RESUMO
The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Glicoproteínas de Membrana , Proteínas de Transporte Vesicular , Proteínas do Envelope Viral/metabolismo , Compostos de Alumínio/farmacologia , Animais , Proteínas de Bactérias , Transporte Biológico/efeitos dos fármacos , Células CHO , Proteínas de Transporte/fisiologia , Linhagem Celular , Permeabilidade da Membrana Celular , Polaridade Celular , Cricetinae , Epitélio/metabolismo , Fluoretos/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Proteínas Sensíveis a N-Etilmaleimida , Estreptolisinas/farmacologiaRESUMO
In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.
Assuntos
Autofagia/fisiologia , Proteínas de Membrana/deficiência , Fagossomos/fisiologia , Proteínas/metabolismo , Células-Tronco/fisiologia , Animais , Proteína 12 Relacionada à Autofagia , Compartimento Celular , Embrião de Mamíferos/citologia , Marcação de Genes , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutagênese , Sinais Direcionadores de Proteínas , Células-Tronco/ultraestruturaRESUMO
Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.
Assuntos
Autofagia , Citoplasma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Vacúolos/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Transporte Biológico , Domínio Catalítico , Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismoRESUMO
We characterized Apg8/Aut7p essential for autophagy in yeast. Apg8p was transcriptionally upregulated in response to starvation and mostly existed as a protein bound to membrane under both growing and starvation conditions. Immunofluorescence microscopy revealed that the intracellular localization of Apg8p changed drastically after shift to starvation. Apg8p resided on unidentified tiny dot structures dispersed in the cytoplasm at growing phase. During starvation, it was localized on large punctate structures, some of which were confirmed to be autophagosomes and autophagic bodies by immuno-EM. Besides these structures, we found that Apg8p was enriched on isolation membranes and in electron less-dense regions, which should contain Apg8p-localized membrane- or lipid-containing structures. These structures would represent intermediate structures during autophagosome formation. Here, we also showed that microtubule does not play an essential role in the autophagy in yeast. The result does not match with the previously proposed role of Apg8/Aut7p, delivery of autophagosome to the vacuole along microtubule. Moreover, it is revealed that autophagosome formation is severely impaired in the apg8 null mutant. Apg8p would play an important role in the autophagosome formation.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Fagossomos/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Fúngicas/fisiologia , Microscopia Eletrônica , Fagocitose/fisiologia , Fagossomos/ultraestrutura , Saccharomyces cerevisiae/ultraestruturaRESUMO
The tyrosine kinase inhibitor imatinib (Gleevec, Novartis Pharmaceuticals Corporation; Basel, Switzerland) is a powerful drug for treatment of chronic myelogenous leukemia (CML) and other malignancies. It selectively targets various tyrosine kinases, thereby leading to growth arrest of respective cancer cells. Given its wide application, it is of high importance to know all related underlying molecular mechanisms. We had previously found that imatinib increases the cellular clearance of intracellular protein aggregates by targeting the abl pathway and thereby upregulating lysosomal activity. Here, we describe that imatinib dose dependently activates the cellular autophagy machinery in mammalian cells, independently of tissue type, species origin or immortalization status of cells. Autophagy is an archetypical cellular degradation mechanism implicated in many physiological and pathophysiological conditions. Our data link for the first time the process of autophagy with the mode of action of imatinib. Induction of autophagy might represent an additional mechanism of imatinib to induce growth arrest, promote apoptosis in cancer cells and eventually even promote tumour regression.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Mesilato de Imatinib , Lisossomos/efeitos dos fármacos , Camundongos , Fagossomos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidoresRESUMO
Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.
Assuntos
Adenosina Trifosfatases , Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Fusão de Membrana/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fagossomos/fisiologia , Proteínas de Saccharomyces cerevisiae , Vacúolos/fisiologia , Proteínas de Transporte Vesicular , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Centrifugação com Gradiente de Concentração , Proteínas Fúngicas/fisiologia , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina , Glicoproteínas de Membrana/fisiologia , Proteínas Sensíveis a N-Etilmaleimida , Proteínas Qb-SNARE , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.
Assuntos
Adenosina Trifosfatases/metabolismo , Endossomos/metabolismo , Proteínas Fúngicas/química , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Substituição de Aminoácidos/genética , Animais , Transporte Biológico , Biomarcadores/análise , Linhagem Celular , Tamanho Celular , Precipitação Química , Regulação para Baixo , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/ultraestrutura , Fator de Crescimento Epidérmico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Camundongos , Ratos , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Transfecção , Transferrina/metabolismo , ATPases Vacuolares Próton-Translocadoras , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas de Transporte VesicularRESUMO
We have investigated the effects of bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, on the secretion and intracellular distribution of prolactin in cultured rat pituitary tumor cells (GH3 cells). Pulse-labeling the cells with L-[35S]methionine for 5 min and subsequently incubating in chase medium containing 1 microM bafilomycin A1 showed inhibition of basal secretion of labeled prolactin. The inhibition of the secretion by the drug was clearly observed when it was added within 7.5 min after the pulse-labeling, whereas inhibition was barely observed when added at 22 min. When the pulse-labeled cells were chased with 1 microM bafilomycin A1 for 1 h and then washed and incubated in the presence of 10 micrograms/ml brefeldin A (BFA) for 2 h, BFA barely affected the secretion during the latter 2 h period. This result suggested that the labeled prolactin had passed through a BFA-sensitive step(s) in the intracellular transport during the treatment with 1 microM bafilomycin A1. Immunofluorescence microscopy and immunogold electron microscopy revealed that, when the cells were incubated with 0.1 to 1.0 microM bafilomycin A1, small, dense secretory granules containing prolactin decreased markedly, and numerous large vacuoles appeared, which also contained prolactin, and were partially coated with clathrin-like materials. The Golgi apparatus itself was preserved except for some dilatation of the trans-Golgi cisternae and trans-Golgi network (TGN) where these vacuoles were likely to be formed. These results suggest that acidification in immature secretory granules generated by a vacuolar type H(+)-ATPase is necessary for the intracellular transport of prolactin, maturation and concentration processes of the secretory granules.
Assuntos
Antibacterianos/farmacologia , Macrolídeos , Prolactina/metabolismo , Animais , Antibacterianos/imunologia , Transporte Biológico/efeitos dos fármacos , Brefeldina A , Ciclopentanos/farmacologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Complexo de Golgi/química , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Microscopia Imunoeletrônica , Neoplasias Hipofisárias , Prolactina/análise , Ratos , Células Tumorais CultivadasRESUMO
A wide variety of phospholipase A2 inhibitors blocks intra-Golgi protein transport reconstituted in a cell-free system. Phospholipase A2 activity detectable under the protein transport assay conditions is actually inhibited by the inhibitors. There is a good correlation between the inhibition of protein transport and that of phospholipase A2 activity. Prolactin secretion from GH3 cells is also blocked by a membrane-permeable phospholipase A2 inhibitor, suggesting the physiological relevance to inhibition of protein transport in vitro by phospholipase A2 inhibitors.
Assuntos
Complexo de Golgi/metabolismo , Glicoproteínas de Membrana , Fosfolipases A/análise , Proteínas do Envelope Viral/metabolismo , Acetilglucosamina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Brefeldina A , Sistema Livre de Células , Células Cultivadas , Ciclopentanos/farmacologia , Ácidos Graxos/farmacologia , Complexo de Golgi/efeitos dos fármacos , Masoprocol/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Prolactina/metabolismo , ortoaminobenzoatos/farmacologiaRESUMO
We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.
Assuntos
Membrana Celular/enzimologia , Isomerases/análise , Pâncreas/enzimologia , Animais , Grânulos Citoplasmáticos/enzimologia , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/enzimologia , Ouro , Complexo de Golgi/enzimologia , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Membrana Nuclear/enzimologia , Pâncreas/ultraestrutura , Isomerases de Dissulfetos de Proteínas , Ratos , Ratos Endogâmicos , Proteína Estafilocócica ARESUMO
We investigated quantitatively the distribution of protein disulfide isomerase (PDI) in rat hepatocytes by immunocytochemistry using a post-embedding protein A-gold technique. In hepatocytes, gold particles were mainly localized in the intracisternal space of the rough and smooth endoplasmic reticulum (ER) and nuclear envelopes. Autolysosomes engulfing ER were occasionally densely labeled, especially in rat hepatocytes previously treated with leupeptin in vivo, suggesting that the autophagosome-autolysosome system may be an important route for degradation of PDI. A few gold particles were also found on the plasma membranes. Localization of gold particles on the other subcellular organelles, such as Golgi apparatus, peroxisomes, and nuclear matrix, was sparse and at the control level. The predominant localization of PDI on the intracisternal surface of the ER and nuclear envelope supports a potential role of PDI in the formation of disulfide bonds of nascent polypeptides, thus accelerating formation of the higher-order structure of secretory and membrane proteins and rendering the translocation process irreversible.
Assuntos
Isomerases/análise , Fígado/enzimologia , Animais , Membrana Celular/enzimologia , Retículo Endoplasmático/enzimologia , Ouro , Immunoblotting , Imuno-Histoquímica , Leupeptinas/farmacologia , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Lisossomos/enzimologia , Masculino , Microscopia Eletrônica , Microssomos Hepáticos/enzimologia , Membrana Nuclear/enzimologia , Isomerases de Dissulfetos de Proteínas , Ratos , Ratos Endogâmicos , Proteína Estafilocócica ARESUMO
We investigated the distribution of heat shock protein 47 (hsp47) in cultured chicken embryonic chondrocytes and epiphyseal chondrocytes of tibial bones from 1-day-old to 6-week-old chickens. Northern blot and immunoblot analyses revealed that hsp47 exists in epiphyseal cartilage and cultured chondrocytes. Confocal laser immunofluorescence microscopy showed that hsp47 was localized mainly in the many granular structures found in the cytoplasm that contain Type II collagen. Epiphyseal cartilage and cultured chondrocytes were embedded in LR White resin and hsp47 was detected by protein A-immunogold electron microscopy. Gold particles were localized exclusively in the cisternal space of the endoplasmic reticulum (ER), and the labeling density of the cisternal space of the dilated ER was always higher than that of the non-dilated ER. In all the differentiating zones of epiphyseal cartilage, the labeling density was highest in the hypertrophic cells. These findings suggest that hsp47 plays an important role(s) in the synthesis, processing, and assembly of Type II collagen.
Assuntos
Cartilagem/química , Retículo Endoplasmático/química , Proteínas de Choque Térmico/análise , Animais , Northern Blotting , Cartilagem/crescimento & desenvolvimento , Cartilagem/ultraestrutura , Células Cultivadas , Embrião de Galinha , Galinhas , Colágeno/análise , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Testes de Precipitina , RNA Mensageiro/análiseRESUMO
We investigated the intracellular distribution of protein disulfide isomerase (PDI) in rat epiphyseal chondrocytes by immunocytochemistry, using a post-embedding protein A-gold technique. Gold particles were localized primarily in the cisternal space of the rough endoplasmic reticulum (ER) and nuclear envelopes. The ER cisternae of the chondrocytes in all the differentiating epiphyseal zones--resting, proliferative, pre-hypertrophic, and hypertrophic--were equally and highly labeled. The labeling density of the cisternal space of the dilated ER, probably reflecting marked accumulation of secretory proteins such as procollagen, was always higher than that of the non-dilated ER. In the dilated cisternal space, gold particles were freely and evenly distributed, without preferential binding to the luminal surface of the ER membranes. We suggest that PDI catalyzes the formation of disulfide bonds of various secretory proteins, perhaps type II procollagen, in the cisternal space of the ER in epiphyseal chondrocytes. The exclusive localization of gold particles in the cisternal space of the ER and nuclear envelopes and the lack of gold particles in the Golgi apparatus, including cis-Golgi cisternae, indicate that PDI is an ER-soluble protein in the chondrocytes and is presumably sorted out in some pre-Golgi compartment and not transported to the Golgi apparatus.
Assuntos
Cartilagem/enzimologia , Dissulfetos/metabolismo , Isomerases/metabolismo , Envelhecimento , Animais , Western Blotting , Cartilagem/ultraestrutura , Divisão Celular , Retículo Endoplasmático/enzimologia , Imuno-Histoquímica , Membrana Nuclear/enzimologia , Isomerases de Dissulfetos de Proteínas , Ratos , Ratos EndogâmicosRESUMO
PURPOSE: The authors investigated the phagocytic processes of the rod outer segments (ROS) in rat retinal pigment epithelium (RPE) cells, and the appearance of lysosomal enzymes, acidification, and degradation of the contents in the phagolysosomes. In particular, they examined the effect of bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, on the degradation of ROS in the RPE cells in vivo. METHODS: A lysosomal enzyme (cathepsin D), a lysosomal membrane protein (LGP107), and opsin were localized in the RPE cells by the immunogold electron microscopic technique. Bafilomycin A1 was injected into the vitreous, and acidification of the phagosomes was measured in vivo by injecting 3-[2,4-dinitroanilino]3'amino-N-methyldipropylamine (DAMP) in the vitreous and detecting the accumulation of DAMP in the phagolysosomes using anti-dinitrophenol antibody. RESULTS: Opsin was abundantly detected in phagosomes that did not contain cathepsin D, but the immunolabeling of opsin rapidly disappeared soon after the appearance of cathepsin D. By double staining with cathepsin D and DAMP, it was shown that the pH of the phagosomes dramatically decreased after fusion with lysosomes. When bafilomycin A1 was injected into the vitreous, many large phagolysosomes containing cathepsin D appeared in the RPE cells, in which the immunoreactivity of opsin was well preserved. CONCLUSIONS: Degradation of opsin and acidification proceeded almost parallel with the appearance of cathepsin D in the phagolysosomes. Bafilomycin A1 did not inhibit the fusion of phagosomes with lysosomes, but it increased intraphagosomal pH and markedly inhibited the degradation of ROS in the phagolysosomes. This result indicates that vacuolar-type H(+)-ATPase is essential for acidifying the lumen of phagolysosomes and subsequent protein degradation of ROS in the RPE cells.
Assuntos
Macrolídeos , Fagocitose , Fagossomos/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Animais , Antibacterianos/farmacologia , Catepsina D/metabolismo , Contagem de Células , Dinitrobenzenos/metabolismo , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Proteínas de Membrana Lisossomal , Lisossomos/metabolismo , Masculino , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/ultraestrutura , Epitélio Pigmentado Ocular/ultraestrutura , ATPases Translocadoras de Prótons/metabolismo , Ratos , Ratos Sprague-Dawley , Segmento Externo da Célula Bastonete/ultraestrutura , Opsinas de Bastonetes/metabolismo , Sialoglicoproteínas/metabolismoRESUMO
Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells [Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M., & Pastan, I. (1986) Cancer Res. 46, 5941-5946]. We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H(+)-ATPase as model systems. Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP. Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K+, but was inhibited by ammonium chloride (10 mM) and nigericin plus K+. Quinidine (5 microM), verapamil (5 microM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake. Daunomycin was also taken up by liposomes reconstituted with F-type H(+)-ATPase. Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not. The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A1 or concanamycin A, or by increasing the internal pH by addition of nigericin. Melittin and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide dissipated the delta pH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells. These results indicate that the delta pH established by vacuolar-type ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.
Assuntos
Trifosfato de Adenosina/farmacologia , Antineoplásicos/metabolismo , Daunorrubicina/metabolismo , Organelas/metabolismo , Cloreto de Amônio/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Bovinos , Células Cultivadas , Grânulos Cromafim/efeitos dos fármacos , Grânulos Cromafim/metabolismo , Resistência a Medicamentos , Concentração de Íons de Hidrogênio , Camundongos , Nigericina/farmacologia , Proteolipídeos/efeitos dos fármacos , Proteolipídeos/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Quinidina/farmacologia , Reserpina/farmacologia , Valinomicina/farmacologia , Vanadatos/farmacologia , Verapamil/farmacologiaRESUMO
Nordihydroguaiaretic acid (NDGA) blocks intra-Golgi protein transport in a cell-free system and prolactin secretion from GH3 cells [Tagaya, M., Henomatsu, N., Yoshimori, T., Yamamoto, A., Tashiro, Y., and Fukui, T. (1993) FEBS Lett. 324, 201-204]. To determine which intracellular secretory pathway(s) is inhibited by NDGA, we investigated its effect on the transport of the vesicular stomatitis virus-encoded glycoprotein in BHK-21 cells. NDGA blocked protein transport from the endoplasmic reticulum to the Golgi apparatus, and from the trans-Golgi network to the plasma membrane. In addition, it retarded the brefeldin A-induced retrograde transport of mannosidase II to the endoplasmic reticulum. Although NDGA had an inhibitory effect on protein synthesis, it induced the expression of BiP, a chaperone located in the endoplasmic reticulum. The induction of BiP may be a consequence of the inhibition of protein transport by NDGA.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico , Manosidases/metabolismo , Masoprocol/farmacologia , Glicoproteínas de Membrana , Proteínas do Envelope Viral/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Brefeldina A , Proteínas de Transporte/biossíntese , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Cricetinae , Ciclopentanos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Chaperona BiP do Retículo Endoplasmático , Glicoproteínas/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Chaperonas Moleculares/biossíntese , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
The N-terminal half of the beta-subunit of rat brain Na,K-ATPase was expressed in HeLa cells transfected with the plasmid pSV2TKneo beta N containing the truncated beta-subunit cDNA to study the assembly and transport of alpha-beta complex. Immunoprecipitation from extracts of metabolically labeled transformed cells demonstrated that the truncated beta-subunit polypeptide (beta N) was neither transported to the plasma membrane nor assembled into an alpha-beta complex with the endogenous alpha-subunit. Cell fractionation experiments showed that the beta N truncated subunit remained unassembled within rough microsomes, suggesting that it never exited from the endoplasmic reticulum (ER). The assembly of the endogenous alpha-and beta-subunits in the beta N-expressing cells was significantly inhibited compared with control cells or with the transformants that did not express the beta N. These results suggest that the N-terminal portion of the beta-subunit interferes with the normal assembly of the endogenous complex which normally takes place in the ER.
Assuntos
ATPase Trocadora de Sódio-Potássio/genética , Animais , Transporte Biológico , Membrana Celular/metabolismo , DNA/química , Retículo Endoplasmático/metabolismo , Expressão Gênica , Células HeLa/enzimologia , Humanos , Microssomos/metabolismo , Testes de Precipitina , Conformação Proteica , Ratos , ATPase Trocadora de Sódio-Potássio/biossíntese , Transfecção , Transformação GenéticaRESUMO
A review is given of the methods of drying standard reference materials, and handling them after drying. The status of the Faraday as an international reference standard for titrimetric analysis is also discussed.
RESUMO
Iron in Mohr's salt, electrolytic iron and iron ores has been determined by precision coulometric titration with electrolytically generated manganese (III) fluoride, with biamperometric end-point detection. The titration curve indicated the irreversibility of the electrode reaction of manganese(III) fluoride. Total iron in several standard samples of iron ores was determined with standard deviations of about 0.012%.
RESUMO
The water content of dried potassium dichromate and sodium carbonate, which had been heated at various temperatures and then cooled in a "desiccated" atmosphere of argon, was measured by a coulometric microdetermination method. The amounts of water in the dried standard reference materials depended mainly on the heating temperature and only a little on the desiccant used. The dichromate and carbonate contained less than 50 ppm of water when they were heated at temperatures higher than 200 degrees and 250 degrees respectively and then cooled in an atmosphere of argon desiccated with magnesium perchlorate. Sulphuric acid was not advisable as the desiccant for sodium carbonate.