1. Cérebro mitocondrial Isolation (Adaptado de Brown MR et al. 9) <ol><li> Sacrificar o rato usando métodos aprovados pelo Animal Care Institucional e Comitê de Uso (IACUC). </li><li> Corte a cabeça do animal por decapitação, cortar a pele e expor o crânio. </li><li> Abra o crânio delicadamente cortando com uma tesoura ou rongeur. Remover o cérebro. </li><li> Picar finamente o cérebro sem cerebelo em tampão de isolamento (ver Tabela 1) e transferi-lo para a 5 ml de vidro / teflon homogeneizador (veja lista de equipamentos). </li><li> Homogeneizar o tecido suavemente 10 vezes (sem bolhas), cerca de 5 min. </li><li> Centrifugar amostra a 1500 × g durante 10 min a 4 ° C numa centrífuga de bancada. </li><li> Salve o sobrenadante (mitocondrial e sinaptossomas) e descartar o pellet (material nuclear e de restos celulares). Centrifugar a 16.000 × g durante 10 min a 4 ° C numa centrífuga de bancada. </li><li> Descartarsobrenadante e re-suspensão da pelota em 500 ul de tampão de isolamento. Disrupt sinaptosomas com uma embarcação rompimento celular (veja lista de equipamentos). Aplicar uma pressão de 1200 psi, durante 10 min, seguido por descompressão rápida. </li><li> Camada a mistura em gradientes de Ficoll (ver Tabela 2), em lugar do rotor SW-50.1 e centrifugar a 126.500 x g durante 20 min a 4 ° C numa ultracentrifugadora (ver lista de equipamento). O sedimento é purificado a mitocôndria, a camada entre as diferentes densidades de Ficoll sinaptossomas é ininterrupto. </li><li> Lavar pelete por centrifugação em tampão de isolamento a 16.000 × g durante 10 min a 4 ° C numa centrífuga de bancada. </li></ol> 2. Submitocondriais vesículas (SMV) Isolamento (Adaptado de Chan et al. 10) <ol><li> Re-suspender as mitocôndrias em 200 ul de tampão de isolamento combinada com um volume igual de 1% de digitonina e permitir a sentar-se em gelo durante 15 min. </li><li> Adicionar mais isolamento Buffer e centrifugar a 16000 x g durante 10 min a 4 ° C numa centrífuga de bancada. Faça isso duas vezes. </li><li> Re-suspender o pellet em 200 mL tampão de isolamento e adicionar 2 mL de 10% Lubrol PX (C12E9). Misture e permitir a sentar-se no gelo durante 15 min. </li><li> Camada de mistura em tampão de isolamento, no lugar do rotor SW-50.1 e centrifugar a 182.000 x g a 4 ° C durante 1 hora. </li><li> Lavagem final dos aglomerados por centrifugação numa centrífuga de mesa superior em tampão de isolamento a 16.000 × g durante 10 min a 4 ° C. </li></ol> 3. A gravação eletrofisiológico <ol><li> Um equipamento típico electrofisiologia inclui um amplificador, um computador PC 1440A equipado com um interface de conversor de analógico para digital Digidata em conjunto com o software pClamp10.0, manipuladores, um microscópio, uma mesa de isolamento de vibrações, gaiola de Faraday. </li><li> Tubos capilares de vidro de borosilicato são inseridos em um Flaming / Brown Micropipeta Puller modelo P-87. Um programa de pipeta-extrator é otimizado paragerar pipetas com resistências entre 80 a 100 mohms. </li><li> SMVs são colocadas numa solução fisiológica intracelular (ATP é adicionado no momento apropriado, durante a gravação) (Tabela 3). Patch clamp pipetas são cheias com a mesma solução (sem ATP). As gravações são feitas através da formação de um selo giga-ohm para SMVs à temperatura ambiente. As vesículas são visualizadas por microscopia de contraste de fase com um microscópio invertido Nikon ou Zeiss. </li><li> O potencial de membrana é mantida a tensões que variam de -100 mV a +100 mV, por períodos de 10 segundos. As gravações são filtradas em 5 kHz utilizando os circuitos de amplificador. Nível de ruído elétrico ou não-específico de rua de menos de 1 pA são desejáveis ​​para a gravação bem sucedida. </li><li> Os dados são analisados ​​com o software Clampfit 10.0, por exemplo, para determinar a frequência e amplitude de eventos de canal único. Dependência de tensão é determinado traçando uma relação de tensão atual. </li></ol>

<ol>
	<li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Duchen, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+calcium+in+health+and+disease.">Mitochondria and calcium in health and disease.</a> Cell Calcium. 44, 1-5 (2008).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mitochondrial+membrane+permeability+in+pathogenesis,+autophagy+and+control+of+metabolism.">Modulation of mitochondrial membrane permeability in pathogenesis, autophagy and control of metabolism.</a> J. Gastroenterol. Hepatol. 22, S31-S37 (2007).</li><li>Cox, G. B., Jans, D. A., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis.+The+mechanism+of+ATP+synthase.+Conformational+change+by+rotation+of+the+beta-subunit.">Hypothesis. The mechanism of ATP synthase. Conformational change by rotation of the beta-subunit.</a> Biochim. Biophys. Acta. 768, 201-208 (1984).</li><li>Cox, G. B., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+ATP+synthase:+a+reassessment+of+the+functions+of+the+b+and+a+subunits.">The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits.</a> Biochim. Biophys. Acta. 849, 62-69 (1986).</li><li>Cory, S., Huang, D. C., Adams, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bcl-2+family:+roles+in+cell+survival+and+oncogenesis.">The Bcl-2 family: roles in cell survival and oncogenesis.</a> Oncogene. 22, 8590-8607 (2003).</li><li>Vander Heiden, M. G., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+proteins:+regulators+of+apoptosis+or+of+mitochondrial+homeostasis.">Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis.</a> Nat. Cell Biol. 1, 209-216 (1999).</li><li>Alavian, K. N., Li, H., Collis, L., Bonanni, L., Zeng, L., Sacchetti, S., Lazrove, E., Nabili, P., Flaherty, B., Graham, M., Chen, Y., Messerli, S. M., Mariggio, M. A., Rahner, C., McNay, E., Shore, G. C., Smith, P. J. S., Hardwick, J. M., Jonas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13 (10), 1224-1233 (2011).</li><li>Brown, M. R., Sullivan, P. G., Dorenbos, K. A., Modafferi, E. A., Geddes, J. W., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+disruption+of+synaptoneurosomes:+an+alternative+method+to+isolate+brain+mitochondria.">Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.</a> Journal of Neuroscience Methods. 137, 299-303 (2004).</li><li>Chan, T. L., Greenawalt, J. W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+ultrastructural+properties+of+a+mitochondrial+inner+membrane+fraction+deficient+in+outer+membrane+and+matrix+activities.">Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.</a> J. Cell Biol. 45 (2), 291-305 (1970).</li><li>Young, H. K. o., Delannoy, M., Hullihen, J., Chiu, W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP+Synthasomes.">Mitochondrial ATP Synthasomes.</a> J. Biol. Chem. 278 (14), 12305-12309 (2003).</li></ol>

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Os métodos aqui descritos permitem o isolamento de mitocôndrias puro no fim da etapa 1 e vesículas (submitocondriais SMVs) após a etapa 2 a partir de cérebro inteiro sem distinção de célula phenotypes.SMVspurified por este método são essencialmente isentos de contaminação por outros organelos subcelulares, como mostrado na A Figura 1 e o nosso trabalho anterior (Alavian KN et al. 8) e mantêm a sua integridade estrutural e funcional antes da congelação. Após congelaç?...

<table><tbody><tr><td>Potter-Elvehjem Tissue Grinder withPTFEPestle</td><td>Krackeler Scientific, Inc.</td><td>1-7725T-5</td><td></td></tr><tr><td>Eppendorf Centrifuge 5424 </td><td>Eppendorf</td><td>5424 000.410</td><td></td></tr><tr><td>4639 Cell Disruption Vessel</td><td>Parr Instrument Company</td><td>4639</td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>F5415</td><td></td></tr><tr><td>Polycarbonate centrifuge tubes</td><td>Beckman Coulter</td><td>P20314</td><td></td></tr><tr><td>SW-50.1 rotor </td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>L8-70M Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>D5628</td><td></td></tr><tr><td>Lubrol PX (C12E9) </td><td>Calbiochem</td><td>205534</td><td></td></tr><tr><td>Axopatch 200B </td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Digidata 1440A </td><td>Molecular Device</td><td></td><td></td></tr><tr><td>pClamp10.0</td><td>Molecular Device</td><td></td><td></td></tr><tr><td>Manipulator</td><td>Sutter Instrument</td><td></td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>World Precision Instruments</td><td>1308325</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller Model P-87</td><td>Sutter Instrument</td><td></td><td></td></tr></tbody></table>

f1fo atpase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes

As mitocôndrias são envolvidos em várias funções celulares importantes incluindo o metabolismo, uma sobrevivência, desenvolvimento e, a sinalização de cálcio 2. Duas das mais importantes funções mitocondriais estão relacionadas com a produção eficiente de ATP, a moeda de energia da célula, pela fosforilação oxidativa, e na mediação de sinais para a morte programada das células 3. A enzima primariamente responsável pela produção de ATP é a F1FO-ATP sintase, também chamada ATP sintase 4-5. Nos últimos anos, o papel da mitocôndria na morte celular por apoptose e necrose tem recebido uma atenção considerável. Na morte celular por apoptose, Bcl-2, tais como as proteínas da família Bax entrar na membrana mitocondrial externa, e oligomerizar permeabilizar a membrana externa, libertando factores pró-apoptóticos para o citosol 6. Na morte celular necrótica clássico, tal como a produzida por isquemia ou excitotoxicidade em neurónios, um large, aumento pouco regulada em matriz de cálcio contribui para a abertura de um poro da membrana interior, a poro de transição da permeabilidade mitocondrial ou PTPm. Este despolariza a membrana interna e provoca deslocamentos osmóticos, contribuindo para a ruptura da membrana externa, a libertação de factores pró-apoptóticos e disfunção metabólica. Muitas proteínas, incluindo Bcl-xL 7 interagir com F1FO ATP sintase, modulando a sua função. Bcl-xL, interage directamente com a subunidade beta da sintase ATP F1FO, e esta interacção diminui uma condutância de vazamento dentro do F1FOATPasecomplex, aumentando a rede de transporte de H + por F1FO durante F1FO ATPase 8 e aumentando assim a eficiência mitocondrial. Para o estudo da atividade e modulação da ATP sintase, que isolado do cérebro de roedores submitocondriais vesículas (SMVs) contendo F1FO ATPase. Os SMVs manter a integridade estrutural e funcional do F1FO ATPase como mostrado na Alavian et ai. Aqui, nós descrevemos um métodoque temos utilizado com sucesso no isolamento de SMVs de cérebro de rato e delinear a técnica de patch clamp para analisar a actividade de canal (ião vazamento condutância) dos SMVs.

O primeiro passo do nosso protocolo permite o isolamento de mitocôndrias purificadas como mostrado por Western blot na figura 1. Na Figura 2 é mostrado um exemplo de uma gravação de remendo vesícula submitocondriais derivado do cérebro. Usando a configuração de patch de dentro para fora, demonstramos a atividade do canal modulado pela ATP. O controle (CTL) de gravação (esquerda) mostra a atividade multi-canal de condutância com uma condutância pico de 600 pS em média. que f...

Watch this Scientific Journal Video about F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes at JoVE.com

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Um método para isolar vesículas submitocondriais enriquecidas em F1FO ATP sintase a partir de complexos de cérebro de rato é descrito. Essas vesículas permite o estudo da actividade de F1FO complexo ATPase ea sua modulação por meio da técnica de gravação de patch clamp.

<sub&gt; 1</sub&gt; F<sub&gt; Ó</sub&gt; ATPase Vesicle Preparação e Técnica para Execução de Remendo Recordings Grampo de membranas de vesículas submitocondriais

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

f1fo-atpase-vesicle-preparation-technique-for-performing-patch-clamp

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

10.2K visualizações.  Yale University. Um método para isolar vesículas submitocondriais enriquecidas em F1FO ATP sintase a partir de complexos de cérebro de rato é descrito. Essas vesículas permite o estudo da actividade de F1FO complexo ATPase ea sua modulação por meio da técnica de gravação de patch clamp.

Vídeo: F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Livre de detergente ultra reconstituição das proteínas de membrana em lípidos Bilayers usando Fusogenic Proteoliposomes complementares carregada.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Bioquímica

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

Isolamento de F1-ATPase do Protista parasita Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

Remodelação de membrana das vesículas gigantes em resposta a gradientes de íons cálcio localizadas

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Química

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Uma plataforma de membrana modelo para reconstituir a dinâmica da membrana mitocondrial

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Avaliação da Localização da Proteína Subocondrial em Budding Levedura Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Isolamento de locais de contato mitocondrial

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Autor em destaque: Revelando locais de contato mitocondrial e insights arquitetônicos 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Um método aprimorado para isolar locais de contato mitocondrial

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparação, purificação e utilização de lipossomas contendo ácido graxo

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Bioengenharia

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstituição de um canal de Kv para membranas lipídicas de Estudos estruturais e funcionais

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

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Reconstituição de um canal de Kv para membranas lipídicas de Estudos estruturais e funcionais