RESUMEN
We report a pH-responsive photothermal ablation agent (pH-PTT) based on cyanine dyes for photothermal therapy (PTT). The nanoparticles formed by BSA and pH-PTT preferentially accumulated in the Golgi apparatus of cancer cells compared to normal cells, and thus can be specifically activated by the acidic Golgi apparatus in cancer cells for effective PTT both ex vivo and in vivo.
Asunto(s)
Carbocianinas/química , Colorantes/química , Aparato de Golgi/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Procesos Fotoquímicos , Temperatura , Carbocianinas/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colorantes/farmacología , Aparato de Golgi/química , Aparato de Golgi/efectos de los fármacos , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Nanopartículas/química , Imagen Óptica , FototerapiaRESUMEN
Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Aparato de Golgi/metabolismo , Guanosina Difosfato Fucosa/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Arabidopsis/clasificación , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Pared Celular/química , Pared Celular/metabolismo , Clonación Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucanos/biosíntesis , Aparato de Golgi/química , Proteínas de Transporte de Monosacáridos/metabolismo , Pectinas/biosíntesis , Filogenia , Células Vegetales/química , Células Vegetales/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xilanos/biosíntesisRESUMEN
We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein ß-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER-Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.
Asunto(s)
Técnicas Citológicas , Glicoproteínas/química , Glicoproteínas/metabolismo , Membranas Intracelulares/metabolismo , Oligosacáridos/análisis , Oligosacáridos/metabolismo , Lectinas de Plantas/química , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Galanthus/química , Glicoproteínas/análisis , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/química , Yohexol , Lens (Planta)/química , Oligosacáridos/química , Pisum sativum/química , Transporte de Proteínas , SacarosaRESUMEN
Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6-dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose- and time-dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N-ST-YFP and TGN labelled with VHA-a1-GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.
Asunto(s)
Arabidopsis/efectos de los fármacos , Pared Celular/efectos de los fármacos , Celulosa/biosíntesis , Citoesqueleto/efectos de los fármacos , Nitrilos/farmacología , Arabidopsis/anatomía & histología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Transporte Biológico , Pared Celular/química , Pared Celular/ultraestructura , Celulosa/antagonistas & inhibidores , Citoesqueleto/química , Relación Dosis-Respuesta a Droga , Aparato de Golgi/química , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Microfibrillas/química , Microscopía Confocal , Microscopía Electrónica de Transmisión , Pectinas/química , Células Vegetales/química , Células Vegetales/ultraestructura , Raíces de Plantas/química , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Semillas/química , Semillas/crecimiento & desarrollo , Espectroscopía Infrarroja por Transformada de Fourier , Factores de TiempoRESUMEN
Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid-binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends on PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.
Asunto(s)
Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Aparato de Golgi/química , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Miosinas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Antibodies were produced against fragments of the microtubule-binding domain and the motor domain of the dynein heavy chain from Dictyostelium discoideum to probe whole cell extracts of root meristem cells of wheat Triticum aestivum. In plant extracts, these antibodies cross-reacted with a polypeptide of high molecular weight (>500kDa). The antibodies bound to protein A-Sepharose precipitated high molecular weight polypeptide from cell extracts. Immunofluorescence showed that the antibodies identified various aggregates inside cells, localized at the perinuclear area during interphase to early prophase, at the spindle periphery and polar area during mitosis, and in the interzonal region during phragmoplast development. Some aggregates were also co-labeled by markers for the Golgi apparatus. Thus, we found in higher plant cells a high molecular weight antigen cross-reacting with the antibodies to motor and microtubule-binding domains of dynein heavy chains. This antigen is associated with aggregates distributed in the cytoplasm in cell cycle-dependent manner. A subset of these aggregates belongs to the Golgi complex.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Dineínas/inmunología , Aparato de Golgi/química , Proteínas de Plantas/inmunología , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas , Dictyostelium/inmunología , Aparato de Golgi/inmunología , Interfase , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/inmunología , Proteínas de Plantas/análisis , Profase , Triticum/inmunologíaRESUMEN
Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.
Asunto(s)
Encéfalo/citología , Proliferación Celular , Retículo Endoplásmico/química , Aparato de Golgi/química , Membranas Intracelulares/química , Animales , Western Blotting , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Encéfalo/ultraestructura , Retículo Endoplásmico/ultraestructura , Femenino , Glioblastoma/química , Glioblastoma/metabolismo , Glioblastoma/patología , Aparato de Golgi/ultraestructura , Humanos , Inmunohistoquímica , Membranas Intracelulares/ultraestructura , Lípidos de la Membrana/análisis , Proteínas de la Membrana/análisis , Fósforo/análisis , Ratas , Ratas WistarRESUMEN
Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.
Asunto(s)
Proteínas 14-3-3/análisis , Membranas Intracelulares/química , Lilium/química , Proteínas de Plantas/análisis , Polen/química , Proteínas 14-3-3/fisiología , Ácido Anhídrido Hidrolasas/metabolismo , Biomarcadores , Complejo IV de Transporte de Electrones/metabolismo , Retículo Endoplásmico/química , Aparato de Golgi/química , Lilium/fisiología , Lilium/ultraestructura , Mitocondrias/química , NADH Deshidrogenasa/metabolismo , Proteínas de Plantas/fisiología , Polen/fisiología , Polen/ultraestructura , Unión Proteica , ATPasas de Translocación de Protón/metabolismoRESUMEN
Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3' untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas/genética , Selenio , Selenocisteína/genética , Adenosina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Compartimento Celular , Retículo Endoplásmico/química , Aparato de Golgi/química , Humanos , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Selenoproteínas , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Pectin methylesterases catalyse the demethylesterification of cell wall polygalacturonans. In dicot plants, these ubiquitous cell wall enzymes are involved in important developmental processes including cellular adhesion and stem elongation. Here, I highlight recent studies that challenge the accepted views of the mechanism and function of pectin methylesterases, including the co-secretion of pectins and pectin methylesterases into the apoplasm, new action patterns of mature pectin methylesterases and a possible function of the pro regions of pectin methylesterases as intramolecular chaperones.
Asunto(s)
Arabidopsis/enzimología , Hidrolasas de Éster Carboxílico/fisiología , Pectinas/metabolismo , Fenómenos Fisiológicos de las Plantas , Arabidopsis/química , Calcio/farmacología , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Pared Celular/química , Pared Celular/enzimología , Aparato de Golgi/química , Aparato de Golgi/enzimología , Modelos Moleculares , Chaperonas Moleculares/fisiología , Familia de Multigenes , Pectinas/química , Conformación ProteicaRESUMEN
The potent antioxidant properties of rosemary (Rosmarinus officinalis) extracts have been attributed to its major diterpene, carnosic acid. Carnosic acid has received considerable attention in food science and biomedicine, but little is known about its function in the plant in vivo. We recently found that highly oxidized diterpenes increase in rosemary plants exposed to drought and high light stress as a result of the antioxidant activity of carnosic acid (S. Munné-Bosch, K. Schwarz, L. Alegre [1999] Plant Physiol 121: 1047-1052). To elucidate the significance of the antioxidant function of carnosic acid in vivo we measured the relative amounts of carnosic acid and its metabolites in different compartments of rosemary leaves. Subcellular localization studies show that carnosic acid protects chloroplasts from oxidative stress in vivo by following a highly regulated compartmentation of oxidation products. Carnosic acid scavenges free radicals within the chloroplasts, giving rise to diterpene alcohols, mainly isorosmanol. This oxidation product is O-methylated within the chloroplasts, and the resulting form, 11,12-di-O-methylisorosmanol, is transferred to the plasma membrane. This appears to represent a mechanism of a way out for free radicals from chloroplasts. Carnosic acid also undergoes direct O-methylation within the chloroplasts, and its derived product, 12-O-methylcarnosic acid, accumulates in the plasma membrane. O-methylated diterpenes do not display antioxidant activity, but they may influence the stability of the plasma membrane. This study shows the relevance of the compartmentation of carnosic acid metabolism to the protection of rosemary plants from oxidative stress in vivo.
Asunto(s)
Diterpenos/análisis , Extractos Vegetales/análisis , Hojas de la Planta/química , Rosmarinus/química , Abietanos , Membrana Celular/química , Diterpenos/química , Retículo Endoplásmico/química , Aparato de Golgi/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Estructura Molecular , Extractos Vegetales/química , Fracciones Subcelulares/químicaRESUMEN
By using Western blotting, immunofluorescence and immunogold labeling, a novel alpha-actinin-like protein was found in pollen and pollen tubes of Lilium davidii, a model system for cytoskeleton and Golgi apparatus study of plant. As measured by Western blotting, the molecular mass of the a-actinin-like protein was about 80 kDa. Under confocal laser scanning microscopy after immunofluorescence labeling, the distribution of the alpha-actinin-like protein appeared punctated in the cytoplasm of the pollen and pollen tubes. When double labeled, the protein was co-localized with Golgi 58K protein. In addition, some fraction of the alpha-actinin-like protein was found to co-distribute with F-actin bundles in the pollen tubes. Additional studies with immuno-gold labeling and transmission electron microscopy revealed that the alpha-actinin-like protein bound mainly to the membranes of Golgi-associated vesicles. When the pollen tubes were treated with Brefeldin A (BFA), the a-actinin-like proteins were dispersed into the cytoplasm, and the growth of pollen tubes was inhibited. After BFA was removed, the protein was reversibly recovered on the Golgi apparatus. These results suggest that the novel alpha-actinin-like protein is a BFA-sensitive protein on the membranes of Golgi-associated vesicles, and may participate in Golgi-associated vesicles budding and/or sorting, together with actin microfilaments.
Asunto(s)
Actinina/análisis , Vesículas Citoplasmáticas/química , Aparato de Golgi/química , Proteínas de Plantas/análisis , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/ultraestructura , Actinas/análisis , Brefeldino A/farmacología , Vesículas Citoplasmáticas/ultraestructura , Aparato de Golgi/ultraestructura , Lilium , Microscopía Confocal , Microscopía Inmunoelectrónica , Polen/química , Inhibidores de la Síntesis de la Proteína/farmacologíaRESUMEN
Ultrastructural analysis of garlic roots treated for 24 h with sodium selenate or sodium selenite at the concentrations 80, 160, 320 microM revealed the presence of selenium deposits in meristematic cells. They appeared as small and large granules or aggregates of electron-dense material. Many small granules were localised in plastids but some in mitochondria, endoplasmic reticulum as well as in Golgi apparatus, nucleus and cytoplasm. Sometimes the large granules were seen in cytoplasm but aggregates of electron-dense material only in vacuoles. It seems possible that these deposits represent a non-dissolved form of selenium, i.e. elemental selenium or its complexes with other ions.
Asunto(s)
Allium/química , Raíces de Plantas/química , Selenio/análisis , Selenio/farmacología , Absorción , Allium/metabolismo , Allium/ultraestructura , Núcleo Celular/química , Citoplasma/química , Gránulos Citoplasmáticos/química , Retículo Endoplásmico/química , Aparato de Golgi/química , Microscopía Electrónica , Mitocondrias/química , Raíces de Plantas/ultraestructura , Ácido Selénico , Compuestos de Selenio/metabolismo , Compuestos de Selenio/farmacología , Selenito de Sodio/metabolismo , Selenito de Sodio/farmacologíaRESUMEN
Antibodies directed against long chain acyl-CoAs (having 16 and 18 carbon atoms) have been prepared and are reported for the first time. A modified ELISA procedure adapted to these amphiphilic molecules has been developed: it is a rapid, simple and sensitive test permitting to detect as little as 3 pmol of acyl-CoA. These antibodies represent a new tool for studying long-chain acyl-CoAs. Their use in an immunochemical approach for the study of protein-acyl-CoA interactions is presented.
Asunto(s)
Acilcoenzima A/inmunología , Anticuerpos/inmunología , Acilcoenzima A/análisis , Acilcoenzima A/química , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo , Aciltransferasas/metabolismo , Animales , Membrana Celular/química , Colorimetría , Ensayo de Inmunoadsorción Enzimática , Aparato de Golgi/química , Aparato de Golgi/enzimología , Inmunización , Immunoblotting , Cebollas , ConejosRESUMEN
The dietary uptake of alpha-tocopherol and coenzyme Q was investigated in rats. Rats were fed diets supplemented with alpha-tocopherol or coenzyme Q10 (1 g/kg diet) or an unsupplemented control diet. In control rat tissues, the content of coenzyme Q was 4-11 times higher than that of alpha-tocopherol, but in plasma, the ratio was reversed. Among the subcellular fractions of rat liver homogenate, Golgi vesicles and lysosomes had the highest alpha-tocopherol concentration, and high concentrations of coenzyme Q were observed in the outer and inner mitochondrial membranes as well as in lysosomes, Golgi vesicles and plasma membranes. The uptake of alpha-tocopherol into the liver and plasma reached a maximal level after only 2 d of supplementation, whereas in the kidney, heart, muscle and brain, the levels continued to increase throughout the 6-wk treatment period. In contrast, dietary coenzyme Q was taken up into the liver and plasma only, and not into the other organs. This lipid appeared mainly in the Golgi system, whereas alpha-tocopherol exhibited a more general cellular distribution. The decay of the supplied alpha-tocopherol was slow in the various organs, but the disappearance of coenzyme Q was rapid from both liver and plasma. Pretreatment of rats with alpha-tocopherol increased the levels of both endogenous and exogenous coenzyme Q in the liver and plasma. These results demonstrate that the uptake of alpha-tocopherol from the diet is an extensive and general phenomenon at both the tissue and cellular levels, in contrast to the selective and restricted uptake of coenzyme Q.
Asunto(s)
Hígado/citología , Hígado/metabolismo , Ubiquinona/farmacocinética , Vitamina E/farmacocinética , Animales , Transporte Biológico/fisiología , Encéfalo/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Riñón/química , Riñón/metabolismo , Hígado/ultraestructura , Lisosomas/química , Lisosomas/metabolismo , Masculino , Mitocondrias/química , Mitocondrias/metabolismo , Músculos/química , Músculos/metabolismo , Miocardio/química , Miocardio/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Ubiquinona/análisis , Ubiquinona/sangre , Vitamina E/análisis , Vitamina E/sangreRESUMEN
Little is known about the integral membrane proteins that participate in the early secretory pathway of mammalian cells. The complementary DNA encoding a 28-kilodalton protein (p28) of the cis-Golgi was cloned and sequenced. The protein was predicted to contain a central coiled-coil domain with a carboxyl-terminal membrane anchor. An in vitro assay for endoplasmic reticulum-Golgi transport was used to show that p28 participates in the docking and fusion stage of this transport event. Biochemical studies established that p28 is a core component of the Golgi SNAP receptor (SNARE) complex.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/química , Glicoproteínas de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/análisis , Línea Celular , Clonación Molecular , ADN Complementario/genética , Ácido Egtácico/farmacología , Aparato de Golgi/metabolismo , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Proteínas SNARE , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Virus de la Estomatitis Vesicular Indiana , Proteínas del Envoltorio Viral/metabolismoRESUMEN
We have identified and characterized Cab45, a novel 45-kD protein from mouse 3T3-L1 adipocytes. Cab45 is ubiquitously expressed, contains an NH2-terminal signal sequence but no membrane-anchor sequences, and binds Ca2+ due to the presence of six EF-hand motifs. Within the superfamily of calcium-binding proteins, it belongs to a recently identified group of proteins consisting of Reticulocalbin (Ozawa, M., and T. Muramatsu. 1993. J. Biol. Chem. 268:699-705) and ERC 55 (Weis, K., G. Griffiths, and A.I. Lamond. 1994. J. Biol Chem. 269:19142-19150), both of which share significant sequence homology with Cab45 outside the EF-hand motifs. In contrast to reticulocalbin and ERC-55 which are soluble components of the endoplasmic reticulum, Cab45 is a soluble protein localized to the Golgi. Cab45 is the first calcium-binding protein localized to the lumenal portion of a post-ER compartment; Cab45 is also the first known soluble protein resident in the Golgi lumen. Cab45 can serve as a model protein to determine the mechanism of retention of soluble proteins in the Golgi compartment.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Glicoproteínas/análisis , Aparato de Golgi/química , Células 3T3 , Adipocitos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Brefeldino A , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Clonación Molecular , Ciclopentanos/farmacología , ADN Complementario/genética , Expresión Génica , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADNRESUMEN
The influence of docosahexaenoic acid (DHA) and sardine oil diets on the ultrastructure of jejunal absorptive cells was studied. Adult male Crj:CD-1 (ICR) mice were fed a fat-free semisynthetic diet supplemented with 5% (by weight) purified DHA ethyl ester, refined sardine oil, or palm oil. The mice received the DHA or palm oil diets for 7 days (groups 1 and 2) and the refined sardine oil or palm oil diets for 30 days (groups 3 and 4). There were significant ultrastructural changes in the jejunal absorptive cells between the mice fed on the palm oil diet and those receiving the DHA and sardine oil diets. The endoplasmic reticulum and Golgi apparatus of some jejunal absorptive cells in the mice fed on the palm oil diet for 7 and 30 days developed vacuolation on the upper site of the nucleus. In contrast, many granules, which appeared to be lipid droplets, were observed in the endoplasmic reticulum and Golgi apparatus of the jejunal absorptive cells in the DHA and sardine oil diet groups. These results suggest that ultrastructural differences in the jejunal absorptive cells between mice in the omega-3 fatty acid and palm oil diet groups may be associated with the changes in lipid metabolism.
Asunto(s)
Ácidos Docosahexaenoicos/farmacología , Aceites de Pescado/farmacología , Absorción Intestinal , Yeyuno/ultraestructura , Animales , Retículo Endoplásmico/química , Aparato de Golgi/química , Yeyuno/efectos de los fármacos , Lípidos/análisis , Lisosomas/química , Masculino , Ratones , Ratones Endogámicos ICR , Microscopía ElectrónicaRESUMEN
A 100-kDa polypeptide with microtubule-interacting properties was identified in a Golgi vesicle-enriched fraction from Corylus avellana pollen. The k71s23 antibody (directed to the kinesin heavy chain from bovine brain) [Tiezzi et al., 1992: Cell Motil. Cytoskeleton 21:132-137] localized the polypeptide on the external surface of membrane-bounded organelles. Some 100-kDa-containing vesicles copelleted with microtubules (polymerized from purified bovine brain tubulin) either in presence or absence of 5 mM AMPPNP, but they could be released by 10 mM ATP or 0.5 M KCl. The pollen microtubule-interacting protein, salt-extracted from membranes and partially purified by gel filtration, exhibited an ATPase activity (16.2 nmolPi/mg/min) which could be stimulated about 2-fold (32.5 nmolPi/mg/min) by addition of bovine brain microtubules. We suppose that the 100-kDa polypeptide is part of a molecular complex showing properties of the kinesin class.
Asunto(s)
Aparato de Golgi/química , Cinesinas/análisis , Polen/química , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Aparato de Golgi/metabolismo , Aparato de Golgi/fisiología , Inmunohistoquímica , Cinesinas/metabolismo , Cinesinas/fisiología , Microscopía Inmunoelectrónica , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/fisiología , Peso Molecular , Orgánulos/química , Orgánulos/metabolismo , Orgánulos/fisiología , Polen/ultraestructura , Árboles , Tubulina (Proteína)/análisis , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/fisiologíaRESUMEN
Overlapping cDNAs encoding the entire human ERGIC-53, a 53 kDa membrane protein of the ER-Golgi intermediate compartment, have been isolated and their nucleotide sequence determined. The isolated cDNA is about 2.7 kb in length. The deduced polypeptide chain for ERGIC-53 consists of 510 amino acids (M(r) 54217) including an N-terminal signal sequence of 30 amino acids, a single putative transmembrane segment of 18 amino acids, and a short cytoplasmic domain of 12 amino acids. Surprisingly, the cytoplasmic segment contains two lysines positioned three and four residues from the C-terminus. Such a double lysine motif is known to function as a retention signal for a group of membrane proteins associated with the ER. Expression of a full-length cDNA of ERGIC-53 in Vero cells revealed intracellular localization similar but not always identical to the endogenously expressed ERGIC-53. The presence of an ER retention motif in a protein of the ER-Golgi intermediate compartment has important implications for the retention mechanism mediated by this signal.