Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 75
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Cell Sci ; 132(4)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670475

RESUMO

The endoplasmic reticulum (ER) is a major membrane-bound organelle in all eukaryotic cells. This organelle comprises morphologically distinct domains, including the nuclear envelope and peripheral sheets and tubules. The tubules are connected by three-way junctions into a network. Several membrane proteins have been implicated in network formation; curvature-stabilizing proteins generate the tubules themselves, and membrane-anchored GTPases fuse tubules into a network. Recent experiments have shown that a tubular network can be formed with reconstituted proteoliposomes containing the yeast membrane-fusing GTPase Sey1 and a curvature-stabilizing protein of either the reticulon or REEP protein families. The network forms in the presence of GTP and is rapidly disassembled when GTP hydrolysis of Sey1 is inhibited, indicating that continuous membrane fusion is required for its maintenance. Atlastin, the ortholog of Sey1 in metazoans, forms a network on its own, serving both as a fusion and curvature-stabilizing protein. These results show that the reticular ER can be generated by a surprisingly small set of proteins, and represents an energy-dependent steady state between formation and disassembly. Models for the molecular mechanism by which curvature-stabilizing proteins cooperate with fusion GTPases to form a reticular network have been proposed, but many aspects remain speculative, including the function of additional proteins, such as the lunapark protein, and the mechanism by which the ER interacts with the cytoskeleton. How the nuclear envelope and peripheral ER sheets are formed remain major unresolved questions in the field. Here, we review reconstitution experiments with purified curvature-stabilizing proteins and fusion GTPases, discuss mechanistic implications and point out open questions.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Proteolipídeos/metabolismo , Citoesqueleto de Actina/ultraestrutura , Fenômenos Biomecânicos , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Fusão de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Membrana Nuclear/ultraestrutura , Proteolipídeos/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo
2.
Methods Mol Biol ; 1805: 51-71, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971712

RESUMO

F-type adenosine triphosphate (ATP) synthase is a membrane-bound macromolecular complex, which is responsible for the synthesis of ATP, the universal energy source in living cells. This enzyme uses the proton- or sodium-motive force to power ATP synthesis by a unique rotary mechanism and can also operate in reverse, ATP hydrolysis, to generate ion gradients across membranes. The F1Fo-ATP synthases from bacteria consist of eight different structural subunits, forming a complex of ∼550 kDa in size. In the bacterium Ilyobacter tartaricus the ATP synthase has the stoichiometry α3ß3γδεab2c11. This chapter describes a wet-lab working protocol for the purification of several tens of milligrams of pure, heterologously (E. coli-)produced I. tartaricus Na+-driven F1Fo-ATP synthase and its subsequent efficient reconstitution into proteoliposomes. The methods are useful for a broad range of subsequent biochemical and biotechnological applications.


Assuntos
Bioquímica/métodos , Fusobactérias/enzimologia , ATPases Translocadoras de Prótons/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Cromatografia de Afinidade , Escherichia coli/enzimologia , Hidrólise , Plasmídeos/genética , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , ATPases Translocadoras de Prótons/ultraestrutura
3.
Structure ; 26(1): 161-170.e3, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29249608

RESUMO

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems.


Assuntos
Membrana Celular/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/ultraestrutura , Proteínas de Membrana Transportadoras/ultraestrutura , Proteolipídeos/ultraestrutura , Membrana Celular/química , Membrana Celular/metabolismo , Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Detergentes , Tomografia com Microscopia Eletrônica/instrumentação , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Ressonância Magnética Nuclear Biomolecular/instrumentação , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de Proteína , Proteolipídeos/química , Proteolipídeos/metabolismo
4.
Biochim Biophys Acta Biomembr ; 1860(2): 378-383, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28993151

RESUMO

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.


Assuntos
Microscopia Crioeletrônica/métodos , Bicamadas Lipídicas/química , Maleatos/química , Proteínas de Membrana/química , Poliestirenos/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/ultraestrutura , Conformação Proteica , Multimerização Proteica , Proteolipídeos/química , Proteolipídeos/ultraestrutura
5.
Proc Natl Acad Sci U S A ; 114(34): 9110-9115, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28739947

RESUMO

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.


Assuntos
Proteínas de Fusão de Membrana/metabolismo , Fusão de Membrana , Neurônios/metabolismo , Membranas Sinápticas/metabolismo , Animais , Cálcio/metabolismo , Microscopia Crioeletrônica/métodos , Proteínas de Fusão de Membrana/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios Proteicos , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptotagmina I/química , Sinaptotagmina I/metabolismo
6.
Biochim Biophys Acta Bioenerg ; 1858(9): 763-770, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28634030

RESUMO

The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from Escherichia coli, cytochrome bo3, for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055-16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P-side of single HCOs. Proton transport activity of cytochrome bo3 was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH6.4-8.4, while proton release at the P-side had an optimum pH of ~7.4, suggesting that the pH optimum is related to proton release from the proton exit site. Our previous single-enzyme experiments identified rare, long-lived conformation states of cytochrome bo3 where protons leak back under turn-over conditions. Here, we analyzed and found that ~23% of cytochrome bo3 proteoliposomes show ΔpH half-lives below 50s after stopping turnover, while only ~5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo3 exhibit such fast decays. These single-enzyme results confirm our model in which HCO exhibit heterogeneous pumping rates and can adopt rare leak states in which protons are able to rapidly flow back.


Assuntos
Citocromos/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Proteolipídeos/metabolismo , Bombas de Próton/metabolismo , Transporte Biológico , Grupo dos Citocromos b , Citocromos/genética , Técnicas Eletroquímicas/instrumentação , Transporte de Elétrons , Escherichia coli/enzimologia , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Corantes Fluorescentes , Lipossomos/metabolismo , Microscopia de Fluorescência , Oxirredução , Proteolipídeos/ultraestrutura , Bombas de Próton/genética , Prótons
7.
Nature ; 543(7644): 257-260, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28225760

RESUMO

Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation, but how exactly they mediate network formation and whether they are all required are unclear. Here we reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p (refs 6, 7) and the curvature-stabilizing protein Yop1p (refs 8, 9) from Saccharomyces cerevisiae form a tubular network upon addition of GTP. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favours the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon and receptor expression-enhancing proteins (REEPs) from different species. Atlastin, the vertebrate orthologue of Sey1p, forms a GTP-hydrolysis-dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.


Assuntos
Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Hidrólise/efeitos dos fármacos , Fusão de Membrana , Proteínas de Membrana Transportadoras/ultraestrutura , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Proteínas de Transporte Vesicular/ultraestrutura
8.
ACS Nano ; 10(1): 556-61, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26615669

RESUMO

Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-to-ATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control.


Assuntos
Trifosfato de Adenosina/biossíntese , Materiais Biomiméticos/química , Complexo de Proteína do Fotossistema II/química , Proteolipídeos/química , ATPases Translocadoras de Prótons/química , Prótons , Materiais Biomiméticos/metabolismo , Cloroplastos/química , Cloroplastos/efeitos da radiação , Cloroplastos/ultraestrutura , Luz , Microesferas , Fotofosforilação/efeitos da radiação , Complexo de Proteína do Fotossistema II/metabolismo , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , ATPases Translocadoras de Prótons/metabolismo , Termodinâmica
9.
Biochim Biophys Acta ; 1848(2): 643-53, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25450342

RESUMO

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/química , Hidrogéis/química , Modelos Estatísticos , Proteolipídeos/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Animais , Ligação Competitiva , Transporte Biológico , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Difusão , Corantes Fluorescentes , Humanos , Cinética , Lepidópteros/química , Técnicas de Patch-Clamp , Ligação Proteica , Proteolipídeos/ultraestrutura , Rodamina 123 , Sefarose/química , Transgenes , Verapamil/farmacologia
10.
J Biol Chem ; 289(43): 29912-26, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25160627

RESUMO

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41(int)-Cys) and show that it folds into an elongated ∼ 12-nm-long extended structure based on small angle x-ray scattering data. Gp41(int)-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41(int)-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140(CA018) in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140(CA018) was higher than that induced by gp41(int)-Cys, the majority of animals immunized with gp41(int)-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/imunologia , Feminino , Cobaias , Proteína gp41 do Envelope de HIV/química , Humanos , Soros Imunes/imunologia , Imunização , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Difração de Nêutrons , Estrutura Terciária de Proteína , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Espalhamento a Baixo Ângulo
11.
Plant Cell ; 25(7): 2661-78, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23839788

RESUMO

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Cloroplastos/ultraestrutura , Immunoblotting , Membranas Intracelulares/ultraestrutura , Lipídeos/análise , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Fosforilação , Fotossíntese , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tilacoides/ultraestrutura
12.
Elife ; 1: e00109, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23240085

RESUMO

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 µM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001.


Assuntos
Cálcio/metabolismo , Fusão de Membrana , Proteolipídeos/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/metabolismo , Potenciais de Ação , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Transporte Biológico , Cálcio/farmacologia , Expressão Gênica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteolipídeos/ultraestrutura , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Proteína 25 Associada a Sinaptossoma/genética , Sinaptotagmina I/genética , Sintaxina 1/genética , Sintaxina 1/metabolismo , Fatores de Tempo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo
13.
J Cell Mol Med ; 15(1): 31-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20716122

RESUMO

Since the discovery and implication of N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full-length recombinant t-SNAREs and v-SNARE present in opposing liposomes, leading to the assembly of a t-/v-SNARE ring complex. Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution. Similar to the t-/v-SNARE ring complex formed when 50 nm v-SNARE liposomes meet a t-SNARE-reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t-SNARE-reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.


Assuntos
Membrana Celular/metabolismo , Neurônios/metabolismo , Proteínas SNARE/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Bicamadas Lipídicas , Lipossomos , Fusão de Membrana , Microscopia de Força Atômica , Neurônios/ultraestrutura , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Proteínas SNARE/química , Proteínas SNARE/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura
14.
FEBS Lett ; 584(20): 4339-43, 2010 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-20875821

RESUMO

Soybean nodulin 26 (nod26), a member of the aquaporin superfamily, is the major protein component of the symbiosome membrane that encloses nitrogen-fixing bacteroids in root nodules. Previous work has demonstrated that nod26 facilitates the transport of water and glycerol, although a potential additional role as a channel for fixed ammonia efflux has been hypothesized. In the present study it is shown that recombinant nod26 reconstituted into proteoliposomes facilitates NH(3) transport in an Hg(2+)-sensitive manner with a reduced activation energy, hallmarks of protein-facilitated transport characteristic of aquaporins. Comparison of the predicted single-channel transport rates of nod26 suggests a 4.9-fold preference for ammonia compared to water.


Assuntos
Amônia/metabolismo , Aquaporinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Aquaporinas/genética , Transporte Biológico/efeitos dos fármacos , Western Blotting , Clonagem Molecular , Cinética , Proteínas de Membrana/genética , Cloreto de Mercúrio/farmacologia , Microscopia Eletrônica de Transmissão , Permeabilidade/efeitos dos fármacos , Proteínas de Plantas/genética , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/metabolismo , Água/metabolismo
15.
Biophys J ; 99(2): 553-60, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20643074

RESUMO

Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE)-mediated lipid mixing can be efficiently recapitulated in vitro by the incorporation of purified vesicle membrane (-v) SNARE and target membrane (t-) SNARE proteins into separate liposome populations. Despite the strong correlation between the observed activities in this system and the known SNARE physiology, some recent works have suggested that SNARE-mediated lipid mixing may be limited to circumstances where membrane defects arise from artifactual reconstitution conditions (such as nonphysiological high-protein concentrations or unrealistically small liposome populations). Here, we show that the previously published strategies used to reconstitute SNAREs into liposomes do not significantly affect either the physical parameters of the proteoliposomes or the ability of SNAREs to drive lipid mixing in vitro. The surface density of SNARE proteins turns out to be the most critical parameter, which controls both the rate and the extent of SNARE-mediated liposome fusion. In addition, the specific activity of the t-SNARE complex is significantly influenced by expression and reconstitution protocols, such that we only observe optimal lipid mixing when the t-SNARE proteins are coexpressed before purification.


Assuntos
Lipídeos/química , Proteínas SNARE/metabolismo , Animais , Microscopia Crioeletrônica , Cinética , Fusão de Membrana , Camundongos , Peso Molecular , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Ratos , Proteínas SNARE/ultraestrutura , Propriedades de Superfície
16.
Methods Mol Biol ; 606: 351-61, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20013407

RESUMO

Atomic Force Microscopy (AFM) is a useful tool for the visualization of soft biological samples in a nanoscale resolution. In the study presented here, the surface morphology ofP-selectin and Transferrin modified proteoliposomes were investigated in air and under water. The proteins were visualized without pre-functionalization or staining.


Assuntos
Microscopia de Força Atômica/métodos , Proteolipídeos/ultraestrutura , Lipossomos/química , Lipossomos/ultraestrutura , Selectina-P/química , Proteolipídeos/química , Propriedades de Superfície , Transferrina/química
17.
Proc Natl Acad Sci U S A ; 106(42): 17626-33, 2009 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-19826089

RESUMO

Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.


Assuntos
Fusão de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Adenosina Trifosfatases/metabolismo , Guanosina Trifosfato/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas de Transporte Vesicular/metabolismo
18.
J Cell Mol Med ; 13(10): 4161-5, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19737333

RESUMO

To enable fusion between biological membranes, t-SNAREs and v-SNARE present in opposing bilayers, interact and assemble in a circular configuration forming ring-complexes, which establish continuity between the opposing membranes, in presence of calcium ions. The size of a t-/v-SNARE ring complex is dictated by the curvature of the opposing membrane. Hence smaller vesicles form small SNARE-ring complexes, as opposed to large vesicles. Neuronal communication depends on the fusion of 40-50 nm in diameter membrane-bound synaptic vesicles containing neurotransmitters at the nerve terminal. At the presynaptic membrane, 12-17 nm in diameter cup-shaped neuronal porosomes are present where synaptic vesicles transiently dock and fuse. Studies demonstrate the presence of SNAREs at the porosome base. Atomic force microscopy (AFM), electron microscopy (EM), and electron density measurement studies demonstrate that at the porosome base, where synaptic vesicles dock and transiently fuse, proteins, possibly comprised of t-SNAREs, are found assembled in a ring conformation. To further determine the structure and arrangement of the neuronal t-/v-SNARE complex, 50 nm t-and v-SNARE proteoliposomes were mixed, allowing t-SNARE-vesicles to interact with v-SNARE vesicles, followed by detergent solubilization and imaging of the resultant t-/v-SNARE complexes formed using both AFM and EM. Our results demonstrate formation of 6-7 nm membrane-directed self-assembled t-/v-SNARE ring complexes, similar to, but twice as large as the ring structures present at the base of neuronal porosomes. The smaller SNARE ring at the porosome base may reflect the 3-4 nm base diameter, where 40-50 nm in diameter v-SNARE-associated synaptic vesicle transiently dock and fuse to release neurotransmitters.


Assuntos
Membrana Celular/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Proteínas SNARE/química , Microscopia de Força Atômica , Neurônios/ultraestrutura , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas SNARE/ultraestrutura
19.
Nature ; 459(7250): 1091-7, 2009 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-19458617

RESUMO

Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.


Assuntos
Endossomos/fisiologia , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cricetinae , Citosol/metabolismo , Cães , Endossomos/metabolismo , Humanos , Microscopia Eletrônica , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/metabolismo
20.
J Struct Biol ; 160(3): 324-31, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17977016

RESUMO

Screening trials to determine the presence of two-dimensional (2D) protein crystals suitable for three-dimensional structure determination using electron crystallography is a very labor-intensive process. Methods compatible with fully automated screening have been developed for the process of crystal production by dialysis and for producing negatively stained grids of the resulting trials. Further automation via robotic handling of the EM grids, and semi-automated transmission electron microscopic imaging and evaluation of the trial grids is also possible. We, and others, have developed working prototypes for several of these tools and tested and evaluated them in a simple screen of 24 crystallization conditions. While further development of these tools is certainly required for a turn-key system, the goal of fully automated screening appears to be within reach.


Assuntos
Cristalografia/métodos , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica de Transmissão/métodos , Robótica/instrumentação , Automação , Cristalização , Proteínas de Membrana/química , Microscopia Eletrônica de Transmissão/instrumentação , Coloração Negativa/métodos , Proteolipídeos/ultraestrutura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA