RESUMEN
In most eukaryotic cells microtubules undergo post-translational modifications such as acetylation of α-tubulin on lysine 40, a widespread modification restricted to a subset of microtubules that turns over slowly. This subset of stable microtubules accumulates in cell protrusions and regulates cell polarization, migration and invasion. However, mechanisms restricting acetylation to these microtubules are unknown. Here we report that clathrin-coated pits (CCPs) control microtubule acetylation through a direct interaction of the α-tubulin acetyltransferase αTAT1 (refs 8, 9) with the clathrin adaptor AP2. We observe that about one-third of growing microtubule ends contact and pause at CCPs and that loss of CCPs decreases lysine 40 acetylation levels. We show that αTAT1 localizes to CCPs through a direct interaction with AP2 that is required for microtubule acetylation. In migrating cells, the polarized orientation of acetylated microtubules correlates with CCP accumulation at the leading edge, and interaction of αTAT1 with AP2 is required for directional migration. We conclude that microtubules contacting CCPs become acetylated by αTAT1. In migrating cells, this mechanism ensures the acetylation of microtubules oriented towards the leading edge, thus promoting directional cell locomotion and chemotaxis.
Asunto(s)
Acetiltransferasas/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Microtúbulos/metabolismo , Acetilación , Complejo 2 de Proteína Adaptadora/metabolismo , Biocatálisis , Movimiento Celular , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Células HeLa , Humanos , Microtúbulos/química , Unión Proteica , Tubulina (Proteína)/metabolismoRESUMEN
Numb is an endocytic protein that is proposed to influence clathrin-coated pit assembly, although its mode of action and the mechanisms that regulate its activity are unknown. In this study, we show that Numb binds to and is phosphorylated by adaptor-associated kinase 1 (AAK1), a key endocytic kinase. We find that AAK1 redistributes Numb to perinuclear endosomes when overexpressed, while kinase depletion causes Numb to accumulate at the plasma membrane. Overexpression of a Numb point mutant (T102A) that lacks the AAK1 phosphorylation site potently disrupts transferrin and low-density lipoprotein internalization but does not impact EGF uptake. Consistent with Numb redistribution results, we find that T102A Numb no longer localizes to perinuclear endosomes. Instead, it is enriched at the plasma membrane where it shows elevated levels of colocalization with coated pit markers. Collectively, these observations demonstrate that Numb endocytic activity is regulated by AAK1 and that phosphorylation may be a critical step in promoting coated pit maturation.
Asunto(s)
Clatrina/fisiología , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Endocitosis/fisiología , Endosomas/fisiología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/enzimología , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The tyrosine kinase receptor Tie2 is expressed on endothelial cells, and together with its ligand angiopoietin-1 (Ang1), is important for angiogenesis and vascular stability. Upon activation by Ang1, Tie2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold electron microscopy, we show that on the surface of endothelial cells, Tie2 is arranged in variably sized clusters containing dimers and higher order oligomers. Clusters of Tie2 were expressed on the apical and basolateral plasma membranes, and on the tips of microvilli. Upon activation by Ang1, Tie2 co-localized with the clathrin heavy chain at the apical and basolateral plasma membranes and within endothelial cells indicating that Tie2 internalizes through clathrin-coated pits. Inhibiting cellular endocytosis by depleting cellular potassium or by acidifying the cytosol blocked the internalization of Tie2 in response to Ang1. Our results suggest that one pathway mediating the internalization of Tie2 in response to Ang1 is through clathrin-coated pits.
Asunto(s)
Angiopoyetina 1/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endotelio Vascular/enzimología , Receptor TIE-2/metabolismo , Angiopoyetina 1/agonistas , Células Cultivadas , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Endotelio Vascular/ultraestructura , HumanosRESUMEN
To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a approximately threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Neuronas/enzimología , Animales , Línea Celular , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/efectos de los fármacos , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Dinamina I , Dinaminas , Proteína Adaptadora GRB2 , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/ultraestructura , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacología , Humanos , Hidrólisis , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/ultraestructura , Cinética , Microscopía Inmunoelectrónica , Microtúbulos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/metabolismo , Ratas , Eliminación de Secuencia/genética , TermodinámicaRESUMEN
In this review, I hope to achieve the following: (a) to document the presence of a lysosome-like proton pump ATPase in many different membrane systems of animal, plant and microbial origin; (b) to glean from the diverse data common characteristics of these ATPases, especially as regards their similarities and differences with mitochondrial-type F1F0 proton pump ATPases; and (c) to consider questions of synthesis and regulation of a cellular proton pump system with such a widespread distribution.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Canales Iónicos/metabolismo , Lisosomas/enzimología , Organoides/enzimología , Protones , Vacuolas/enzimología , Animales , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Hongos/enzimología , Plantas/enzimología , ATPasas de Translocación de Protón/metabolismoRESUMEN
Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, Mr 150 000, 120 000, 48 000 and 32 000. An endogenous protein, Mr 48 000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn2+, Mg2+, Co2+, Ca2+, Zn2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction.
Asunto(s)
Encéfalo/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , Proteínas Quinasas/análisis , Animales , Cationes Bivalentes/farmacología , Bovinos , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Electroforesis en Gel de Poliacrilamida , Histonas/metabolismo , Microscopía Electrónica , Peso Molecular , Protaminas/metabolismoRESUMEN
Purified bovine brain coated vesicles contain protein kinase activity which phosphorylates 165, 54 and 50 kDa protein substrates. These phosphorylations do not seem to be induced by a unique protein kinase: indeed, the three substrates present different localizations in coated vesicles, the phosphorylation sites are either serine or threonine residues and vanadate and ATP[gamma S] have different effects on 32P incorporation in the substrates. Comparison of the coated vesicle protein and phosphorylation patterns from different tissues and animal origins shows that only the 50 kDa protein phosphorylation is always observed, compared to the great diversity in other minor phosphorylations which are observed or not in the various coated vesicles. The possible presence of a 50 kDa phosphoprotein phosphatase is also discussed. It is suggested that the 50 kDa protein with its connected specific kinase and phosphatase seems to constitute a regulatory system present in coated vesicles.
Asunto(s)
Encéfalo/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , Proteínas Quinasas/análisis , Glándulas Suprarrenales/enzimología , Animales , Encéfalo/enzimología , Bovinos , Electroforesis en Gel de Poliacrilamida , Hígado/enzimología , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilación , Ratas , Porcinos , Temperatura , Factores de TiempoRESUMEN
We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+ transport specific activity was shown by column chromatograpy to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+ transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 15 mM), but was relatively insensitive to cation replacement as long as Cl- was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol greater than potassium gluconate greater than KCl) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+ transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl- salts and KNO3. Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+ transport activity similar to that of the coated vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Encéfalo/ultraestructura , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , ATPasas de Translocación de Protón/metabolismo , Naranja de Acridina/metabolismo , Animales , Encéfalo/enzimología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Bovinos , Cloruros/farmacología , Clorpromazina/farmacología , Diciclohexilcarbodiimida/farmacología , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Peso Molecular , Ouabaína/farmacología , Valinomicina/farmacologíaRESUMEN
Coated vesicles have been found to contain much higher tyrosinase and gamma-glutamyl transpeptidase activities than premelanosomes. This indicates that similar to tyrosinase, gamma-glutamyl transpeptidase, an enzyme responsible for pheomelanogenesis, is highly concentrated in coated vesicles after its maturation in Golgi associated endoplasmic reticulum (GERL). Furthermore, in the pre- and post-dopaquinone melanogenic pathway, coated vesicles convert dopachrome to colorless indole compounds more quickly than in premelanosomes because of their higher dopachrome conversion factor activity. Melanosomes have been found to exhibit indole conversion factor activity, while coated vesicles show indole blocking factor activity. In moderately tyrosinase-rich premelanosomes, the levels of dopachrome conversion factor and indole blocking factor are lower than in coated vesicles or melanosomes. High levels of indole blocking factor in coated vesicles may indicate why melanin polymer formation does not occur there in vivo despite their high tyrosinase activity.
Asunto(s)
Invaginaciones Cubiertas de la Membrana Celular/fisiología , Endosomas/fisiología , Indolquinonas , Melaninas/biosíntesis , Melanoma Experimental/fisiopatología , Organoides/fisiología , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Indoles/metabolismo , Microscopía Electrónica , Monofenol Monooxigenasa/metabolismo , Organoides/enzimología , Quinonas/metabolismo , Fracciones Subcelulares/enzimología , gamma-Glutamiltransferasa/metabolismoRESUMEN
Bovine brain coated vesicles display free calcium-dependent phospholipase C activity. Gpp(NH)p and GTP gamma S inhibited phospholipase C at nanomolar concentrations. Increasing concentrations of Gpp(NH)p and GTP gamma S reversed the inhibitory effects and stimulated phospholipase C activity. Preincubation of coated vesicles with pertussis toxin blocked the poorly-hydrolyzable GTP-analogs' inhibitory effects on phospholipase C. These data indicate that guanine nucleotides exert a dual regulatory control of phospholipase C in coated vesicles and that the inhibitory pathway is mediated by a pertussis toxin-sensitive G-protein.
Asunto(s)
Encéfalo/enzimología , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Proteínas de Unión al GTP/fisiología , Nucleótidos de Guanina/farmacología , Fosfolipasas de Tipo C/fisiología , Animales , Bovinos , Membrana Celular/enzimología , Técnicas In Vitro , Toxina del Pertussis , Transducción de Señal , Fosfolipasas de Tipo C/antagonistas & inhibidores , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The V-ATPases are a novel class of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. They play an important role in receptor-mediated endocytosis, intracellular membrane traffic, macromolecular processing and degradation and coupled transport, as well as functioning in the plasma membrane of certain specialized cell types. The V-ATPases are multisubunit complexes that are organized into a peripheral V1 complex responsible for ATP hydrolysis and an integral V0 domain responsible for proton translocation. Regulation of vacuolar acidification is critical to its role in membrane traffic and other cellular processes. We are currently investigating several mechanisms of regulation of vacuolar acidification, including disulfide bond formation between cysteine residues located at the catalytic site, control of assembly of the peripheral and integral domains, and differential targeting of V-ATPases to different intracellular destinations using their interaction with organelle-specific adaptin complexes.
Asunto(s)
Invaginaciones Cubiertas de la Membrana Celular/enzimología , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Vesículas Sinápticas/enzimología , Animales , Clatrina/metabolismo , Sustancias Macromoleculares , Modelos Biológicos , Modelos Estructurales , Vacuolas/enzimologíaRESUMEN
The mechanism(s) whereby membrane translocations are energized are poorly understood. Our work has focused on transmembrane microsomal and plasma membrane redox constituents as a means to energize membranes via alternative mechanisms complementary to ATP-driven processes. One such component is NADH-ascorbate free radical (mono- or semidehydroascorbate) oxidoreductase. This activity is associated with the trans or exit face of the Golgi apparatus, transport vesicles that move between the Golgi apparatus and the plasma membrane, and with the plasma membrane itself. Various lines of evidence, mostly indirect, link this activity to membrane translocations. Included is an apparent activation of the reductase in membranes when coated with clathrin, a single large polypeptide chain involved in exocytosis and in receptor-mediated and absorptive endocytosis. The results are consistent with a role of the ascorbate free radical as an acceptor for electron transport-mediated transfer of electrons from NADH perhaps to oxygen by coated membranes as a part of a mechanism to drive membrane translocations via generation of a proton gradient or of a membrane potential. Additionally, plasma membrane redox may be important in the regulation of cell growth, but a strict dependence on ascorbate free radical for the latter seems less likely than with internal endomembranes, where redox function may strictly depend upon the restricted pool of regeneratable acceptor that the ascorbate free radical provides.
Asunto(s)
Ácido Ascórbico/metabolismo , Membrana Celular/metabolismo , Metabolismo Energético , Animales , Fraccionamiento Celular , Clatrina/fisiología , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Transporte de Electrón , Retículo Endoplásmico/enzimología , Radicales Libres , Aparato de Golgi/enzimología , Histocitoquímica , Hígado/enzimología , Hígado/ultraestructura , Masculino , Microscopía Electrónica , NAD/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , RatasRESUMEN
An equilibrium kinetics model is proposed to described some of the enzymatic properties of the cyclic GMP-stimulated phosphodiesterase activity associated with brain clathrin coated vesicles. The model assumes the presence of pharmacologically distinct regulatory and catalytic domains in the enzyme. The model contemplates that random fashion occupancy of the regulatory site by the substrate, cyclic GMP, induces a conformational change which leads to the generation of an actived catalytic state. Therefore, cyclic GMP is a positive allosteric modulator of the coated vesicle enzyme. Experimental data revealed that occupancy or activation of the regulatory site was not essential for catalysis to occur since hydrolysis occurred after loss (200%) of the activation by cyclic GMP. This constitutes an example of non-essential substrate activation. Analysis of this PDE following activation by cGMP and after loss of the regulation, activation capacity of the enzyme allows the calculation of the various kinetic parameters inherent in the model.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Encéfalo/enzimología , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Modelos Biológicos , Regulación Alostérica , Animales , Encéfalo/ultraestructura , Bovinos , GMP Cíclico/metabolismo , Activación Enzimática , CinéticaAsunto(s)
Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , ATPasas de Translocación de Protón/aislamiento & purificación , Animales , Encéfalo/enzimología , Encéfalo/ultraestructura , Bovinos , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad/métodos , Clatrina/aislamiento & purificación , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Indicadores y Reactivos , Cinética , Peso Molecular , ATPasas de Translocación de Protón/metabolismo , Ultracentrifugación/métodosAsunto(s)
Invaginaciones Cubiertas de la Membrana Celular/enzimología , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Vacuolas/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Sustancias Macromoleculares , Modelos Estructurales , Datos de Secuencia Molecular , Vacuolas/ultraestructuraRESUMEN
Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , Animales , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , RatasRESUMEN
Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.
Asunto(s)
Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , ATPasas de Translocación de Protón/aislamiento & purificación , Animales , Encéfalo/enzimología , Encéfalo/ultraestructura , Bovinos , Electroforesis en Gel de Poliacrilamida , Etilmaleimida/farmacología , Peso MolecularRESUMEN
We have studied the polypeptide pattern of cathepsin D associated with coated vesicle fractions prepared from human placenta. In these fractions cathepsin D was about 35-fold enriched in the precursor polypeptides as compared to the unfractionated tissue extract. The enrichment was more prominent if the vesicles were fractionated in the presence of Triton X-100. Adsorption of exogenously added metabolically labelled cathepsin D precursor to the fractionated material was negligible. It is likely that the precursor and may be also the mature cathepsin D polypeptides are present in the matrix of the coated vesicles. This finding substantiates the idea that coated vesicles participate in the transport of newly synthesized cathepsin D into the lysosomes.
Asunto(s)
Catepsinas/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Endosomas/enzimología , Placenta/enzimología , Catepsina D , Catepsinas/aislamiento & purificación , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Femenino , Humanos , Lisosomas/enzimología , Microscopía Electrónica , EmbarazoRESUMEN
A cycle of clathrin assembly and disassembly drives the formation of coated vesicles, intermediates in intracellular protein transport. The heavy chain of clathrin is needed for assembly, but the function of the clathrin light chains has remained obscure. An enzyme has now been purified which uses ATP hydrolysis to power the release of clathrin from coated vesicles, presumably recycling the coat protein for repeated rounds of vesicle budding. This 'uncoating ATPase' requires clathrin light chains for its action.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Clatrina/fisiología , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Endosomas/fisiología , Animales , Bovinos , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Sustancias Macromoleculares , Peso MolecularRESUMEN
The polyhedral surface lattice of coated vesicles consists of three-legged hexameric protein complexes called triskelions which constitute the basic assembly unit. The triskelion is a molecular complex of molecular weight 630,000 (Mr 630K) composed of three clathrin heavy chains (subunit 180K) and three light chains (subunits 33K and 36K) (refs 2,3). The presence of additional coated vesicle-specific proteins in the 100-130K and 50-55K range have been reported. We previously described the presence of a cyclic nucleotide- and Ca2+-independent protein kinase activity in coated vesicles which was confirmed by others. This protein kinase specifically phosphorylates the 50K protein (pp50). In this report, we show that the coated vesicle kinase and its 50K protein substrate are part of a stable multimolecular system. In addition we show that the clathrin-light chain complex stimulates the pp50 phosphorylation and only light chains are implicated in this stimulation and that the pp50 phosphorylation does not seem to be affected by the vesicle.