Asunto(s)
Proteínas Portadoras/química , Membrana Celular/metabolismo , Proteínas Fúngicas/química , Fosfatos/metabolismo , Simportadores de Protón-Fosfato , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Simportadores , Secuencia de Aminoácidos , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Fosfatos/deficiencia , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Proteínas Cotransportadoras de Sodio-FosfatoRESUMEN
Hydropathy analysis of the amino acid sequence of the Pho84 phosphate permease of Saccharomyces cerevisiae suggests that the protein consists of 12 transmembrane domains connected by hydrophilic loops. The Pho84 protein has been modified by a gene fusion approach, yielding two different N-terminal His-tagged chimeras which can be expressed in Escherichia coli, purified and functionally reconstituted into defined proteoliposomes. The continuous epitopes in the N- and C-terminal sequences of the Pho84 chimeras were shown to be accessible in proteoliposomes containing the purified active Pho84 proteins. Site-specific proteolysis of the immunoreactive N-terminal sequence in the reconstituted protein suggests a unidirectional insertion into liposomes.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Fúngicas/química , Proteínas de Transporte de Membrana/química , Simportadores de Protón-Fosfato , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Aminoácidos/química , Transporte Biológico Activo , Proteínas Portadoras/farmacocinética , Membrana Celular/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteolípidos/metabolismo , Proteínas Recombinantes de Fusión/química , Factores de TiempoRESUMEN
The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Simportadores de Protón-Fosfato , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Transporte Biológico , Proteínas Portadoras/biosíntesis , Cationes Bivalentes , Ácido Edético , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Liposomas , Potenciales de la Membrana , Proteínas de Unión a Fosfato , Fosfatos/metabolismo , PlásmidosRESUMEN
The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane. Hydropathy analysis suggests a secondary structure arrangements of the proteins in 12 transmembrane domains. The derepressible Pho84 and Pho89 transporters appear to have characteristic similarities with the phosphate transporters of Neurospora crassa. The Pho84 protein catalyzes a proton-coupled phosphate transport at acidic pH, while the Pho89 protein catalyzes a sodium-dependent phosphate uptake at alkaline pH. The Pho84 transporter can be stably overproduced in the cytoplasmic membrane of Escherichia coli, purified and reconstituted in a functional state into proteoliposomes.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Fosfato , Simportadores de Protón-Fosfato , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Simportadores , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo , Proteínas Portadoras/química , Proteínas Fúngicas/química , Humanos , Proteínas de Transporte de Membrana/química , Datos de Secuencia Molecular , Organofosfatos/metabolismo , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIIRESUMEN
Membrane vesicles with an inside-out orientation were isolated from the plasma membrane of Saccharomyces cerevisiae by an improved aqueous two-phase partitioning technique. The activity of the orthovanadate-sensitive H+-pumping ATPase, the plasma membrane marker, was highly enriched by the partitioning technique. The obtained results suggest that the membrane vesicles produced were predominantly oriented inside-out. The isolated plasma membrane vesicles displayed cross-reactions with antibodies raised against synthetic peptide corresponding to the N-terminal (residues 1-10) and the C-terminal (residues 578-597) regions of the plasma membrane phosphate transporter encoded by the PHO84 gene and the H+-pumping ATPase of S. cerevisiae. The purified membrane vesicles catalyzed a derepressible inhibitor-sensitive phosphate uptake at levels comparable with the situation in intact cells of S. cerevisiae indicating that transport of phosphate across the membrane is both functional and bidirectional. The PHO84 transporter harbored in isolated plasma membranes could moreover be enriched in a high state of purity by immunoaffinity chromatography using immobilized anti-PHO84 antibodies.
Asunto(s)
Proteínas Portadoras/metabolismo , ATPasas de Translocación de Protón/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Anticuerpos , Transporte Biológico , Western Blotting , Proteínas Portadoras/análisis , Fraccionamiento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Complejo IV de Transporte de Electrones/metabolismo , Cinética , Datos de Secuencia Molecular , NADPH-Ferrihemoproteína Reductasa/metabolismo , Orgánulos/metabolismo , Orgánulos/ultraestructura , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas de Unión a Fosfato , Fosfatos/metabolismo , Saccharomyces cerevisiae/ultraestructura , Vanadatos/farmacologíaRESUMEN
The plasma membrane high-affinity phosphate permease of Saccharomyces cerevisiae has been overproduced as a stable membrane-bound chimeric protein in Escherichia coli. Construction of a chimera between the permease and a peptide containing 10 consecutive histidine residues allowed selective binding of the chimera to a chelating column charged with Ni2+, and elution with imidazole in a high state of purity. Approximately 5 mg purified His10-permease was obtained from 3 g (wet mass) cells. The purified phosphate permease chimera catalyzes uncoupler-sensitive phosphate transport after reconstitution into proteoliposomes.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Fosfato , Saccharomyces cerevisiae/genética , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Recombinante , Escherichia coli/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de Transporte de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Bacterial membranes from Klebsiella pneumoniae were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Inverted membrane vesicles derived from these cells catalyzed a reduction of NAD+ or 3-acetylpyridine-NAD+ by NADPH, which showed a maximal activity of about 260 nmoles/minute per milligram protein at pH 7-8. In the presence of a protonic uncoupler the specific activity was stimulated about two-fold in this pH range. The presence of detergents did not further increase the specific activity of enzyme. The Klebsiella pneumoniae transhydrogenase activity was sensitive to phenylarsine oxide and palmityl-Coenzyme A, both of which are agents known to inhibit the mammalian enzyme. The Ki-value for palmityl-Coenzyme A with respect to NADPH was about 1.25 microM. Antibodies raised against beef heart transhydrogenase crossreacted with a 54 kD protein in the Klebsiella pneumonia membrane.