RESUMEN
Golgi-localized, γ-ear-containing, ADP ribosylation factor-binding (GGA) proteins are monomeric adaptors implicated in clathrin-mediated vesicular transport between the trans Golgi network and endosomes, characterized mainly from cell culture analysis of lysosomal sorting. To provide the first demonstration of GGA's role in vivo, we used Drosophila which has a single GGA and a single lysosomal sorting receptor, lysosomal enzyme receptor protein (LERP). Using RNAi knockdowns, we show that the Drosophila GGA is required for lysosomal sorting. We further identified authentic components of the Drosophila lysosomal sorting system--the sorting receptor LERP, the sorting adaptor GGA and the lysosomal cargo cathepsins B1, D and L--to show that GGA depletion results in lysosomal dysfunction. Abnormal lysosomal morphology, missorting of lysosomal cathepsins and impaired lysosomal proteolysis show disturbed LERP trafficking after GGA depletion. GGA is highly expressed in the mushroom bodies and the pigment cells of the retina, and increasing or decreasing the levels of GGA in the eyes leads to retinal defects. Reduced GGA levels also enhance an eye defect caused by overexpression of the autophagy-associated protein Blue cheese (Bchs), implicating GGA in autophagic processes. This shows that Drosophila provides an excellent whole-animal model to gain new insights into the function of GGA in the physiological environment of a multicellular organism.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Catepsinas/metabolismo , Clatrina/metabolismo , Drosophila , Proteínas de Drosophila/genética , Endosomas/genética , Endosomas/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Lisosomas/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Transporte de Proteínas , Proteolisis , Interferencia de ARN , Retina/metabolismo , Vesículas Transportadoras/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 mu1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Cricetulus , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Proteínas Relacionadas con Receptor de LDL/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Interferencia de ARN , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Spinach fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a redox-modulated chloroplast enzyme and part of the Calvin cycle, and three different Cys mutants were expressed in E. coli. The properties of the purified proteins were compared to those of native and recombinant chloroplast FBPase from the red alga Galdieria sulphuraria. In spinach chloroplast FBPase, Cys(155) and Cys(174) are engaged in the formation of the disulfide bridge. The corresponding mutants are active when expressed in E. coli, while C179S is inactive and can be reductively activated as can the wild-type enzyme. The active C174S mutant, however, could be inactivated by oxidation, and reactivated, but only by reduction, not alternatively with high pH and high Mg(2+) as is the case for the wild-type enzyme. In the sequence of Galdieria FBPase, the Cys that corresponds to Cys(179) in the spinach enzyme is lacking. However, the Galdieria FBPase, in contrast to the spinach Cys(179) mutant, does not show any indication for a comparable redox modulation of its activity. Instead, oxidation only leads to partial inactivation without any qualitative changes in enzyme properties. Upon reduction, the lost activity can be recovered.
Asunto(s)
Cloroplastos/enzimología , Fructosa-Bifosfatasa/genética , Rhodophyta/enzimología , Spinacia oleracea/genética , Secuencia de Aminoácidos , Cisteína/química , Disulfuros , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Escherichia coli/metabolismo , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/metabolismo , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Magnesio , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Alineación de Secuencia , Spinacia oleracea/enzimologíaRESUMEN
The pleiotropic effects of Calloselasma rhodostoma venom is caused by various toxins, among them kistrin and ancrod, which block platelet activation triggered by RGD-dependent integrins and the blood clotting cascade, respectively. Here, we demonstrate that rhodocetin, another component of this venom, acts as alpha2beta1 integrin inhibiting disintegrin and antagonizes important cellular responses to type I collagen. Cell adhesion, migration, and collagen lattice contraction in vitro were specifically inhibited by rhodocetin, whereas expression of collagen-degrading matrix metalloproteases was differently modulated. Moreover, cell invasion of HT1080 fibrosarcoma cells into a type I collagen matrix, but not into a fibrin gel or a basement membrane-extracted matrigel was efficiently blocked by rhodocetin. Unlike its natural ligand collagen, rhodocetin failed to cluster alpha2beta1 integrin, despite similar binding affinities. Hence, in the absence of focal adhesions cells do not attach firmly to rhodocetin and do not respond with any of alpha2beta1-triggered cell reactions, except for MMP-1 production. Therefore, this disintegrin may be a valuable tool to specifically target stromal tumor invasion and to manipulate other alpha2beta1 integrin-mediated functions, such as excessive scar contraction and fibrosis. Rhodocetin might be therapeutically useful because of its lack of interference with RGD-dependent integrins, low molecular mass, high solubility, and biochemical stability.
Asunto(s)
Fenómenos Fisiológicos Celulares/efectos de los fármacos , Venenos de Crotálidos/farmacología , Fibrosarcoma/patología , Integrina alfa2beta1/fisiología , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Colágeno Tipo I , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Invasividad Neoplásica , Células Tumorales CultivadasRESUMEN
Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, gamma-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish.
Asunto(s)
Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Animales , Catepsina D/metabolismo , Bovinos , Pollos/genética , Clonación Molecular , Peces/genética , Perfilación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Xenopus/genética , Pez Cebra/genéticaRESUMEN
Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Lisosomas/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Arginina/química , Catepsina D/química , Catepsina L , Catepsinas/química , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía , Secuencia Conservada , Cisteína Endopeptidasas/química , Citoplasma/metabolismo , Drosophila melanogaster , Fibroblastos/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación , Inmunoprecipitación , Lectinas/metabolismo , Ligandos , Mananos/química , Manosafosfatos/metabolismo , Ratones , Microscopía Confocal , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Unión Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Mensajero/metabolismo , Receptor IGF Tipo 2 , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole. Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins. A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail. Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor. Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast. Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.