RESUMEN

Simulations of lipid membranes typically make use of periodic boundary conditions to mimic macroscopically sized membranes and allow for comparison to experiments performed e.g. on planar lipid membranes or on unilamellar lipid vesicles. However, the lateral periodicity partly suppresses membrane fluctuations or membrane remodeling, processes that are of particular importance in the study of asymmetric membranes-i.e. membranes with integral or associated proteins and/or asymmetric lipid compositions. Here, we devised a simple albeit powerful lipid bicelle model system that (i) displays similar structural, dynamical, and mechanical properties compared to infinite periodic lipid membrane systems and allows (ii) for the study of asymmetric lipid bilayer systems and (iii) the unperturbed formation of local spontaneous curvature induced by lipids or proteins in molecular dynamics simulations. In addition, the system is characterized by largely unbiased thermal fluctuations as opposed to standard bilayer systems. Application of the bicelle system for an asymmetric lipid composition resembling the plasma membrane reveals that the cholesterol density for a tension-free plasma membrane with a vanishing spontaneous curvature is larger by 28% within the extracellular leaflet compared to the cytosolic leaflet.

Asunto(s)

Membrana Dobles de Lípidos , Simulación de Dinámica Molecular , Membrana Dobles de Lípidos/química , Membrana Celular/química , Liposomas Unilamelares , Modelos Biológicos

RESUMEN

The (local) curvature of cellular membranes acts as a driving force for the targeting of membrane-associated proteins to specific membrane domains, as well as a sorting mechanism for transmembrane proteins, e.g., by accumulation in regions of matching spontaneous curvature. The latter measure was previously experimentally employed to study the curvature induced by the potassium channel KvAP and by aquaporin AQP0. However, the direction of the reported spontaneous curvature levels as well as the molecular driving forces governing the membrane curvature induced by these integral transmembrane proteins could not be addressed experimentally. Here, using both coarse-grained and atomistic molecular dynamics (MD) simulations, we report induced spontaneous curvature values for the homologous potassium channel Kv 1.2/2.1 Chimera (KvChim) and AQP0 embedded in unrestrained lipid bicelles that are in very good agreement with experiment. Importantly, the direction of curvature could be directly assessed from our simulations: KvChim induces a strong positive membrane curvature (≈0.036 nm-1) whereas AQP0 causes a comparably small negative curvature (≈-0.019 nm-1). Analyses of protein-lipid interactions within the bicelle revealed that the potassium channel shapes the surrounding membrane via structural determinants. Differences in shape of the protein-lipid interface of the voltage-gating domains between the extracellular and cytosolic membrane leaflets induce membrane stress and thereby promote a protein-proximal membrane curvature. In contrast, the water pore AQP0 displayed a high structural stability and an only faint effect on the surrounding membrane environment that is connected to its wedge-like shape.

Asunto(s)

Acuaporinas , Simulación de Dinámica Molecular , Acuaporinas/química , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/metabolismo , Transporte de Proteínas

RESUMEN

BACKGROUND & AIMS: Mycobacterium bovis Bacillus Calmette-Guérin (BCG), killed by extended freeze-drying (EFD), induces secretion of interleukin-10 and reduces lung inflammation in a mouse model of asthma. We investigated the effects of EFD BCG in mouse models of inflammatory bowel disease. METHODS: EFD BCG was administered subcutaneously to mice with colitis induced by dextran sodium sulfate (DSS), oxazolone, or adoptive transfer of CD4(+)CD45RB(high)Foxp3(-) T cells from C57Bl/6 Foxp3GFP mice to RAG2(-/-) mice. RESULTS: EFD BCG, administered either before induction of DSS and oxazolone colitis or after development of acute or chronic DSS-induced colitis, reduced symptom scores, loss of body weight, and inflammation. Although transfer of CD4(+)CD45RB(high)Foxp3(-) cells induced colitis in RAG2(-/-) mice, administration of EFD BCG at the time of the transfer converted Foxp3(-) T cells to Foxp3(+) T cells and the mice did not develop colitis. EFD BCG protected mice from colitis via a mechanism that required expansion of T regulatory cells and production of interleukin-10 and transforming growth factor ß. EFD BCG activated the retinoid X receptor (RXR)-α-peroxisome proliferator-activated receptor (PPAR)-γ heterodimer, blocked translocation of nuclear factor κB to the nucleus, and reduced colonic inflammation; it did not increase the number of colon tumors that formed in mice with chronic DSS-induced colitis. CONCLUSIONS: EFD BCG controls severe colitis in mice by expanding T regulatory cell populations and PPAR-γ and might be developed to treat patients with inflammatory bowel disease.

Asunto(s)

Vacuna BCG/farmacología , Colitis/prevención & control , Colon/metabolismo , Factores de Transcripción Forkhead/metabolismo , Mycobacterium bovis , Linfocitos T Reguladores/metabolismo , Animales , Vacuna BCG/administración & dosificación , Colitis/inducido químicamente , Colitis/inmunología , Colon/patología , Sulfato de Dextran , Liofilización , Interleucina-1/sangre , Interleucina-10/metabolismo , Interleucina-6/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Oxazolona , PPAR gamma/metabolismo , Peroxidasa/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Pérdida de Peso

RESUMEN

The proton-translocating plant vacuolar H(+)-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells.

Asunto(s)

Arabidopsis/enzimología , Transferencia Resonante de Energía de Fluorescencia/métodos , Mesembryanthemum/enzimología , ATPasas de Translocación de Protón/metabolismo , Protoplastos/metabolismo , Vacuolas/metabolismo , Arabidopsis/genética , Mesembryanthemum/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

BACKGROUND: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. RESULTS: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. CONCLUSIONS: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

Asunto(s)

Caryophyllaceae/citología , Proteínas de Plantas/análisis , Fracciones Subcelulares/enzimología , ATPasas de Translocación de Protón Vacuolares/análisis , Secuencia de Aminoácidos , Arabidopsis , Caryophyllaceae/enzimología , Caryophyllaceae/genética , ADN Complementario/genética , Retículo Endoplásmico/enzimología , Epítopos/análisis , Transferencia Resonante de Energía de Fluorescencia , Inmunohistoquímica , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Cebollas/citología , Hojas de la Planta/citología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/análisis , Estructura Terciaria de Proteína , Subunidades de Proteína , Protoplastos , Proteínas Recombinantes de Fusión/análisis , Saccharomyces cerevisiae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/inmunología , Zea mays/citología , Zea mays/enzimología

RESUMEN

Plant cells are characterized by a highly active secretory system that includes the large central vacuole found in most differentiated tissues. The plant vacuolar H+-ATPase plays an essential role in maintaining the ionic and metabolic gradients across endomembranes, in activating transport processes and vesicle dynamics, and, hence, is indispensable for plant growth, development, and adaptation to changing environmental conditions. The review summarizes recent advances in elucidating the structure, subunit composition, localization, and regulation of plant V-ATPase. Emerging knowledge on subunit isogenes from Arabidopsis and rice genomic sequences as well as from Mesembryanthemum illustrates another level of complexity, the regulation of isogene expression and function of subunit isoforms. To this end, the review attempts to define directions of future research on plant V-ATPase.

Asunto(s)

Proteínas de Plantas/química , ATPasas de Translocación de Protón Vacuolares/química , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Proteínas de Plantas/genética , Subunidades de Proteína/síntesis química , Subunidades de Proteína/genética , ATPasas de Translocación de Protón Vacuolares/genética

RESUMEN

The vacuolar-type ATPase (V-ATPase) and the vacuolar H(+)-pyrophosphatase are electrogenic proton pumps at plant endomembranes that create the proton motive force required for secondary activated transport and metabolite accumulation during development and adaptation to a variety of adverse growth conditions. Twelve distinct vacuolar H(+)-ATPase (VHA) subunits are suggested to constitute the functional V-ATPase complex. Starting from the available expressed sequence tag (EST) sequences and by homology screening, the complete set of 12 VHA subunits was cloned as cDNAs from the halophyte Mesembryanthemum crystallinum, vha-A-H, -a,-c, -d and -e. Transcript levels of all 12 VHA subunits as well as of tonoplast pyrophosphatase and P-ATPase were analysed in root and leaf tissue under conditions of osmotic (700 mM mannitol), heat and cold stress, and salinity. Distinct coordinated changes of stress-induced expression were observed for most subunits in roots and leaves, with mostly paralleled changes in transcript levels of all subunits. In some cases, contrasting responses were seen for vha-B and -c transcript amounts.

Asunto(s)

Mesembryanthemum/enzimología , ATPasas de Translocación de Protón Vacuolares/genética , Secuencia de Aminoácidos , Clonación Molecular , Mesembryanthemum/genética , Datos de Secuencia Molecular , ATPasas de Translocación de Protón Vacuolares/biosíntesis