and rab8 proteins, whereas no phosphorylation was observed in the presence of prostaglandin E1, which stimulates cAMP-dependent protein kinase and inhibits the secretion process. These findings provide evidence that members of the subfamily of rab proteins, rab6 and rab8, are localized in platelets to one type of specific secretory vesicle, the alpha granule, and would suggest their possible implication in the secretion process through phosphorylation mechanisms.
Assuntos
Plaquetas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Trombina/farmacologia , Proteínas rab de Ligação ao GTP , Sequência de Aminoácidos , Plaquetas/efeitos dos fármacos , Fracionamento Celular , Membrana Celular/metabolismo , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Endotélio Vascular/metabolismo , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Fosforilação , Ativação Plaquetária , Frações Subcelulares/metabolismo , Veias Umbilicais , Proteínas rab3 de Ligação ao GTPRESUMO
Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e., dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, regions of cerebral cortex and the cerebellum exhibited striking laminar distributions of rab3A immunoreactivity. In the hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentate granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral cortical hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.
Assuntos
Química Encefálica/fisiologia , Proteínas do Tecido Nervoso/análise , Proteínas Proto-Oncogênicas/análise , Animais , Córtex Cerebral/química , Imuno-Histoquímica , Vias Neurais/química , Ratos , Ratos Sprague-Dawley , Núcleos Talâmicos/química , Proteínas rab3 de Ligação ao GTPRESUMO
In a previous study we demonstrated that a homeobox peptide corresponding to the 60 amino acid long DNA-binding region of the Drosophila antennapedia homeo-protein was capable of crossing the plasma membrane of cells in culture. This finding has led us to investigate whether chimeric molecules encompassing the homeobox would behave in a similar manner. We demonstrate here that a peptide of 93 amino acids composed of the homeobox and of the C terminus of Rab3, a small GTP-binding protein, crosses the membrane of myoblasts, myotubes and neurons and is conveyed to their nuclei. This transport is highly efficient, is observed in all the cells present in the culture and occurs at 37 degrees C and 12 degrees C without quantitative peptide degradation. Beyond its theoretical implications for our current views on cellular interactions, this finding could have technical repercussions on the development of drugs with intracellular targets.