RESUMEN

Lysobisphosphatidic acid (LBPA), or bis(monoacylglycerol)phosphate, is a very interesting lipid, that is mainly found in late endosomes. It has several intriguing characteristics, which differ from those of other animal glycerophospholipids, that may be related to its specific functions, particularly in the metabolism of cholesterol. Its phosphodiester group is bonded at the sn-1 (sn-1') positions of the glycerols rather than at sn-3 (sn-3'); the position of the two fatty acid chains is still under debate but, increasingly, arguments favor the sn-2, sn-2' position in the native molecule, whereas isolation procedures or acidic conditions lead to the thermodynamically more stable sn-3, sn-3' structure. Because of these peculiar features, it can be expected that LBPA shape and interactions with membrane lipids and proteins are related to its structure at the molecular level. We applied quantum mechanical methods to study the structures and stabilities of the 2,2' and 3,3' LBPA isomers, using a step-by-step procedure from glycerol to precursors (in vitro syntheses) and to the final isoforms. The structures of the two positional LBPA isomers are substantially different, showing that the binding positions of the fatty acid chains on the glycerol backbone determine the shape of the LBPA molecule and thus, possibly, its functions. The 3,3' LBPA structures obtained are more stable with respect to the 2,2' form, as expected from experiment. If one argues that the in vivo synthesis starts from the present glycerol conformers and considering the most stable bis(glycero)phosphate structures, the 2,2' isoform should be the most probable isomer.

Asunto(s)

Lisofosfolípidos/química , Monoglicéridos/química , Isomerismo , Lisofosfolípidos/síntesis química , Espectroscopía de Resonancia Magnética , Conformación Molecular , Monoglicéridos/síntesis química , Teoría Cuántica , Termodinámica

RESUMEN

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors.

Asunto(s)

Citosol/metabolismo , Endosomas/metabolismo , Fusión de Membrana/fisiología , Nucleocápside/metabolismo , Animales , Transporte Biológico/fisiología , Bovinos , Línea Celular , Cricetinae , Citosol/ultraestructura , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/ultraestructura , Células Epiteliales/virología , Fibroblastos/virología , Células HeLa , Humanos , Lisofosfolípidos/fisiología , Fusión de Membrana/efectos de los fármacos , Microscopía Electrónica , Microscopía Fluorescente , Monoglicéridos , Fosfatos de Fosfatidilinositol/fisiología , Fosfoproteínas/genética , Fosfoproteínas/fisiología , ARN Viral/biosíntesis , ARN Viral/metabolismo , Transducción de Señal/fisiología , Nexinas de Clasificación , Factores de Tiempo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiología , Virus de la Estomatitis Vesicular Indiana/fisiología , Replicación Viral/genética

RESUMEN

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

Asunto(s)

Endosomas/metabolismo , Receptores ErbB/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de Señal/fisiología , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Endocitosis/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Células Eucariotas/metabolismo , Células HeLa , Peroxidasa de Rábano Silvestre , Humanos , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión , Transducción de Señal/efectos de los fármacos

RESUMEN

This investigation was undertaken to test whether anti-LBPA antibodies and IgG from patients with APS interfere with intracellular beta2GPI distribution in EAhy926 endothelial cells and with the coagulation system. Cell incubation with anti-LBPA MoAb or with patients' IgG resulted in antibody binding to late endosomes and caused beta2GPI redistribution and accumulation within perinuclear vesicular structures reminiscent of late endosomes. This finding suggests that aPI may contribute to the pathogenic mechanisms of APS by modifying the intracellular traffic of proteins, by interactions between aPl and LBPA, beta2GPI and/or LBPA-beta2GPI complexes. The anticoagulant activity of anti-LBPA MoAb was analyzed in a sensitized activated partial thromboplastin time (aPTT) system and in a dilute Russell's viper venom time (dRVVT). A significant, concentration-dependent effect of the antibody on both aPTT and dRVVT prolongation was found. These observations suggest that LBPA is an important lipid target for aPl with potential functional implications for the immunopathogenesis of APS.

Asunto(s)

Anticuerpos Antifosfolípidos/inmunología , Anticuerpos Monoclonales/farmacología , Síndrome Antifosfolípido/metabolismo , Enfermedades Autoinmunes/metabolismo , Coagulación Sanguínea/efectos de los fármacos , Endosomas/metabolismo , Endotelio Vascular/efectos de los fármacos , Glicoproteínas/metabolismo , Lisofosfolípidos/inmunología , Anticuerpos Antifosfolípidos/farmacología , Anticuerpos Monoclonales/inmunología , Síndrome Antifosfolípido/inmunología , Enfermedades Autoinmunes/inmunología , Compartimento Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Membrana Celular/química , Relación Dosis-Respuesta Inmunológica , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Humanos , Microscopía Confocal , Monoglicéridos , Orgánulos/química , Tiempo de Tromboplastina Parcial , Transporte de Proteínas , Tiempo de Protrombina , beta 2 Glicoproteína I

RESUMEN

Organelles in the endocytic pathway are composed of a mosaic of structural and functional regions. These regions consist, at least in part, of specialized protein-lipid domains within the plane of the membrane, or of protein complexes associated with specific membrane lipids. Whereas some of these molecular assemblies can be found in more than one compartment, a given combination seems to be unique to each compartment, indicating that membrane organization might be modular.

Asunto(s)

Endocitosis/fisiología , Animales , Transporte Biológico , Endosomas/fisiología , Humanos , Mamíferos , Modelos Biológicos , Saccharomyces cerevisiae/fisiología , Transducción de Señal/fisiología

RESUMEN

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

Asunto(s)

Macrólidos , Mycobacterium tuberculosis , Fagosomas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Tuberculosis Pulmonar/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab5/metabolismo , Androstadienos/farmacología , Animales , Antibacterianos/farmacología , Anticuerpos/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Glicosilación , Lipopolisacáridos/farmacología , Lisofosfolípidos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Microinyecciones , Microesferas , Monoglicéridos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Qa-SNARE , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Vesículas Transportadoras/metabolismo , Tuberculosis Pulmonar/inmunología , Wortmanina

RESUMEN

Internalization of receptors and other cell surface components is well known to occur via clathrin-mediated endocytosis, although other less well characterized pathways are also involved. Internalized receptors are then delivered to early endosomes, where they are sorted to be recycled back to the plasma membrane for reutilization or transported to late endosomes/lysosomes for degradation. Endocytosis has long been considered as a constitutive, housekeeping function of animal cells that occurs independently of the cellular environment in contrast to regulated secretion. Here, we will discuss recent studies that are uncovering the existence of cross-talk between signaling molecules and components of the transport machinery, indicating that endocytosis can be modulated by signaling pathways.

Asunto(s)

Clatrina/metabolismo , Endocitosis/fisiología , Transporte de Proteínas/fisiología , Transducción de Señal/fisiología , Actinas/metabolismo , Secuencias de Aminoácidos , Animales , Modelos Biológicos , Proteínas de Unión al GTP rab5/metabolismo

RESUMEN

Antiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards beta2-glycoprotein 1 (beta2GP1)phospholipid complexes, we investigated the localisation of beta2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti-beta2GP1 antibodies, we detected beta2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti-beta2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti-beta2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti-beta2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

Asunto(s)

Endosomas/química , Endotelio Vascular/citología , Glicoproteínas/metabolismo , Anticuerpos/metabolismo , Anticuerpos/farmacología , Anticuerpos Antifosfolípidos , Anticoagulantes/inmunología , Anticoagulantes/metabolismo , Síndrome Antifosfolípido/etiología , Endotelio Vascular/ultraestructura , Glicoproteínas/inmunología , Humanos , Microscopía Fluorescente , Receptor IGF Tipo 2/efectos de los fármacos , Receptor IGF Tipo 2/metabolismo , Venas Umbilicales/citología , beta 2 Glicoproteína I

RESUMEN

Early endocytic membrane traffic is regulated by the small GTPase Rab5, which cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle depends on GDI, which functions as a Rab vehicle in the aqueous environment of the cytosol. Here, we report that formation of the GDI:Rab5 complex is stimulated by a cytosolic factor that we purified and then identified as p38 MAPK. We find that p38 regulates GDI in the cytosolic cycle of Rab5 and modulates endocytosis in vivo. Our observations reveal the existence of a cross-talk between endocytosis and the p38-dependent stress response, thus providing molecular evidence that endocytosis can be regulated by the environment.

Asunto(s)

Endocitosis , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Inhibidores de Disociación de Guanina Nucleótido/genética , Humanos , Peróxido de Hidrógeno/farmacología , Imidazoles/farmacología , Cinética , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/aislamiento & purificación , Mutación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Piridinas/farmacología , Proteínas Recombinantes de Fusión , Serina/genética , Serina/metabolismo , Proteínas de Transporte Vesicular , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).

Asunto(s)

Colesterol/metabolismo , Endosomas/metabolismo , Fosfoproteínas/metabolismo , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/metabolismo , Línea Celular , Cricetinae , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Mutagénesis Sitio-Dirigida , Proteína Niemann-Pick C1 , Fosfoproteínas/genética , Unión Proteica , Tirosina/metabolismo

RESUMEN

Some lysosomal storage diseases result from the accumulation of lipids in degradative compartments of the endocytic pathway. Particularly striking is the example of the Niemann-Pick (NP) syndrome. NP syndromes types A and B are characterized by the accumulation of sphingomyelin, whereas cholesterol typically accumulates in NP type C. These two different lipids, sphingomyelin and cholesterol, are normal constituents of specific lipid microdomains called rafts. Because accumulation of raft lipids is observed not only in NP diseases but also in many other lipidoses, we forward the hypothesis that lysosomal storage diseases can be caused by the accumulation of lipid rafts in late endosomes/lysosomes.

Asunto(s)

Endosomas/metabolismo , Enfermedades por Almacenamiento Lisosomal/fisiopatología , Lisosomas/metabolismo , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Endocitosis/fisiología , Endosomas/química , Humanos , Enfermedades por Almacenamiento Lisosomal/etiología , Lisosomas/química , Lípidos de la Membrana/química , Microdominios de Membrana/química , Transporte de Proteínas

RESUMEN

To evaluate patients with gallbladder polyps and to compare them with patients with chronic acalculous cholecystitis, 301 patients with chronic acalculous disease of the gallbladder, of which 45 had polyp disease of the gallbladder, were reviewed out of 7181 cholecystectomies performed from June 1985 through June 1995. Of the 45 patients, 30 (Group A) were diagnosed preoperatively by ultrasound and 15 (Group B) postoperatively on pathologic examination. In each group, the most common polyp was cholesterol type (19/45) with multiple lesions in 10 of these 19 patients. Chronic cholecystitis was present elsewhere in the gallbladder in 40 per cent of Group A and 80 per cent of Group B patients (P = 0.02). Forty-three patients had polyps less than 5 mm in diameter, one a 1.5-cm gallbladder cholesterol polyp, and one a 1.3-cm tubulovillous polyp with a focus of carcinoma in situ. During this same period, 17 patients had primary malignancy of the gallbladder, none of which were found in polypoid lesions. In Group A patients there were significantly fewer preoperative tests than in typical acalculous patients [2.3 versus 3.8 (P<0.03)], including upper endoscopy (P<0.02) and hepatobiliary scintigraphy (P<0.00001). Of the patients with polyps, 42 of 45 (93.3%) had resolution of symptoms postoperatively with a mean follow-up of 178.9+/-505.0 days (range 1-2438 days). Most patients with biliary tract symptoms and a small (<5-mm) gallbladder polyp underwent fewer preoperative diagnostic tests than patients with chronic acalculous cholecystitis. This abbreviated preoperative workup appears warranted in view of the high incidence of symptom resolution.

Asunto(s)

Colecistectomía , Neoplasias de la Vesícula Biliar/cirugía , Pólipos/cirugía , Adulto , Colecistitis/complicaciones , Enfermedad Crónica , Neoplasias de la Vesícula Biliar/complicaciones , Humanos , Persona de Mediana Edad , Pólipos/complicaciones , Estudios Retrospectivos

RESUMEN

We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.

Asunto(s)

Endosomas/metabolismo , Endosomas/ultraestructura , Receptores de Transferrina/metabolismo , ATPasas de Translocación de Protón Vacuolares , Animales , Caveolina 1 , Caveolinas/metabolismo , Caveolinas/ultraestructura , Línea Celular , Colesterol/metabolismo , Perros , Endocitosis , Células Epiteliales/química , Células Epiteliales/citología , Células Epiteliales/fisiología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/química , ATPasas de Translocación de Protón/metabolismo , Receptores de Transferrina/genética , Esfingomielinas/metabolismo , Fracciones Subcelulares/química , Transfección , Transferrina/metabolismo

RESUMEN

In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation of the C-terminal tail distal to the rapid endocytosis motif (YKYSKV), caused the truncated chimera to be transported to, and accumulated within, early endosomes. This truncated chimera did not reach recycling early endosomes labelled with internalised transferrin, to any significant extent, but was accessible to biotinylated HRP internalised for 5 min (or for longer periods at 19 degrees C). Coinfection of these chimeras showed that they follow the same route from the TGN to the early endosomes. We conclude that the sequence distal to the endocytosis motif contains the signals which are required for efficient transport to late endosomes. Our results also suggest that the YKYSKV sequence close to the CI-MPR transmembrane segment is sufficient for targeting to sorting early endosomes.

Asunto(s)

Avidina/metabolismo , Endosomas/metabolismo , Receptor IGF Tipo 2/metabolismo , Secuencias de Aminoácidos , Animales , Avidina/química , Avidina/genética , Transporte Biológico , Biotinilación , Brefeldino A/farmacología , Cationes , Bovinos , Membrana Celular/metabolismo , Pollos , Cricetinae , Reactivos de Enlaces Cruzados/farmacología , Citoplasma/metabolismo , Dimerización , Endocitosis/fisiología , Endosomas/efectos de los fármacos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Povidona/farmacología , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Virus de los Bosques Semliki/genética , Dióxido de Silicio/farmacología , Factores de Tiempo

RESUMEN

An expeditious route to synthetic lysobisphosphatidic acid S,S-1, its enantiomer, and regioisomers is reported. Synthetic difficulties concerning lipid stability and stereochemistry are bypassed using a phosphite triester approach in combination with multiple silyl protection. Spectroscopic studies evidence that acyl group migration in S,S-1 is accelerated by nonpolar solvents and inhibited by pyridine.

Asunto(s)

Bioquímica/métodos , Lisofosfolípidos/síntesis química

RESUMEN

Recent studies show that small trans-membrane proteins of approximately 22-24 kDa (the p24 family), which are grouped into 4 sub-families by sequence homology (p23, p24, p25 and p26), are involved in the early secretory pathway. In this study, we have investigated the mutual requirements of ectopically expressed members of the p24 family for targeting to their proper cellular destination. We find that coexpression of p23 and p24 is both necessary and sufficient for each protein to be transported to the cis-Golgi network/Golgi complex. Proteins from other subfamilies did not substitute for either p23 or p24, even after multiple coexpression. However, trafficking of the p23/p24 couple was facilitated by coexpression of proteins from other sub-families. In addition, we find that the sequence resembling an endoplasmic reticulum retrieval signal present in the cytoplasmic domain of p23 (but not p24) is dispensable. In contrast, the conserved coiled-coil region in the lumenal domain is absolutely required in both p23 and p24 for proper targeting of the p23/p24 couple. These data demonstrate that p23 and p24 must interact with each other to reach their destination, but that this strict requirement is combined with a mutual dependence amongst p24 proteins. We speculate that p24 proteins can form different oligomeric complexes, which contribute to confer specialized sorting/trafficking properties to membranes of the early secretory pathway, perhaps serving as membrane organizers.

Asunto(s)

Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Transporte Biológico/fisiología , Clonación Molecular , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína

RESUMEN

In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular-multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel-Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel-Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel-Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel-Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel-Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel-Palade bodies.

Asunto(s)

Antígenos CD/metabolismo , Endocitosis/fisiología , Endotelio Vascular/citología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Androstenos/farmacología , Anticuerpos Monoclonales/metabolismo , Anticolesterolemiantes/farmacología , Antígenos CD/inmunología , Compartimento Celular , Línea Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Cinética , Orgánulos/metabolismo , Selectina-P/metabolismo , Fosfolípidos/metabolismo , Glicoproteínas de Membrana Plaquetaria/inmunología , Tetraspanina 30 , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo , Factor de von Willebrand/inmunología , Factor de von Willebrand/metabolismo

RESUMEN

Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR.

Asunto(s)

Endosomas/metabolismo , Receptores ErbB/metabolismo , Treonina/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Células CHO , Células Cultivadas , Cricetinae , Regulación hacia Abajo , Endocitosis , Ionóforos/farmacología , Ligandos , Monensina/farmacología , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Relación Estructura-Actividad , Transfección , Ubiquitinas/metabolismo

RESUMEN

Coat proteins of the COP family were recently shown by us and others to be involved in membrane transport in the endocytic pathway, in addition to their known functions in the biosynthetic pathway. We have also shown that membrane association of endosomal COPs depends on the acidic endosomal pH, in contrast to biosynthetic COPs. In this paper, we report that both membrane recruitment of endosomal COPs and in vitro biogenesis of transport intermediates destined for late endosomes, depend on a cytosolic factor, which we identified as the small GTP-binding protein ARF1. Our data indicate that ARF1 does not act via activation of an endosomal phospholipase D. We also find that ARF1 membrane association is regulated by the endosomal pH, and that this controls the pH-dependent association of endosomal COPs. These studies thus show that ARF1 regulates COP functions in the endocytic pathway, and indicate that ARF1 acts as the cytosolic component of a transmembrane pH-sensing mechanism.

Asunto(s)

Factor 1 de Ribosilacion-ADP/metabolismo , Proteína Coatómero/metabolismo , Endocitosis/fisiología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Animales , Cricetinae , Citosol/metabolismo , Endosomas/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Unión Proteica

RESUMEN

In previous studies we have shown that p23, a member of the p24-family of small transmembrane proteins, is highly abundant in membranes of the cis-Golgi network (CGN), and is involved in sorting/trafficking in the early secretory pathway. In the present study, we have further investigated the role of p23 after ectopic expression. We found that ectopically expressed p23 folded and oligomerized properly, even after overexpression. However, in contrast to endogenous p23, exogenous p23 molecules did not localize to the CGN, but induced a significant expansion of characteristic smooth ER membranes, where they accumulated in high amounts. This ER-derived, p23-rich subdomain displayed a highly regular morphology, consisting of tubules and/or cisternae of constant diameter, which were reminiscent of the CGN membranes containing p23 in control cells. The expression of exogenous p23 also led to the specific relocalization of endogenous p23, but not of other proteins, to these specialized ER-derived membranes. Relocalization of p23 modified the ultrastructure of the CGN and Golgi membranes, but did not affect anterograde and retrograde transport reactions to any significant extent. We conclude (i) that p23 has a morphogenic activity that contributes to the morphology of CGN-membranes; and (ii) that the presence of p23 in the CGN is necessary for the proper organization of the Golgi apparatus.

Asunto(s)

Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Proteínas de la Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Microscopía Inmunoelectrónica , Pliegue de Proteína