Este trabajo fue apoyado financieramente por una beca de investigación de la Escuela Nacional de Investigación Combinación holandés, Catálisis Controlado por Diseño Química (NRSC-Catálisis). Dr. HY Steensma y el Dr. GPH van Heusden de la Universidad de Leiden son reconocidos por su apoyo a la expresión recombinante de S. cerevisiae Nar1.

1. Preparación de la instalación y Soluciones <ol><li> Preparar la solución tampón que contiene Tris 100 mM, NaCl 250 mM y 10% de glicerol a pH 8,5. Purgar el tampón por lavado con argón. Introducir el tampón en una cámara anaeróbica. </li><li> Divida la memoria intermedia en dos porciones. A una porción (tampón A) añadir 1 mM DL-ditiotreitol (DTT, M r 154,25 g / mol) y 1 mM de L-cisteína. La otra parte (tampón B) no contiene agentes reductores. </li><li> Preparar soluciones oxidantes y reductores. Hacer 1 ml de soluciones de 2, 20 y 200 mM ferricianuro de potasio (FIC, M r 329,26 g / mol) y de ditionito de sodio (SD, M r 174,11 g / mol) en tampón anaeróbica B. </li><li> Preparar la mezcla mediador como sigue: <ol><li> Añadir 160 M de cada uno de los siguientes mediadores redox y NaCl 50 mM a tampón 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), etosulfato fenacina (PSE-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 (M r 235,17 g / mol, E &#39;0 -0,051 V), Indigodisulphonate (M r 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 (M r 328,28 g / mol, E&#39; 0 -0,225 V), fenosafranina (M r 322,8 g / mol, E &#39;0 -0,252 V), safranina O (M r 350,84 g / mol, E &#39;0 -0,280 V), rojo neutro (M r 288,8 g / mol, E&#39; 0 -0,340 V), bencilo viológeno (M r 409,4 g / mol, E &#39;0 -0,350 V), y metil viológeno (M r 257,16 g / mol, E&#39; 0 -0,440 V). </li><li> Envuelva la botella en papel de aluminio para proteger el mediadors de la luz. </li></ol></li><li> Preparar 10 mg / ml S. cerevisiae proteína Nar1 strep-etiquetado (181 M, M r 55,2 kDa) en tampón A. expreso S. cerevisiae Nar1 en E. coli como una proteína Strep-etiquetados utilizando un vector de expresión pET24a. Se purifica la proteína en una cámara anaeróbica utilizando medios de cromatografía Strep-Tactin (IBA) y purificar usando un sistema de purificación de proteínas tales como purificador Akta (GE Healthcare). </li><li> Preparar la configuración de titulación redox en una cámara anaeróbica como sigue: <ol><li> Conectar un electrodo de referencia Ag / AgCl y un electrodo de alambre de platino para el recipiente de valoración. Conecte los dos electrodos a un voltímetro que pueden medir desde mV hasta V. </li><li> Inserte una pequeña barra de agitación. </li><li> Coloque el recipiente de valoración por encima de un agitador magnético. </li><li> Llene el recipiente de valoración con Tampón B, solución mezcla mediador y solución de proteína de hasta 2 ml, es decir, a concentraciones finales de proteína de 72 mM y 38 mM de cadamediador. Nota: dependiendo de la concentración de proteínas y el mediador requieren concentraciones de las cantidades de las tres soluciones debe ser ajustado. </li><li> Se agita durante 30 minutos y encienda el voltímetro. </li></ol></li><li> Preparar 10 tubos de vidrio de cuarzo EPR limpias. Introducir los tubos en la cámara anaeróbica. </li><li> Llenar una pequeña (circa 200 ml) Dewar de nitrógeno líquido con nitrógeno líquido. </li></ol> 2. Realice Valoración redox <ol><li> Espere hasta que el potencial de la solución es estable. Nota: En la práctica esto significa que el potencial se desplaza menos de 1 mV por minuto. </li><li> Anote el potencial dado por el voltímetro. </li><li> Dibuje la primera muestra de 200 l e inyectar en un tubo de EPR. </li><li> Se tapa el tubo con un tapón de goma y retire el tubo de la cámara anaeróbica. </li><li> Congelar el tubo en nitrógeno líquido de la siguiente manera: ¡Precaución! Esto tiene que ser hecho lentamente, de lo contrario el tubo se rompa. <ol><li> Primero inserte el tip del tubo en el nitrógeno líquido. </li><li> Espere hasta que un silbido se escucha. Nota: Esto puede tardar hasta 10 segundos. </li><li> Introduzca lentamente el resto del tubo de modo que la parte de la muestra está completamente sumergida. </li></ol></li><li> Añadir una pequeña alícuota 1-2 l de la solución 2 mM SD a la solución en el recipiente de valoración para reducir el potencial de la solución a -0,6 V. Nota: Si hay más de 10 l tiene que añadir, cambiar a la solución de 20 mM. Como es muy difícil de contrapesar a un valor potencial específica, los valores reales registrados en el voltímetro se anotan y se usaron para construir la curva de titulación. </li><li> Dibuja una muestra como se describe en el paso 2.3 a 2.5. </li><li> Anote el potencial real propuesta por el voltímetro. </li><li> Añadir oxidante hasta que el potencial se ha incrementado en alrededor de 50 mV. Comience agregando una pequeña alícuota 2/1 l de la solución 2 mM FIC. Si más de 10 l tiene que ser añadido, cambiar a la solución 20 mM. </li><li> Anote how gran parte de las soluciones se ha agregado. Nota: Esto es necesario para corregir la dilución de la muestra durante la valoración. </li><li> Dibuje una muestra como se describe en el 2/3 a 2/5 pasos. </li><li> Anote el potencial real propuesta por el voltímetro. </li><li> Repita los pasos 2,9 y 2,12 hasta el rango potencial de -0,6 V a 0,2 V ha sido cubierto y los 10 tubos de EPR se llenan cada uno con 200 l. Nota: Como se reduce el volumen con cada retirada puede ser necesario ajustar el posicionamiento de los electrodos de tener un buen contacto con la solución sin interferencia de la agitación. Puede ser difícil para medir el potencial después de la retirada de la última muestra y por lo tanto el potencial de que la muestra puede ser menos precisa. </li><li> Opcionalmente, almacenar las muestras EPR congelados en nitrógeno líquido hasta que las medidas de EPR pueden tener lugar. </li></ol> 3. Medida de titulación muestras utilizando EPR Espectroscopia y análisis de datos <ol><li> Record EPR espectros de los diferentesmuestras de titulación de la siguiente manera: <ol><li> Utilice una bomba de alto vacío para evacuar un criostato de cuarzo a &lt;10 -5 bar. </li><li> Conectar la refrigeración por agua del imán EPR. Abrir el flujo de aire seco a través de la cavidad de EPR. </li><li> Encienda la fuente de alimentación para el imán y el equipo de la EPR. </li><li> Ejecutar un programa de calibración de frecuencia. Establezca los parámetros de medición del EPR. Conecte el criostato al dewar helio líquido. Se enfría la cavidad a 9-16 K. </li><li> Introducir la muestra EPR en la cavidad. Registre el espectro EPR. </li></ol></li><li> Identificar las diferentes especies de EPR y utilizar el mejor espectro para cuantificar las señales. Lograr EPR cuantificación por doble integración de los espectros y la comparación con el espectro de una solución estándar externa de cobre mM CuSO 10 mM HCl 4/10 / 2 M NaClO 4. 8 </li><li> Hacer la curva de valoración de la siguiente manera: <ol><li> Trazar la amplitud de la señal de EPR en un g-valor que es característico para el paraespecies magnéticas de interés frente al potencial. </li><li> Cambie la escala de amplitud de la señal de EPR a un giros / escala molécula mediante la cuantificación EPR desde el paso 3.2. </li><li> Corregir la amplitud de la señal de cada muestra para la dilución que se ha producido debido a las adiciones de agente oxidante o reductor. </li><li> Corregir el potencial para el potencial punto medio del electrodo de referencia Ag / AgCl. Nota: El electrodo de referencia Ag / AgCl (con solución saturada de KCl) tiene un potencial punto medio de 0.194 V frente al electrodo de hidrógeno estándar (SHE) a 25 ° C. Por lo tanto, si el potencial en el voltímetro lee alrededor de -0,6 V, con el potencial de referencia a la que ella está -0,4 V. </li></ol></li><li> Montar la señal a la ecuación de Nernst de la siguiente manera: Por una especie que es paramagnético en el estado reducido: <img alt="Ecuación 1" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq1.jpg" width="200" /> Para una especie que es paramagnético en el oestado xidized: <img alt="Ecuación 2" fo:content-width="2in" src="/files/ftp_upload/51611/51611eq2.jpg" width="200" /> Para una especie que es paramagnético en un estado intermedio: <img alt="Ecuación 3" fo:content-width="2.2in" src="/files/ftp_upload/51611/51611eq3.jpg" width="220" /> Estas ecuaciones son válidas para las reacciones redox en los que se transfiere un electrón, por lo que n = 1. E = potencial en voltios E m = potencial de punto medio en Volt F = constante de Faraday = 96480 C · mol -1 R = constante de los gases = 8.314 J · K -1 mol -1 T = Temperatura en grados Kelvin </li></ol>

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Los autores no tienen nada que revelar.

La serie de 13 mediadores redox permite un equilibrio redox en la solución en el rango de -0,45 a 0,3 V frente a SHE (Tabla 1 y Figura 3). Bien por encima y por debajo de estos potenciales de todos los mediadores son o bien totalmente reducidos o completamente oxidados y, por tanto, no más lejos de equilibrio con los centros redox activos de la proteína pueden ocurrir. Este es un concepto importante, como en la literatura titulaciones redox veces se llevan a cabo con sólo dos o tres...

Llama la atención que los procesos químicos más fundamentales que sustentan la vida en este planeta, la fotosíntesis, la fijación de nitrógeno y la respiración, son catalizadas por grandes complejos de proteínas que contienen una amplia gama de cofactores redox orgánicos e inorgánicos. Se ha estimado que aproximadamente el 30% de todas las proteínas contienen uno o más cofactores metálicos. 1,2 identificación y caracterización de los cofactores redox se pueden establecer utilizando la electroquímica directa (por ejemplo, proteína de voltametría película) o valoraciones redox. Las dos técnicas son complementarias en su naturaleza y aplicabilidad. Voltamperometría ofrece determinación rápida de potenciales de punto medio y la cinética de transferencia de electrones de cofactores que pueden reaccionar con una superficie de electrodo. 3,4 lo general, esto funciona bien para las proteínas de transferencia de electrones, tales como citocromo c o ferredoxina. Y a veces funciona para las proteínas más complejas que han sido inmovilizados a una superficie del electrodo. El conocimiento detallado de lanaturaleza de cofactores en la proteína tiene que estar disponible, ya que el voltamograma no dará ninguna información directa sobre la identidad del cofactor. Valoraciones redox son más laborioso para ejecutar y requieren cantidades mg de proteína. Sin embargo, ofrecen información sobre el potencial punto medio y la identidad del cofactor. 5 Además, en una sola titulación múltiples cofactores en una proteína pueden ser monitoreados. El principio de una titulación redox es que la proteína activa redox o enzima se reduce o se oxida químicamente. Con el fin de asegurarse de que los cofactores reaccionan con los mediadores reductor u oxidante redox se utilizan. Estos mediadores redox también reaccionan con un electrodo de modo que el potencial de la solución se puede medir. Los mediadores actúan como un tampón redox y se equilibran entre los cofactores en la proteína y el electrodo. Después de poising químicamente el potencial en el valor deseado, se extrae una muestra y rápidamente congelados en nitrógeno líquido en AWait posterior análisis con técnicas espectroscópicas. Espectroscopia EPR es particularmente útil a este respecto, ya que puede ser usado para medir cuantitativamente centros metálicos paramagnéticos o radicales orgánicos. La valoración redox se puede realizar en dos direcciones: de menor a mayor o de mayor a menor potencial punto medio. La elección depende de la estabilidad de los cofactores en estudio. A menudo es prudente comenzar a bajo potencial y añadir oxidante para escalonadamente aumentar el potencial. Esto se llama una titulación oxidativo y se describe aquí. Aquí mostramos la valoración redox del cluster hierro-azufre que contiene proteína Nar1 de Saccharomyces cerevisiae. Esta proteína está implicada, probablemente como un andamio de proteínas, en la citosólica de hierro-azufre biosintética clúster maquinaria (CIA vía) 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

Resonancia Paramagnética Electrónica (EPR) supervisa valoraciones redox son un poderoso método para determinar el potencial punto medio de cofactores en proteínas y para identificar y cuantificar los cofactores en su estado redox detectable. La técnica es complementaria a la electroquímica directa (voltametría) se aproxima, ya que no ofrece información sobre las tasas de transferencia de electrones, pero no establecer el estado redox identidad y de los cofactores en la proteína en estudio. La técnica es ampliamente aplicable a cualquier proteína que contiene una resonancia paramagnética electrónica (EPR) cofactor detectable. Una titulación típica requiere proteína de 2 ml con una concentración de cofactor en el intervalo de 1-100 mM. La proteína se valora con un reductor químico (ditionito de sodio) o el oxidante (ferricianuro potásico) para contrapesar la muestra en un determinado potencial. Un alambre de platino y un electrodo de referencia de Ag / AgCl se conectan a un voltiometro para medir el potencial de la solución de proteína. Un conjunto de 13 diferentes mediadores redox se utiliza para equilibrar entre los cofactores redox de la proteína y los electrodos. Las muestras se extraen a diferentes potenciales y los espectros de Resonancia Paramagnética Electrónica, característicos de los diferentes cofactores redox en la proteína, se midieron. La trama de la intensidad de la señal en comparación con el potencial de la muestra se analiza utilizando la ecuación de Nernst con el fin de determinar el potencial de punto medio del cofactor.

La titulación redox del cluster hierro-azufre que contiene proteína Nar1 a partir de Saccharomyces cerevisiae como resultado la identificación de tres cofactores diferentes: Nar1: un racimo [3FE-4S], un grupo [4Fe-4S] y un centro de Fe mononuclear (Figura 1) . Las señales EPR se caracterizan por sus valores g: para el [3FE-4S] + g z 2.01, 2.00 g de crossover (Figura 1B); para el [4Fe-4S] +, g z 2,02 g y 1,93,...

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 supervisado Redox La titulación de los cofactores 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 vistas.  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.

Video: 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.

La disección de 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 ...

Investigación

Biología

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 cuantitativo de ácidos grasos 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.

El análisis de microarrays para 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.

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura 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.

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la 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.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<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.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<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;

La purificación de la fibrosis quística regulador de conductancia transmembrana de proteínas expresadas en<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.

La medición de mRNA decadencia Tarifas en<em&gt; Saccharomyces cerevisiae</em&gt; Uso<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.