1. Cervello mitocondriale Isolamento (Adattato da Brown MR et al. 9) <ol><li> Sacrifica il topo utilizzando metodi approvati dalla cura degli animali e del Comitato Istituzionale Usa (IACUC). </li><li> Tagliare la testa dell&#39;animale per decapitazione, tagliare la pelle ed esporre il cranio. </li><li> Aprire delicatamente il cranio, tagliando con una forbice o rongeur. Rimuovere il cervello. </li><li> Tritate finemente il cervello senza cervelletto in isolamento Buffer (vedi tabella 1) e trasferirlo in un ml di vetro / teflon omogeneizzatore 5 (vedi elenco attrezzature). </li><li> Omogeneizzare tessuti delicatamente 10 volte (senza bollicine), circa 5 min. </li><li> Centrifugare campione a 1500 xg per 10 min a 4 ° C in una centrifuga da banco. </li><li> Salvare il surnatante (mitocondriale e sinaptosomi) e scartare il pellet (materiale nucleare e detriti cellulari). Centrifugare a 16.000 xg per 10 min a 4 ° C in una centrifuga da banco. </li><li> Scartaresurnatante e risospendere pellet in 500 ml di tampone di isolamento. Disrupt sinaptosomi con una nave distruzione cellulare (vedere inventario). Applicare una pressione di 1200 psi per 10 minuti, seguita da una rapida decompressione. </li><li> Strato il composto su Ficoll gradienti (vedi Tabella 2), posto in SW-50.1 rotore e centrifugare a 126.500 g per 20 minuti a 4 ° C in un ultracentrifuga (vedi elenco attrezzature). Il pellet è mitocondri purificato, lo strato tra le diverse densità Ficoll è sinaptosomi undisrupted. </li><li> Lavare pellet mediante centrifugazione in isolamento Tampone a 16.000 xg per 10 min a 4 ° C in una centrifuga da banco. </li></ol> 2. Submitocondriali vescicole (SMV) Isolamento (Adattato da Chan et al. 10) <ol><li> Risospendere mitocondri in 200 microlitri di buffer di isolamento combinato con un uguale volume di 1% digitonina e lasciar riposare in ghiaccio per 15 min. </li><li> Aggiungere più Isolamento Buffer e centrifugare a 16.000 xg per 10 min a 4 ° C in una centrifuga da banco. Fate questo due volte. </li><li> Risospendere pellet in 200 microlitri di buffer di isolamento e aggiungere 2 ml di 10% Lubrol PX (C12E9). Mescolare e lasciar riposare in ghiaccio per 15 min. </li><li> Miscela strato sul buffer di isolamento, posto in SW-50.1 rotore e centrifugare a 182.000 xg a 4 ° C per 1 ora. </li><li> Lavare pellet finale mediante centrifugazione in una centrifuga da tavolo in isolamento Tampone a 16.000 xg per 10 min a 4 ° C. </li></ol> 3. Registrazione elettrofisiologica <ol><li> Un tipico impianto di elettrofisiologia include un amplificatore, un computer PC dotato di un Digidata 1440A interfaccia convertitore analogico-digitale in combinazione con il software pClamp10.0, manipolatori, un microscopio, un tavolo isolamento dalle vibrazioni, gabbia di Faraday. </li><li> Borosilicato capillari di vetro sono inserite in un Flaming / Brown Micropipetta Puller modello P-87. Un programma di pipetta-estrattore è ottimizzato pergenerare pipette con resistenze tra 80 a 100 MW. </li><li> SMVs vengono poste in una soluzione fisiologica intracellulare (ATP viene aggiunto al momento opportuno durante la registrazione) (Tabella 3). Patch pipette clamp sono riempite con la stessa soluzione (senza ATP). Le registrazioni sono fatte da formare una guarnizione giga-ohm sul SMVs a temperatura ambiente. Le vescicole sono visualizzabili al microscopio a contrasto di fase con un microscopio invertito Nikon o Zeiss. </li><li> Il potenziale di membrana è mantenuta una tensione da -100 mV a + 100 mV per periodi di 10 secondi. Registrazioni vengono filtrati a 5 kHz utilizzando il circuito dell&#39;amplificatore. Livelli di rumore elettrico o non specifici stray inferiore a 1 pA sono desiderabili per la registrazione di successo. </li><li> Dati vengono analizzati con il software Clampfit 10,0 per esempio per determinare la frequenza e l&#39;ampiezza di eventi singolo canale. Dipendenza di tensione è determinato tracciando un rapporto di tensione di corrente. </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>

Nessun conflitto di interessi dichiarati.

I metodi qui descritti permettono l&#39;isolamento di puro mitocondri alla fine del passaggio 1 e vescicole submitocondriali (SMVs) dopo il passo 2 dal cervello intero senza distinzione di cella phenotypes.SMVspurified da questo metodo sono essenzialmente esente da contaminazione da altri organelli subcellulari come mostrato nella figura 1 e nostro precedente lavoro (Alavian KN et al. 8) e mantengono la loro integrità strutturale e funzionale prima del congelamento. Dopo il congelam...

<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

I mitocondri sono coinvolti in molte importanti funzioni cellulari tra cui il metabolismo, la sopravvivenza a 1, di sviluppo e, di segnalazione di calcio 2. Due delle più importanti funzioni mitocondriali sono legati alla produzione efficiente di ATP, la moneta energetica della cellula, dalla fosforilazione ossidativa e la mediazione di segnali per morte cellulare programmata 3. L&#39;enzima principale responsabile della produzione di ATP sintasi è il F1FO-ATP, chiamato anche ATP sintasi 4-5. Negli ultimi anni, il ruolo dei mitocondri nella morte cellulare per apoptosi e necrotiche ha ricevuto notevole attenzione. In morte cellulare per apoptosi, Bcl-2 proteine ​​della famiglia come Bax entrare la membrana mitocondriale esterna, oligomerize e permeabile la membrana esterna, rilasciando fattori pro-apoptotici nel citosol 6. In classico necrosi cellulare, come quello prodotto da ischemia o eccitotossicità nei neuroni, un large, aumento insufficiente regolazione in matrice calcio contribuisce all&#39;apertura di un poro membrana interna, la permeabilità mitocondriale poro di transizione o mPTP. Questo depolarizza la membrana interna e provoca cambiamenti osmotici, contribuendo alla rottura della membrana esterna, rilascio di fattori pro-apoptotici, e disfunzione metabolica. Molte proteine ​​tra cui Bcl-xL 7 interagiscono con F1FO ATP sintasi, modulando la sua funzione. Bcl-xL interagisce direttamente con la subunità beta della F1FO ATP sintasi, e questa interazione decresce una conduttanza di perdita entro il F1FOATPasecomplex, aumentando il trasporto netto di H + da F1FO durante F1FO ATPasi 8 e aumentando quindi l&#39;efficienza mitocondriale. Per studiare l&#39;attività e la modulazione della ATP sintasi, abbiamo isolato dal cervello di roditori submitocondriali vescicole (SMVs) contenenti F1FO ATPasi. Le SMVs mantengono l&#39;integrità strutturale e funzionale del F1FO ATPasi come mostrato in Alavian et al. Qui, descriviamo un metodoche abbiamo usato con successo per l&#39;isolamento di SMVs da cervello di ratto e noi delineare la tecnica patch clamp per analizzare l&#39;attività del canale (conduttanza perdita ion) delle SMVs.

Il primo passo del nostro protocollo consente l&#39;isolamento di mitocondri purificati come mostrato mediante Western blot in Figura 1. In figura 2 è mostrato un esempio di un derivato dal cervello submitocondriali registrazione cerotto vescicola. Utilizzando la configurazione di patch dentro-fuori dimostriamo attività del canale modulato da ATP. Il controllo (CTL) di registrazione (a sinistra) mostra l&#39;attività del canale multi-conduttanza con una conducibilità massima di 600 ...

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

Un metodo per isolare vescicole submitocondriali arricchiti in F1FO ATP sintasi complessi da cervello di ratto è descritto. Queste vescicole permettono lo studio dell&#39;attività del complesso F1FO ATPasi e la sua modulazione utilizzando la tecnica di patch clamp di.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPasi Vesicle Preparazione e tecnica per permettere Bloccare Registrazioni patch di membrane delle vescicole submitocondriali

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 Views.  Yale University. Un metodo per isolare vescicole submitocondriali arricchiti in F1FO ATP sintasi complessi da cervello di ratto è descritto. Queste vescicole permettono lo studio dell'attività del complesso F1FO ATPasi e la sua modulazione utilizzando la tecnica di patch clamp di.

Video: 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.

Privo di detersivo ultraveloce ricostituzione delle proteine di membrana in lipidici utilizzando fusogena proteoliposomi complementari a carico.

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

Ricerca

Biochimica

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 di F1-ATPasi dal Protista parassita 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.

Rimodellamento della membrana di vescicole giganti in risposta a gradienti di ioni calcio localizzata

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

Chimica

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.

Una piattaforma di membrana modello per la ricostituzione delle dinamiche della membrana mitocondriale

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.

Valutazione della localizzazione della proteina submitocondriale nel lievito in erba Saccharomyces cerevisiae

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

Isolamento dei siti di contatto mitocondriali

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.

Autore in primo piano: Svelare i siti di contatto mitocondriali e le intuizioni architettoniche 

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.

Un metodo migliorato per isolare i siti di contatto mitocondriali

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.

Preparazione, purificazione e l'uso dei liposomi contenenti acidi grassi

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

Bioingegneria

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.

La ricostituzione di un canale Kv in membrane lipidiche per gli studi strutturali e funzionali

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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La ricostituzione di un canale Kv in membrane lipidiche per gli studi strutturali e funzionali