Este trabalho foi financiado por uma bolsa de investigação a partir da combinação Escola Nacional de Pesquisa holandesa, Catalysis Controlled by Design Chemical (NRSC-Catálise). Dr. HY Steensma e Dr. GPH van Heusden da Universidade de Leiden são reconhecidos por seu apoio com a expressão recombinante de S. cerevisiae Nar1.

1. Preparação da instalação e Soluções <ol><li> Preparar a solução de tampão contendo Tris 100 mM, NaCl 250 mM e 10% de glicerol a pH 8,5. Eliminar o ar da tampão lavando com argônio. Introduzir o buffer em uma câmara anaeróbica. </li><li> Divida o tampão em duas porções. A uma porção (tampão A) adicionar 1 mM DL-ditiotreitol (DTT, M r 154,25 g / mol) e 1 mM de L-cisteína. A outra porção (tampão B) não contêm agentes redutores. </li><li> Preparar soluções oxidantes e redutoras. Adicione 1 ml de soluções de 2, 20, e ferricianeto de potássio 200 mM (FIC, M r 329,26 g / mol) e de ditionito de sódio (SD, M r 174,11 g / mol) em tampão B. anaeróbio </li><li> Preparar a mistura mediador como se segue: <ol><li> Adicionar 160 | iM de cada um dos seguintes mediadores redox e NaCl a 50 mM para o tampão B: N, N, N &#39;, N&#39; -tetrametil-p-phenylendediamine (TMPD, M r 237,17 g / mol, E &#39;0 0,276 V) , 2,6-diclorofenol indofenol(DCIP, M r 290,08 g / mol, E &#39;0 0,217 V), fenazino etossulfato (PES, M r 334,4 g / mol, E&#39; 0 0,055 V), azul de metileno (M r 319,85 g / mol, E &#39; 0 0,011 V), Resorufina (Mr 235,17 g / mol, E &#39;0 -0,051 V), Indigodisulphonate (Mr 466,35 g / mol, E&#39; 0 -0,125 V), 2-hidroxi-1,4-naphtaquinone ( M r 174,15 g / mol, E &#39;0 -0,145 V), antraquinona-2-sulfonato de (Mr 328,28 g / mol, E&#39; 0 -0,225 V), Phenosafranin (M r 322,8 g / mol, E &#39;0 -0,252 V), safranina O (Mr 350,84 g / mol, E &#39;0 -0,280 V), vermelho neutro (M r 288,8 g / mol, E&#39; 0 -0,340 V), Benzil viologen (M r 409,4 g / mol, E &#39;0 -0,350 V), e metil viologen (M r 257,16 g / mol, E&#39; 0 -0,440 V). </li><li> Enrole a garrafa em uma folha de alumínio para proteger o mediadors da luz. </li></ol></li><li> Prepare a 10 mg / ml de S. proteína Nar1 marcado com strep cerevisiae (181? M, M r 55,2 kDa) em tampão A. Expresso S. cerevisiae Nar1 em E. coli como uma proteína marcado com estreptococos utilizando um vector de expressão pET24a. Purifica-se a proteína numa câmara anaeróbica utilizando meios de cromatografia Strep-tactin (IBA) e purifica-se utilizando um sistema de purificação de proteína, tais como purificador Akta (GE Healthcare). </li><li> Preparar a titulação redox configuração numa câmara anaeróbia como se segue: <ol><li> Conectar um eléctrodo de referência de Ag / AgCl e um eléctrodo de fio de platina para o recipiente de titulação. Ligue ambos os eletrodos para um voltímetro que pode medir de mV até V. </li><li> Insira uma pequena barra de agitação. </li><li> Coloque o recipiente de titulação acima de um agitador magnético. </li><li> Encher o reservatório de titulação com Tampão B, solução mistura mediador e a solução de proteína até 2 ml, ou seja, para concentrações finais de proteína de 72 uM e 38 uM de cadamediador. Nota: dependendo da concentração de proteína e o mediador necessário concentrações das quantidades dos três soluções deve ser ajustado. </li><li> Agita-se durante 30 min e ligar o voltímetro. </li></ol></li><li> Prepare 10 de quartzo tubos EPR vidro limpo. Introduzir os tubos na câmara anaeróbia. </li><li> Encha uma pequena (cerca de 200 ml) Dewar nitrogênio líquido com nitrogênio líquido. </li></ol> 2. Execute Redox Titulação <ol><li> Espere até que o potencial da solução é estável. Nota: Na prática, isto significa que o potencial trações menos de 1 mV por minuto. </li><li> Anote o potencial dado pelo voltímetro. </li><li> Desenhe a primeira amostra de 200 mL e injetar em um tubo de EPR. </li><li> Tapar o tubo com uma rolha de borracha e retirar o tubo da câmara de anaerobiose. </li><li> Congelar o tubo em azoto líquido como se segue: Cuidado! Isto tem de ser feito lentamente, de outra forma o tubo vai quebrar. <ol><li> Primeiro insira o tip do tubo em azoto líquido. </li><li> Espere até que um som de assobio pode ser ouvido. Nota: Isto pode levar até 10 segundos. </li><li> Lentamente inserir o resto do tubo de modo que a parte da amostra está totalmente submerso. </li></ol></li><li> Adicionar uma pequena alíquota 1-2 ul de solução de 2 mM de SD para a solução no recipiente de titulação, para reduzir o potencial da solução para -0.6 V. Nota: Se mais do que 10 ul tem que ser adicionado, ligar-se à solução 20 mM. Como é muito difícil poise a um valor potencial específico, os valores reais registadas no voltímetro são anotados e utilizados para construir a curva de titulação. </li><li> Desenhe uma amostra conforme descrito na etapa 2,3-2,5. </li><li> Anote o potencial real dado pelo voltímetro. </li><li> Adicionar oxidante até que o potencial aumentou em cerca de 50 mV. Começar por adicionar uma pequena alíquota 1-2 ul da solução FIC 2 mM. Se mais do que 10 ul tem que ser adicionado, ligar-se à solução 20 mM. </li><li> Anote how muito das soluções foi adicionado. Nota: Isto é necessário para corrigir a diluição da amostra durante a titulação. </li><li> Desenhar uma amostra, tal como descrito no 2,3-2,5 passos. </li><li> Anote o potencial real dado pelo voltímetro. </li><li> Repita os passos 2,9 e 2,12, até a faixa de potencial de -0,6 V a 0,2 V tem sido coberto e todos os 10 tubos EPR estão cheios cada um com 200 mL. Observação: Como o volume com cada retirada é reduzido o posicionamento dos eléctrodos podem necessitar de ser ajustadas ter um bom contacto com a solução sem interferência da agitação. Pode ser difícil medir o potencial após a retirada da última amostra e, portanto, o potencial de que a amostra pode ser menos exactas. </li><li> Opcionalmente, armazenar as amostras EPR congelados em nitrogênio líquido até que as medidas de RPE pode ter lugar. </li></ol> 3. medir amostras titulação utilizando EPR Espectroscopia e Análise de Dados <ol><li> Grave EPR espectros do diferenteamostras de titulação da seguinte forma: <ol><li> Usar uma bomba de alto vácuo para evacuar um criostato de quartzo a &lt;-5 10 bar. </li><li> Ligue o resfriamento de água do ímã EPR. Abra o fluxo de ar seco através da cavidade de RPE. </li><li> Ligue a fonte de alimentação para o ímã e o computador do EPR. </li><li> Executar um programa de calibração de frequência. Defina os parâmetros de medição EPR. Ligue o criostato para o hélio líquido dewar. Arrefecer a cavidade para 9-16 K. </li><li> Introduzir a amostra na cavidade de RPE. Grave o espectro EPR. </li></ol></li><li> Identificar as diferentes espécies de EPR e utilizar o melhor para quantificar os espectros de sinais. Alcançar EPR quantificação por dupla integração dos espectros e comparação com o espectro de uma solução padrão de cobre externa CuSO 10 mM HCl 10/04 mM / 2 M NaClO4. 8 </li><li> Fazer a curva de titulação como se segue: <ol><li> Traça-se a amplitude do sinal EPR em um valor de g, que é característica para o parágrafomagnéticos espécies de interesse em relação ao potencial. </li><li> Alterar a escala de amplitude do sinal EPR a um gira / escala molécula usando a quantificação EPR a partir do passo 3.2. </li><li> Corrija a amplitude do sinal de cada amostra para a diluição que ocorreu devido às adições de oxidante ou agente redutor. </li><li> Corrigir o potencial para o potencial de meia onda do eléctrodo de referência de Ag / AgCl. Nota: O eléctrodo de referência de Ag / AgCl (com KCl saturada) tem um potencial de ponto médio de 0,194 V versus o Hidrogénio eléctrodo padrão (ELA), a 25 ° C. Portanto, se o potencial no voltímetro lê cerca de -0,6 V, com o potencial de referência para a ELA é -0.4 V. </li></ol></li><li> Montar o sinal para a equação de Nernst como se segue: Para uma espécie que é paramagnético no estado reduzido: <img alt="Equação 1" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq1.jpg" width="200" /> Para uma espécie que é paramagnético para o oestado xidized: <img alt="Equação 2" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq2.jpg" width="200" /> Para uma espécie que é paramagnética em um estado intermediário: <img alt="Equação 3" fo:content-width="2.2in" src="/files/ftp_upload/51611/51611eq3.jpg" width="220" /> Essas equações são válidas para reacções redox em que um elétron é transferido, então n = 1. E = potencial em volts E m = potencial médio em Volt F = constante de Faraday = 96,480 C · mol -1 R = constante de gás = 8,314 J K -1 · · mol -1 T = Temperatura em Kelvin </li></ol>

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A série de 13 mediadores redox permite um equilíbrio redox da solução na gama de 0,3 a -0,45 V versus ELA (Tabela 1 e Figura 3). Bem acima e abaixo destes potenciais mediadores são todos ou completamente reduzida ou completamente oxidada e, consequentemente, não mais de equilíbrio com os centros activos redox da proteína podem ocorrer. Este é um conceito importante, como na literatura, por vezes redox titulações são realizadas com apenas dois ou três mediadores, permitindo ...

É surpreendente que os processos químicos mais fundamentais que sustentam a vida neste planeta, a fotossíntese, fixação de nitrogênio e respiração, são catalisadas por grandes complexos de proteínas que contêm uma vasta gama de cofatores redox orgânicos e inorgânicos. Estimou-se que aproximadamente 30% de todas as proteínas contêm um ou mais co-factores de metal 1,2. Identificação e caracterização dos cofactores redox pode ser estabelecida utilizando electroquímica directa (por exemplo, proteína película voltametria) ou titulações redox. As duas técnicas são complementares em sua natureza e aplicabilidade. Voltammetry oferece determinação rápida de potenciais médios e cinética de transferência de elétrons de co-fatores que podem reagir com a superfície do eletrodo. 3,4 Geralmente isso funciona bem para proteínas de transferência de elétrons, como citocromo c ou ferredoxin. E, por vezes, trabalha para proteínas mais complexas que foram imobilizadas a uma superfície do eletrodo. O conhecimento detalhado donatureza de cofactores na proteína tem de estar disponível, como o voltamograma não dará nenhuma informação directa sobre a identidade do cofactor. Titulações redox são mais trabalhoso para executar e exigem quantidades mg de proteína. No entanto, eles oferecem informação sobre o potencial de meia onda e a identidade do cofactor. 5 Para além disso, em um único titulação múltiplos cofactores numa proteína pode ser monitorizado. O princípio de uma titulação redox é que a proteína ou enzima redox activo é quimicamente reduzida ou oxidada. De modo a certificar-se de que os cofactores reagir com os mediadores redox oxidante ou redutor são utilizados. Estes mediadores redox também reagir com um eléctrodo de modo a que o potencial da solução podem ser medidos. Os mediadores actuam como um tampão redox e equilibrar entre os co-factores na proteína e o eléctrodo. Após poising quimicamente o potencial para o valor desejado, uma amostra é retirada e rapidamente congelados em azoto líquido para await posterior análise com técnicas espectroscópicas. Espectroscopia de EPR é particularmente útil a este respeito uma vez que pode ser utilizado para medir quantitativamente centros metálicos paramagnéticos ou radicais orgânicos. A titulação redox pode ser realizada em duas direções: de baixo para cima ou de cima para baixo potencial médio. A escolha depende da estabilidade dos cofactores em estudo. Muitas vezes, é aconselhável começar pelo baixo potencial e adicionar oxidante para aumentar o potencial de passo a passo. Isso é chamado de titulação oxidativo e é descrito aqui. Aqui nós mostramos a titulação redox do cluster de ferro-enxofre contendo proteína Nar1 de Saccharomyces cerevisiae. Esta proteína está envolvida, provavelmente como uma proteína de andaime, na citosólica ferro-enxofre biossintética aglomerado máquinas (via CIA) 6,7.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reference electrode (Ag/AgCl)</td>
			<td style="width:101px;">Radiometer</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemicals</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EPR spectrometer</td>
			<td style="width:101px;">Bruker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">N,N,N&#8217;,N&#8217;-tetramethyl-p-phenylendediamine (TMPD) &#183;(HCl)2</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">637-01-4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2,6-dichlorophenol indophenol (DCIP), sodium salt</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">620-45-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenazine ethosulfate (PES)</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">10510-77-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene blue</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">122965-43-9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resorufin, sodium salt</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">34994-50-8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Indigodisulfonate (indigo carmine)</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">860-22-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-hydroxy-1,4-naphtaquinone</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">83-72-7</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Anthraquinone-2-sulfonate Na+ H2O</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">153277-35-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenosafranin</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">81-93-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Safranin O</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">477-73-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neutral red</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">553-24-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzyl viologen</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1102-19-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl viologen</td>
			<td style="width:101px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1910-42-5</td>
		</tr>
	</tbody>
</table>

epr monitored redox titration of the cofactors of saccharomyces cerevisiae nar1

Ressonância paramagnética eletrônica (EPR) acompanhou titulações redox são um poderoso método para determinar o potencial médio de cofatores em proteínas e de identificar e quantificar os cofatores em seu estado redox detectável. A técnica é complementar a electroquímica directa (voltametria) aproxima-se, uma vez que não oferece informação sobre as taxas de transferência de electrões, mas não determinar a identidade e estado redox dos cofactores na proteína sob estudo. A técnica é amplamente aplicável a qualquer proteína que contenha uma ressonância paramagnética electrão (EPR) cofactor detectável. Uma titulação típica requer 2 ml de proteína com uma concentração de co-factor na gama de 1-100 uM. A proteína é titulado com um redutor químico (ditionito de sódio) ou oxidante (ferricianeto de potássio), a fim de poise a amostra a um determinado potencial. Um fio de platina e um eléctrodo de referência de Ag / AgCl são conectados a um voltmedidor para medir o potencial da solução de proteína. Um conjunto de 13 diferentes mediadores redox é usado para equilibrar entre os cofactores redox da proteína e os eléctrodos. As amostras são recolhidas em diferentes potenciais e os espectros de ressonância paramagnética eletrônica, característicos dos diferentes cofatores redox na proteína, são medidos. O gráfico da intensidade do sinal em relação ao potencial da amostra é analisada utilizando a equação de Nernst, a fim de determinar o potencial de meia onda de co-factor.

A titulação redox do conjunto do ferro-enxofre contendo proteína Nar1 a partir de Saccharomyces cerevisiae resultou na identificação de três diferentes co-factores: Nar1: uma [3fe-4S] de fragmentação, um [4FE-4S] de fragmentação e um centro de Fe mononucleares (Figura 1) . Os sinais de RPE são caracterizados por suas g-valores: para o [3FE-4S] + g z 2.01, 2.00 g de crossover (Figura 1B); para o [4FE-4S] +, z g 2,...

Watch this Scientific Journal Video about EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1 at JoVE.com

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPR Monitorado Redox Titulação de cofatores de<em&gt; Saccharomyces cerevisiae</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

epr-monitored-redox-titration-cofactors-saccharomyces-cerevisiae

Research

JoVE Journal

Biology

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

13.4K visualizações.  Delft University of Technology. The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

Vídeo: EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Dissecção da Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Biologia

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Phosphoproteomics quantitativa em ácidos graxos estimulada Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray Analysis for Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Um Ensaio de microscopia de fluorescência para Mitophagy Monitoramento no fermento Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expressão e purificação do Fibrose Cística proteína transmembranar regulador da condutância na Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Ensaios para Monitoramento juntamente redobramento de proteínas em<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Purificação da fibrose cística condutância transmembrana Regulador proteína expressa em<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

Medição do mRNA Decay Preços em<em&gt; Saccharomyces cerevisiae</em&gt; Usando<em&gt; Rpb1-1</em&gt; Cepas

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.