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1.
Biochim Biophys Acta Biomembr ; 1861(10): 183021, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31306626

RESUMEN

OmpG is a general diffusion pore in the E. coli outer membrane with a molecular architecture comprising a 14-stranded ß-barrel scaffold and unique structural features. In contrast to other non-specific porins, OmpG lacks a central constriction zone and has an exceptionally wide pore diameter of about 13 Å. The equatorial plane of OmpG harbors an annulus of four alternating basic and acidic patches whose function is only poorly characterized. We have investigated the role of charge distribution for ion selectivity and sugar transport with the help of OmpG variants mutated in the annulus. Substituting the glutamate residues of the annulus for histidines or alanines led to a strong reduction in cation selectivity. Replacement of the glutamates in the annulus by histidine residues also disfavored the passage of pentoses and hexoses relative to disaccharides. Our results demonstrate that despite the wide pore diameter, an annulus only consisting of two opposing basic patches confers reduced cation and monosaccharide transport compared to OmpG wild type. Furthermore, randomization of charged residues in the annulus had the potential to abolish pH-dependency of sugar transport. Our results indicate that E15, E31, R92, R111 and R211 in the annulus form electrostatic interactions with R228, E229 and D232 in loop L6 that influence pH-dependency of sugar transport.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Porinas/química , Arginina/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Ácido Glutámico/metabolismo , Concentración de Iones de Hidrógeno , Porinas/genética , Porinas/metabolismo , Porinas/fisiología , Especificidad por Sustrato/fisiología , Azúcares/metabolismo
2.
J Biol Chem ; 291(34): 17848-60, 2016 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-27339897

RESUMEN

Chloroplasts and mitochondria are unique endosymbiotic cellular organelles surrounded by two membranes. Essential metabolic networking between these compartments and their hosting cells requires the exchange of a large number of biochemical pathway intermediates in a directed and coordinated fashion across their inner and outer envelope membranes. Here, we describe the identification and functional characterization of a highly specific, regulated solute channel in the outer envelope of chloroplasts, named OEP40. Loss of OEP40 function in Arabidopsis thaliana results in early flowering under cold temperature. The reconstituted recombinant OEP40 protein forms a high conductance ß-barrel ion channel with subconductant states in planar lipid bilayers. The OEP40 channel is slightly cation-selective PK+/PCl- ≈ 4:1 and rectifying (i⃗/i⃖ ≅ 2) with a slope conductance of Gmax ≅ 690 picosiemens. The OEP40 channel has a restriction zone diameter of ≅1.4 nm and is permeable for glucose, glucose 1-phosphate and glucose 6-phosphate, but not for maltose. Moreover, channel properties are regulated by trehalose 6-phosphate, which cannot permeate. Altogether, our results indicate that OEP40 is a "glucose-gate" in the outer envelope membrane of chloroplasts, facilitating selective metabolite exchange between chloroplasts and the surrounding cell.


Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Cloroplastos/química , Cloroplastos/química , Membranas Intracelulares/química , Proteínas de la Membrana/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Glucosa/química , Glucosa/genética , Glucosa/metabolismo , Membranas Intracelulares/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo
3.
J Biol Chem ; 290(30): 18621-35, 2015 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-26085089

RESUMEN

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca(2+) leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca(2+) leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic ß-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca(2+) imaging to monitor the effects on ER Ca(2+) leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca(2+) leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca(2+) leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic ß-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease.


Asunto(s)
Diabetes Mellitus/genética , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Señalización del Calcio/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Chaperón BiP del Retículo Endoplásmico , Silenciador del Gen , Proteínas del Choque Térmico HSP40/genética , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Proteínas de la Membrana/genética , Ratones , Unión Proteica , Transporte de Proteínas , Canales de Translocación SEC
4.
Biochim Biophys Acta ; 1850(4): 602-11, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25484312

RESUMEN

BACKGROUND: Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers. METHODS: Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped. RESULTS: Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers. CONCLUSIONS: Allicin increases the permeability of membranes. GENERAL SIGNIFICANCE: Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicin's ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.


Asunto(s)
Beta vulgaris/efectos de los fármacos , Chara/efectos de los fármacos , Commelinaceae/efectos de los fármacos , Ajo/química , Membrana Dobles de Lípidos/metabolismo , Ácidos Sulfínicos/farmacología , Beta vulgaris/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Chara/metabolismo , Commelinaceae/metabolismo , Dimetilsulfóxido/farmacología , Disulfuros , Pigmentos Biológicos/metabolismo , Ácidos Sulfínicos/farmacocinética
5.
Eur J Cell Biol ; 90(9): 721-30, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21684628

RESUMEN

Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.


Asunto(s)
Membranas Intracelulares/metabolismo , Membrana Dobles de Lípidos/metabolismo , Orgánulos/metabolismo , Proteínas/metabolismo , Humanos , Transporte de Proteínas
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