RESUMEN
After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2-VDAC complex, which leads to an accumulation of ROS.
Asunto(s)
Plasmodium falciparum/crecimiento & desarrollo , Protoporfirinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de GABA/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD34/metabolismo , Transporte Biológico , Diferenciación Celular , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Células Eritroides/citología , Células Eritroides/metabolismo , Perfilación de la Expresión Génica , Glutatión/metabolismo , Humanos , Ligandos , Espectrometría de Masas , Parásitos/crecimiento & desarrollo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de GABA/química , Receptores de GABA/genética , Canales Aniónicos Dependientes del Voltaje/química , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed.
Asunto(s)
Retículo Endoplásmico/enzimología , Nucleotidasas/metabolismo , Glándula Submandibular/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidrólisis , Inmunohistoquímica , Cinética , Magnesio/metabolismo , Masculino , Manganeso/metabolismo , Microscopía Electrónica , Microsomas/enzimología , Nucleotidasas/antagonistas & inhibidores , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Glándula Submandibular/efectos de los fármacos , Uridina Difosfato/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
We report the case of an 83-year-old French woman with multiple melanomas showing a severe DNA repair deficiency, corrected after transfection by XPC cDNA. Two biallelic mutations in the XPC gene are reported: an inactivating frameshift mutation in exon 15 (c.2544delG, p.W848X) and a missense mutation in exon 11 (c.2108 C>T, P703L). We demonstrate that these new mutations are involved in the DNA repair deficiency and confirm the diagnosis of xeroderma pigmentosum from complementation group C (XP-C). We speculate that the coexistence of a MC1R variant may be involved in the phenotype of multiple melanomas and that the unusual long-term survival may be related to a lower ultraviolet radiation exposure and to a regular clinical follow-up. This patient appears to be the first French Caucasian XP-C case and one of the oldest living patients with XP reported worldwide.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Mutación del Sistema de Lectura/genética , Melanoma/genética , Mutación Missense/genética , Neoplasias Primarias Múltiples/genética , Neoplasias Cutáneas/genética , Xerodermia Pigmentosa/genética , Anciano de 80 o más Años , Femenino , Humanos , Melanoma/patología , Neoplasias Primarias Múltiples/patología , Fenotipo , Neoplasias Cutáneas/patología , Sobrevivientes , Población Blanca , Xerodermia Pigmentosa/patologíaRESUMEN
Vasoactive intestinal peptide (VIP) exerts many biological functions through interaction with the VPAC1 receptor, a class II G protein-coupled receptor. Photoaffinity labeling studies associated with receptor mapping and three-dimensional molecular modeling demonstrated that the central part of VIP (6-24) interacts with the N-terminal ectodomain of VPAC1 receptor. However, the domain of the VPAC1 receptor interacting with the C-terminus of VIP is still unknown. A photoaffinity probe, Bpa28-VIP, was synthetized by substitution of amidated Asn28 of VIP by amidated photoreactive para-benzoyl-L-Phe (Bpa). Bpa28-VIP was shown to be a hVPAC1 receptor agonist in CHO cells expressing the recombinant VPAC1 receptor. After obtaining a covalent 125I-[Bpa28-VIP]/hVPAC1 complex, it was cleaved by CNBr, PNGase F, and endopeptidase Glu-C and the cleavage products were analyzed by electrophoresis. The data demonstrated that 125I-[Bpa28-VIP] was covalently bonded to the 121-133 fragment within the N-terminal ectodomain of the receptor. This fragment is adjacent to those covalently attached to the central part (6-24) of VIP.
Asunto(s)
Fragmentos de Péptidos/farmacología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Cricetinae , Humanos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Unión Proteica , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/químicaRESUMEN
Germline anomalies of the INK4a-ARF and Cdk4 genes were sought in a series of 89 patients suspected of having a genetic predisposition to melanoma. Patients were selected based on the following criteria: (a) familial melanoma (23 cases), (b) multiple primary melanoma (MPM; 18 cases), (c) melanoma and additional unrelated cancers (13 cases), (d) age at diagnosis less than 25 years (21 cases), and (e) nonphoto-induced melanoma (NPIM; 14 cases). Mutations of INK4a-ARF and Cdk4 were characterised by automated sequencing, and germline deletions of INK4a-ARF were also examined by real-time quantitative PCR. Seven germline changes of INK4a-ARF, five of which were novel, were found in seven patients (8%). Four were very likely to be pathogenic mutations and were found in three high-risk melanoma families and in a patient who had a pancreatic carcinoma in addition to melanoma. Three variants of uncertain significance were detected in one MPM patient, one patient <25 years, and one NPIM patient. No germline deletion of INK4a-ARF was found in 71 patients, and no Cdk4 mutation was observed in the 89 patients. This study confirms that INK4a-ARF mutations are infrequent outside stringent familial criteria, and that germline INK4a-ARF deletions are rarely involved in genetic predisposition to melanoma.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Melanoma/genética , Neoplasias Cutáneas/genética , Proteína p14ARF Supresora de Tumor/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Análisis Mutacional de ADN , ADN de Neoplasias , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Linaje , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Neoplasias Cutáneas/patologíaRESUMEN
Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.
Asunto(s)
Proteínas Portadoras/metabolismo , Cloruros/metabolismo , Mucosa Gástrica/metabolismo , Receptores de GABA-A/metabolismo , Animales , Benzodiazepinonas/metabolismo , Electrofisiología , Mucosa Gástrica/fisiología , Mucosa Gástrica/ultraestructura , Inmunohistoquímica , Isoquinolinas/metabolismo , Ligandos , Masculino , Microscopía Inmunoelectrónica , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/fisiología , Células Parietales Gástricas/ultraestructura , Ensayo de Unión Radioligante , Ratas , Ratas WistarAsunto(s)
ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Sitios de Unión , Citoplasma/enzimología , Cinética , Modelos Moleculares , Fosforilación , Estructura Secundaria de Proteína , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.
Asunto(s)
Receptores de GABA-A/química , Animales , Benzodiazepinonas/metabolismo , Colesterol/metabolismo , Cromatografía , Detergentes/química , Escherichia coli , Técnica de Fractura por Congelación , Isoquinolinas/metabolismo , Ligandos , Membrana Dobles de Lípidos/química , Ratones , Tamaño de la Partícula , Porinas/metabolismo , Unión Proteica/fisiología , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestructura , Proteolípidos/química , Proteolípidos/ultraestructura , Ensayo de Unión Radioligante , Receptores de GABA-A/metabolismo , Receptores de GABA-A/ultraestructura , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , Dodecil Sulfato de Sodio/química , Canales Aniónicos Dependientes del VoltajeRESUMEN
The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, J.-J. & McIntosh, D. B. (2000) J. Biol. Chem. 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved.
Asunto(s)
ATPasas Transportadoras de Calcio/química , Animales , ATPasas Transportadoras de Calcio/metabolismo , ATPasas Transportadoras de Calcio/ultraestructura , Cristalización , Estabilidad de Enzimas , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Cinética , Fosforilación , Vanadatos/farmacologíaRESUMEN
After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Fluoresceína-5-Isotiocianato , Fosfoproteínas/metabolismo , Retículo Sarcoplasmático/enzimología , Transporte Biológico Activo , Polaridad Celular , Citosol , Estabilidad de Enzimas , Fluorescencia , Modelos Químicos , Organofosfatos/metabolismo , Fosfatos/metabolismo , EspectrofotometríaRESUMEN
When gastric H(+),K(+)-ATPase-containing microsomes are solubilized by detergents, a rapid loss of ATPase activity is generally observed. In this article, SDS/PAGE of octa(ethylene glycol)dodecyl monoether (C(12)E(8))- and n-dodecyl beta-d-maltoside-solubilized microsomes and their purifications by affinity chromatography on Reactive Red column reveal that inactivation is due to two main effects. (i) Solubilization activates an aspartic protease that cleaves down the alpha-subunit of the H(+),K(+)-ATPase. Addition of pepstatin A at slightly acidic pH and at low temperature prevents the proteolysis. (ii) A too-harsh delipidation inactivates the ATPase. When n-dodecyl-beta-d-maltoside is the detergent, the soluble H(+), K(+)-ATPase is highly active (2.5 micromol/mg per h at pH 6.0 and 5 degrees C) as long as ATP is added. When C(12)E(8) is used, the detergent induces an inactivation due to delipidation, since addition of lipids restores activity. The two subunits of the H(+), K(+)-ATPase are present in equimolar ratio in the n-dodecyl beta-d-maltoside-purified complex. Moreover, two main types of complex (330 and 660 kDa) were resolved in non-denaturing gels and should be the dimeric (alphabeta)(2) and tetrameric (alphabeta)(4) heterodimers respectively. In conclusion, purification of active, stable, soluble complexes of H(+),K(+)-ATPase with few lipids (a lipid/protein ratio of 0.25, w/w) has been achieved. This material should be useful for further structural studies.
Asunto(s)
Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , Adenosina Trifosfato/farmacología , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Cromatografía de Afinidad , Detergentes , Estabilidad de Enzimas , Glicoles de Etileno/farmacología , Glucósidos/farmacología , Microsomas/enzimología , Pepstatinas/farmacología , Inhibidores de Proteasas , Solubilidad , Porcinos , TemperaturaRESUMEN
Thin three-dimensional crystals of the cytochrome b6 f complex from the unicellular algae Chlamydomonas reinhardtii have been grown by BioBeads-mediated detergent removal from a mixture of protein and lipid solubilized in Hecameg. Frozen-hydrated crystals, exhibiting p22121 plane group symmetry, were studied by electron crystallography and a projection map at 9 A resolution was calculated. The crystals (unit cell dimensions of a=173.5 A, b=70.0 A and gamma=90.0 degrees) showed the presence of dimers, and within each monomer 14 domains of electron density were observed. The combination of the projection map obtained from ice-embedded crystals of cytochrome b6 f with a previous map obtained from negatively stained samples brings new insight in the organization of the complex. For example, it distinguishes some peaks and/or domains that are only extramembrane or transmembrane, and reveals the possible localization of single-stranded transmembrane alpha-helices (Pet subunits). Furthermore, the cross-correlation of our projection map from frozen hydrated samples with the atomic model of the transmembrane part of the cytochrome bc1 complex has allowed us to localize the cytochrome b6 at the dimer interface and to reveal structural differences between the two complexes.
Asunto(s)
Chlamydomonas reinhardtii , Grupo Citocromo b/ultraestructura , Citocromos/ultraestructura , Animales , Cristalografía , Complejo de Citocromo b6f , Citocromos f , Transporte de Electrón , Electrones , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , FotosíntesisRESUMEN
By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.
Asunto(s)
ATPasas Transportadoras de Calcio/aislamiento & purificación , Animales , Fenómenos Biofísicos , Biofisica , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/ultraestructura , Cristalización , Detergentes/aislamiento & purificación , Lípidos , Micelas , Microscopía Electrónica , Poliestirenos , Conformación Proteica , Retículo Sarcoplasmático/enzimologíaRESUMEN
We have developed a novel technique which allows one to direct the two dimensional motion of actin filaments on a myosin coated sheet using a weak electric field parallel to the plane of motion. The filament velocity can be increased or decreased, and even reversed, as a function of orientation and strength of the field. PMMA (poly(methylmethacrylate)) gratings, which act as rails for actin, allow one for the first time to explore three quadrants of the force velocity diagram. We discuss effective friction, duty ratio and stall force at different myosin densities. A discontinuity in the velocity force relationship suggests the existence of dynamical phase transition.
Asunto(s)
Actinas/química , Actinas/fisiología , Animales , Fenómenos Biomecánicos , Fenómenos Biofísicos , Biofisica , Estimulación Eléctrica , Técnicas In Vitro , Microscopía Fluorescente , Movimiento/fisiología , Miosinas/química , Miosinas/fisiología , Polimetil MetacrilatoRESUMEN
We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals. For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein. This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein. When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure. For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent. Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy. Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/química , Canales Iónicos/química , Proteínas Bacterianas/química , Cristalización , Detergentes/metabolismo , Electrofisiología , Canales Iónicos/ultraestructura , Liposomas/metabolismo , Microscopía Electrónica , Técnicas de Placa-Clamp , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.
Asunto(s)
Proteínas Portadoras/metabolismo , Aparato de Golgi/metabolismo , Cinesinas/metabolismo , Proteínas de Unión al GTP rab , Proteínas ras/metabolismo , Adenosina Trifosfatasas/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Retículo Endoplásmico/metabolismo , Aparato de Golgi/química , Aparato de Golgi/ultraestructura , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Cinesinas/análisis , Cinesinas/química , Cinesinas/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Datos de Secuencia Molecular , Peso MolecularRESUMEN
In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.
Asunto(s)
ATPasas Transportadoras de Calcio/aislamiento & purificación , Animales , Fenómenos Biofísicos , Biofisica , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/ultraestructura , Cristalización , Detergentes , Técnicas In Vitro , Lípidos/análisis , Microscopía Electrónica , Proteínas/análisis , Conejos , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/enzimología , SolubilidadRESUMEN
This work establishes the potential of Bio-Beads as a simple alternative to conventional dialysis for removing detergent and for obtaining 2D crystals of integral membrane proteins useful for structure analysis by electron crystallography. Kinetic and equilibrium aspects of removal of different detergents by adsorption onto hydrophobic Bio-Beads SM2 have been systematically investigated and extended to 2D crystallization of different prototypic membrane proteins, including: (a) Ca2+ ATPase from sarcoplasmic reticulum; (b) melibiose permease from Escherichia coli; (c) cytochrome b6f from Chlamydomonas reinhardtii. Different crystals could be produced from all protein preparations, with optical diffraction down to 20-25 A in negative stain.