RESUMEN

The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H(+) ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na(+)/H(+) exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca(2+) ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca(2+) in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).

Asunto(s)

Calcio/metabolismo , Cationes/metabolismo , Endocitosis , Lisosomas/metabolismo , Fenobarbital/metabolismo

RESUMEN

The integrity of the actin cytoskeleton and associated motor proteins are essential for the efficient functioning of clathrin mediated endocytosis at least in polarised cells. Myosin VI, the only motor protein so far identified that moves towards the minus end of actin filaments, is the first motor protein to be shown to associate with clathrin coated pits/vesicles at the plasma membrane and to modulate clathrin mediated endocytosis. Recent kinetic studies suggest that myosin VI may move processively along actin filaments providing clues about its functions in the cell. The possible role(s) of myosin VI in the sequential steps involved in receptor mediated endocytosis are discussed.

Asunto(s)

Clatrina/fisiología , Endocitosis , Proteínas Motoras Moleculares/fisiología , Cadenas Pesadas de Miosina/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Diferenciación Celular , Polaridad Celular , Vesículas Cubiertas por Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Humanos , Proteínas Motoras Moleculares/química , Cadenas Pesadas de Miosina/química , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo

RESUMEN

We have examined the trafficking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immunofluorescence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXPhi and was localized primarily to lysosomes at steady state, significant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and efficiently transported to lysosomes. Analysis of endolyn-CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells.

Asunto(s)

Lisosomas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Secuencias de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/inmunología , Biotinilación , Antígeno CD146 , Antígenos CD8/metabolismo , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Detergentes/metabolismo , Detergentes/farmacología , Perros , Endolina , Glicosilación , Aparato de Golgi/metabolismo , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Microscopía Fluorescente , Octoxinol/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Conejos , Transducción de Señal , Factores de Tiempo , Transfección

RESUMEN

Late endosomes, which have the morphological characteristics of multivesicular bodies, have received relatively little attention in comparison with early endosomes and lysosomes. Recent work in mammalian and yeast cells has given insights into their structure and function, including the generation of their multivesicular morphology. Lipid partitioning to create microdomains enriched in specific lipids is observed in late endosomes, with some lumenal vesicles enriched in lysobisphosphatidic acid and others in phosphatidylinositol 3-phosphate. Sorting of membrane proteins into the lumenal vesicles may occur because of the properties of their trans-membrane domains, or as a result of tagging with ubiquitin. Yeast class E Vps proteins and their mammalian orthologs are the best candidates to make up the protein machinery that controls inward budding, a process that starts in early endosomes. Late endosomes are able to undergo homotypic fusion events and also heterotypic fusion with lysosomes, a process that delivers endocytosed macromolecules for proteolytic degradation.

Asunto(s)

Endocitosis , Endosomas/metabolismo , Endosomas/fisiología , Lisosomas/metabolismo , Animales , Metabolismo de los Lípidos , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas

RESUMEN

Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation-independent mannose 6-phosphate receptors, but those induced by wortmannin were characterised by time-dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.

Asunto(s)

Androstadienos/farmacología , Membrana Celular/metabolismo , Endocitosis , Inhibidores Enzimáticos/farmacología , Membranas Intracelulares/metabolismo , Sacarosa/metabolismo , Animales , Línea Celular , Lisofosfolípidos/metabolismo , Lisosomas/metabolismo , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Monoglicéridos , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Receptor IGF Tipo 2/metabolismo , Receptores de Transferrina/metabolismo , Sacarosa/farmacocinética , Factores de Tiempo , Wortmanina

RESUMEN

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are re-formed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Re-formation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules.

Asunto(s)

Endocitosis/fisiología , Endosomas/fisiología , Lisosomas/fisiología , Animales , Fusión de Membrana/fisiología , Proteínas de la Membrana/fisiología

RESUMEN

Myosin VI is involved in membrane traffic and dynamics and is the only myosin known to move towards the minus end of actin filaments. Splice variants of myosin VI with a large insert in the tail domain were specifically expressed in polarized cells containing microvilli. In these polarized cells, endogenous myosin VI containing the large insert was concentrated at the apical domain co-localizing with clathrin- coated pits/vesicles. Using full-length myosin VI and deletion mutants tagged with green fluorescent protein (GFP) we have shown that myosin VI associates and co-localizes with clathrin-coated pits/vesicles by its C-terminal tail. Myosin VI, precipitated from whole cytosol, was present in a protein complex containing adaptor protein (AP)-2 and clathrin, and enriched in purified clathrin-coated vesicles. Over-expression of the tail domain of myosin VI containing the large insert in fibroblasts reduced transferrin uptake in transiently and stably transfected cells by >50%. Myosin VI is the first motor protein to be identified associated with clathrin-coated pits/vesicles and shown to modulate clathrin-mediated endocytosis.

Asunto(s)

Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis/fisiología , Cadenas Pesadas de Miosina/metabolismo , Secuencia de Aminoácidos , Animales , Células CACO-2 , Línea Celular , Pollos , Clatrina/fisiología , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as "sentinels" of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Asunto(s)

Antígenos CD34/metabolismo , Endosomas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células de Langerhans/metabolismo , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Células Dendríticas/metabolismo , Interacciones Farmacológicas , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Células de Langerhans/fisiología , Lipopolisacáridos/farmacología , Microscopía Confocal , Fenotipo , Primaquina/farmacología , Células Madre/fisiología

RESUMEN

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7. Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMP8 to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Asunto(s)

Proteínas Portadoras/metabolismo , Melanoma Experimental/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Melanoma Experimental/patología , Proteínas de la Membrana/inmunología , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas R-SNARE , Células Tumorales Cultivadas

RESUMEN

Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug.

Asunto(s)

Catepsina D/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , ADN Complementario , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas

RESUMEN

A number of recent studies have highlighted the importance of lipid domains within endocytic organelles in the sorting and movement of integral membrane proteins. In particular, considerable attention has become focussed upon the role of the unusual phospholipid lysobisphosphatidic acid (LBPA). This lipid appears to be directly involved in the trafficking of cholesterol and glycosphingolipids, and accumulates in a number of lysosomal storage disorders. Antibody-mediated disruption of LBPA function also leads to mis-sorting of cation-independent mannose 6-phosphate receptors. We now report that the converse is also true, and that spontaneous loss of cation-independent mannose 6-phosphate receptors from a rat fibroblast cell line led to the formation of aberrant late endocytic structures enriched in LBPA. Accumulation of LBPA was directly dependent upon the loss of the receptors, and could be reversed by expression of bovine cation-independent mannose 6-phosphate receptors in the mutant cell line. Ultrastructural analysis indicated that the abnormal organelles were electron-dense, had a multi-lamellar structure, accumulated endocytosed probes, and were distinct from dense-core lysosomes present within the same cells. The late endocytic structures present at steady state within any particular cell likely reflect the balance of membrane traffic through the endocytic pathway of that cell, and the rate of maturation of individual endocytic organelles. Moreover, there is considerable evidence which suggests that cargo receptors also play a direct mechanistic role in membrane trafficking events. Therefore, loss of such a protein may disturb the overall equilibrium of the pathway, and hence cause the accumulation of aberrant organelles. We propose that this mechanism underlies the phenotype of the mutant cell line, and that the formation of inclusion bodies in many lysosomal storage diseases is also due to an imbalance in membrane trafficking within the endocytic pathway.

Asunto(s)

Fibroblastos/fisiología , Fibroblastos/ultraestructura , Lisofosfolípidos/metabolismo , Orgánulos/fisiología , Receptor IGF Tipo 2/fisiología , Animales , Autofagia , Bovinos , Células Clonales , Endocitosis , Filipina/metabolismo , Monoglicéridos , Ratas , Receptor IGF Tipo 2/genética , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

Asunto(s)

Endosomas/fisiología , Lisosomas/fisiología , Fusión de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Perros , Endocitosis , Endosomas/ultraestructura , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Riñón/fisiología , Riñón/ultraestructura , Hígado/fisiología , Hígado/ultraestructura , Lisosomas/ultraestructura , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Red trans-Golgi/fisiología , Red trans-Golgi/ultraestructura

RESUMEN

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Asunto(s)

Calcio/fisiología , Endosomas/fisiología , Lisosomas/fisiología , Macrólidos , Fusión de Membrana/fisiología , Adenosina Trifosfato/metabolismo , Animales , Antibacterianos/farmacología , Calmodulina/metabolismo , Sistema Libre de Células/fisiología , Quelantes/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Endosomas/ultraestructura , Inhibidores Enzimáticos/farmacología , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Inmunoglobulina A/metabolismo , Inmunoglobulina A/farmacología , Radioisótopos de Yodo , Hígado/citología , Hígado/metabolismo , Lisosomas/ultraestructura , Fusión de Membrana/efectos de los fármacos , Microscopía Electrónica , Ratas

RESUMEN

The process of clathrin-mediated endocytosis from the plasma membrane has been the subject of many biological and biochemical investigations. Recent atomic resolution structures determined by X-ray crystallography now enable the molecular basis for the interactions of some components of the endocytic machinery to be understood in detail.

Asunto(s)

Clatrina/química , Clatrina/metabolismo , Endocitosis/fisiología , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Humanos , Proteínas de la Membrana/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad

RESUMEN

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Asunto(s)

Endosomas/fisiología , Lisosomas/fisiología , Fusión de Membrana , Animales , Endocitosis , Humanos

RESUMEN

Endolyn (endolyn-78) is a membrane protein found in lysosomal and endosomal compartments of mammalian cells. Unlike 'classical' lysosomal membrane proteins, such as lysosome-associated membrane protein (lamp)-1, it is also present in a subapical compartment in polarized WIF-B hepatocytes. The structural features that determine sorting of endolyn are unknown. We have identified a rat endolyn cDNA by expression screening. The cDNA encodes a ubiquitously expressed type I membrane protein with a short cytoplasmic tail of 13 amino acids and many putative sites for N- and O-linked glycosylation in the predicted luminal domain. Endolyn is closely related to two human mucin-like proteins, multi-glycosylated core protein (MGC)-24 and CD164 (MGC-24v), expressed in gastric carcinoma cells and bone marrow stromal and haematopoietic precursor cells respectively. The predicted transmembrane and cytoplasmic tail domains of endolyn, as well as parts of its luminal domain, also show some similarities with lamp-1 and lamp-2. Like these and other known lysosomal membrane proteins, endolyn contains a YXXO motif at the C-terminus of its cytoplasmic tail (where O is a bulky hydrophobic amino acid), but with no preceding glycine. Nonetheless, the last ten amino acids of this tail, when transplanted on to human CD8, caused efficient targeting of the chimaeric protein to endosomes and lysosomes in transfected normal rat kidney cells.

Asunto(s)

Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Moléculas de Adhesión de Célula Nerviosa , Señales de Clasificación de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD , Secuencia de Bases , Antígeno CD146 , Compartimento Celular , Polaridad Celular , Secuencia Conservada , Disulfuros , Endolina , Biblioteca de Genes , Hígado/citología , Hígado/metabolismo , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mucinas/genética , Señales de Clasificación de Proteína/genética , Ratas , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

Asunto(s)

Polaridad Celular/fisiología , Resistencia a Medicamentos/fisiología , Ricina/farmacología , Transporte Biológico , Células CACO-2 , Humanos

RESUMEN

TGN38 is a heavily glycosylated, type I integral membrane protein which is predominantly localized to the trans Golgi network (TGN), but which constitutively traffics between the TGN and the cell surface. The trafficking of TGN38 has been extensively studied in non-polarized cells, and a short, tyrosine-based, peptide motif within the cytosolic domain of the protein has been shown to be necessary and sufficient for its rapid internalization from the cell surface and efficient delivery to the TGN. Such tyrosine-based motifs have also been shown to act as basolateral targeting signals, whilst N-linked glycans (as occur on the extracytosolic domain of TGN38) can act as apical targeting signals. TGN38 has previously been shown to be sorted to the basolateral surface of polarized canine MDCK cells; a polarized cell line in which biosynthetic sorting decisions concerning the eventual destination of apical or basolateral targeted plasma membrane proteins are made at the TGN. We now show that TGN38 is targeted exclusively to the basolateral domain of polarized human Caco-2 cells, a cell line in which newly synthesized membrane proteins destined for either the apical or basolateral plasma membrane may be sorted for delivery to their final destination either at the TGN or at the cell surface. These data also demonstrate that the heavily glycosylated, extracytosolic domain of TGN38 does not contain a dominant apical targeting signal.

Asunto(s)

Glicoproteínas , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana , Animales , Transporte Biológico , Células CACO-2 , Polaridad Celular , Humanos , Ratas

RESUMEN

p53 acts as a tumor suppressor by inducing both growth arrest and apoptosis. p53-induced apoptosis can occur without new RNA synthesis through an unknown mechanism. In human vascular smooth muscle cells, p53 activation transiently increased surface Fas (CD95) expression by transport from the Golgi complex. Golgi disruption blocked both p53-induced surface Fas expression and apoptosis. p53 also induced Fas-FADD binding and transiently sensitized cells to Fas-induced apoptosis. In contrast, lpr and gld fibroblasts were resistant to p53-induced apoptosis. Thus, p53 can mediate apoptosis through Fas transport from cytoplasmic stores.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Membrana Celular/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Receptor fas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Brefeldino A/farmacología , Proteínas Portadoras/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Proteína Ligando Fas , Proteína de Dominio de Muerte Asociada a Fas , Aparato de Golgi/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Músculo Liso Vascular/citología , Mutación , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Inhibidores de Topoisomerasa II , Receptor fas/genética

RESUMEN

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.

Asunto(s)

Antígenos CD/metabolismo , Endocitosis/fisiología , Glicoproteínas , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Sitios de Unión , Catepsina D/metabolismo , Línea Celular , Cloroquina/farmacología , Endosomas/metabolismo , Células HeLa , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas , Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Nocodazol/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetraspanina 30