Gostaríamos de agradecer a Dr. Rich Rogers de assistência técnica com análises de espectrometria de massa e discussões úteis, e as Dras. Rob Moritz, Jeff Ranish, e Hamid Mirzai para discussões úteis. Este trabalho foi financiado pelo NIH Centros de Excelência.

Crescimento celular e media <ol><li> Uma única colônia de células BY4742Δarg4Δlys1 overnight em 100 mL de rich media para um OD 600 de 1,0 sementes, em seguida, em duas culturas 1 litro de um meio de levedura mínimo (0,17% Base de Dados de nitrogênio fermento sem sulfato de amônio ou Aminoácidos, sulfato de amônio 0,5%), contendo uma conjunto completo de aminoácidos, suplementado com L / 20mg de arginina isotopicamente normal ou pesado (13 C 6 15 N 4; Isotec) e lisina (13 C 6 15 N 2; Isotec). </li><li> As células foram cultivadas por 18 horas, para um OD 600 de 1.8. É fundamental que as células passam por pelo menos 9 gerações para alcançar a plena incorporação dos isótopos rotulados. A amostra foi peletizada luz, lavados com água estéril, reseeded em um meio contendo oleato (arginina isotopicamente normal e lisina, oleato de 0,2% (Sigma Chemicals) e 0,5% Tween 40 (Sigma Chemicals)) e estimulado por outros 85 minutos. Isso gera uma isotopicamente leve, com respeito a arginina e lisina, oleato estimulada amostra e uma amostra de referência isotopicamente pesada glucose-grown. </li></ol> Lise celular, Isolamento e fracionamento de Peptídeos <ol start="3"><li> Pesar garrafa centrífuga. Colheita de amostras por centrifugação por 3 minutos. Remova a mídia e aspirar o excesso de líquido. Pesar pellet e garrafa (normalmente rendimento entre 1-2 gramas), em seguida, congelar flash em nitrogênio líquido. Adicionar um volume equivalente a pelota peso de &#39;moer&#39; buffer para as pelotas congelados em nitrogênio líquido (Phosphate Buffered Saline (PBS, Gibco), 10% glicerol, inibidores da protease (Comprimidos Inibidor da Protease SigmaFAST, Sigma) e HALT inibidores de fosfatase (Thermo Scientific )). </li><li> Congelar o navio de moagem em nitrogênio líquido. Aguarde até que o nitrogênio líquido deixa ferver. Transferir a pelota para o navio de moagem, com rolamentos de esferas congelada. </li><li> Moagem de 600 rpm com um ciclo de 1 min 20 sec com uma inversão direção por 3 minutos. Recongelar o navio de moagem em nitrogênio líquido. Repita 4 vezes mais por 15 minutos tempo de moagem total. </li><li> Recolher o grindate congelados em um tubo de 50ml de gelo lugar Falcon seco. Armazenar a -80 ° C até que esteja pronto para uso. </li><li> Fazer uma solução de 10ml de 8M uréia, bicarbonato de amônio 0,1 M, 0,1 M Tris pH 8.6. Adicionar 3 volumes do buffer de uréia para 1 volume do grindate congelados (por exemplo 3mls do buffer de uréia adicionados a um 1 grama pellet com 1 ml de tampão PBS). Sonicate imediatamente a mistura com um sonicador ponta da sonda (2 - 10 pulsos segundo). Manter a ponta na parte inferior do tubo para evitar a formação de espuma da solução. O grindate deve imediatamente entrar em solução. </li><li> Limpar o lisado por centrifugação por 5 minutos a 4 ° C. </li><li> Transfira o sobrenadante para tubos fresco. </li><li> Faça uma nova porção de Tris 0,5 M [2-carboxietil] fosfina (TCEP). Adicionar ao da amostra em 1:100 para uma concentração 5mM final. Incubar a 37 ° C por 1 h. </li><li> Alquilação de cisteínas reduzidas. Deixe esfriar até a temperatura ambiente. Faça uma nova porção da iodoacetamida 1M. Manter no escuro (que envolva o tubo em papel alumínio). Adicionar a uma concentração final de 20mM. Incubar no escuro por 1 h em temperatura ambiente. </li><li> Saciar a iodoacetamida com DTT 20mM de um estoque 1M em temperatura ambiente por 1 h. </li><li> Quantificar a proteína com um padrão ou Bradford ensaio BCA (não descritos). Tomar uma amostra para análise por SDS-PAGE abaixo. </li><li> Para validar a incorporação dos isótopos pesados, use 100 ml do lisado reduzidos e alquilados. Diluir 1:4 com dH 2 O, sonicate e adicione tripsina em 50:1 proteína para tripsina. Digest para um mínimo de 4 horas, amostra seca para baixo em uma velocidade vac e depois desalt com colunas Ultramicrospin C18 (Grupo A Nest). <ol><li> Coluna hidratado com 100 ml de Acetonitrila </li><li> Equilibrar com 200 mL TFA 0,1%. </li><li> Ressuspender amostra seca em 200 mL TFA 0,1%. Carga para coluna. Centrifugar a 200 xg ~ ou a velocidade mínima necessária para o fluxo através da coluna. </li><li> Lavar duas vezes com 200 mL TFA 0,1%. </li><li> Eluir com duas vezes com 50 mL Acetonitrila 60%, TFA 0,1%. </li><li> Amostra seca em uma velocidade vac. </li><li> Amostra ressuspender em 20 mL de ácido fórmico 0,1%. Executar 2 l em um espectrômetro de massa. Verifique incorporação de arginina e lisina isotopicamente pesado por uma mudança Dalton 10 e 8 no espectro m / z respectivos. A incorporação de cada aminoácido deve estar entre 96-98%. </li></ol></li><li> Misture quantidades equivalentes (mg de proteínas) das amostras isotopicamente leves e pesados. </li><li> Diluir a amostra 1:4 com metanol 20%. </li><li> Adicionar tripsina 1:200. Incubar durante a 37 ° C. </li><li> Verificar que a digestão tem ido até a conclusão por PAGE SDS. Executar 2 e 4 mL da amostra predigestion e 8 umd mL 16 da tripsina pós digerir. Visualize por coloração Coomassie que a digestão é completa. Se ele não estiver completo, sonicate a amostra com um breve sonicador ponta da sonda e digerir novamente com tripsina. </li><li> Secar a amostra para baixo em uma velocidade vac. Ao adicionar a seco de 200 mL filtrado dH 2 O e vortex até o pellet entra em solução. Amostra seca para baixo em uma velocidade vac. Ressuspender novamente com dH 2 O. Seco da amostra novamente. </li><li> Ressuspender pellet em 0,1% Ácido Trifluoracetic (TFA; Sigma). Verifique se o pH é de cerca de 3 usando tiras de pH. Se necessário acidificar o meio com TFA 10%. </li><li> Pellet qualquer precipitado. </li><li> Dessalinizar as amostras em um Waters Sep-Pak Vac coluna C18 de 500 mg. Não sobrecarregue as colunas. A quantidade máxima é de 5 mg, mas é melhor carregar um pouco menos. Dividir a amostra entre várias colunas se necessário. <ol><li> Coluna o hidrato com 5 ml de acetonitrila 100%. Centrifugar a 200 xg ~ ou a velocidade mínima necessária para o fluxo através da coluna. </li><li> Equilibrar coluna com TFA 0,1% 5ml. Carga da amostra. Colete de fluxo e de carga novamente. </li><li> Lavar com 5 ml TFA 0,1%. Repetir. </li><li> Eluir com 2ml Acetonitrila 60%, TFA 0,1%. </li><li> Amostra seca para baixo em uma velocidade vac. </li></ol></li></ol> Fracionamento e Isolamento de peptídeo Fosfopeptídeos <ol><li> Peptídeos são fracionado em uma coluna HILIC. Nós usamos um TOSOH TSK Gel-amida-80 4,6 milímetros x 25 cm coluna analítica com uma coluna de guarda. Leve o seu tempo com este passo, execute a coluna por 2 horas a 90% A em seguida, executar dois gradientes em branco antes de colocar a sua amostra. A carga máxima em uma coluna deste tamanho é pensado para ser em torno de XX, que normalmente não carregar mais de 5 mg por corrida. </li><li> Um solvente - 98% ACN/0.1% TFA Solvente B - 2% ACN/0.1% TFA Carga em 90% A mais de 20 &#39;, 90% A 85% A mais de 5&#39;, 85% A 60% A mais de 40 &#39;, 60% A para 0% A mais de 5&#39;, 0% A% a 90 A mais 5 &#39;. </li><li> A taxa de fluxo é 1mL/min e frações foram coletadas em intervalos de 2 minutos, começando às 85% a 60% A um gradiente. </li><li> Combine frações para reduzir o número de fracções a 10. Frações aumentou, provavelmente, maior rendimento de cobertura do phosphoproteome. </li></ol> Enriquecimento de fosfopeptídeos <ol><li> Fosfopeptídeos são enriquecidos com PHOS-Select Gel de Afinidade de Ferro. Nós alíquota nossa resina IMAC para evitar congelar repetido descongelamento. Fazer 50 mls de um ácido acético 250 mM, a solução de carga de 30% ACN. Trazer o pH para 2,7 com HCl. Ressuspender pellets em 500 ml de solução de carga. Verificar o pH, ajuste com HCl ou NaOH, conforme necessário. </li><li> ML de pré-lavagem de 200 contas IMAC em uma coluna apropriada. Usamos colunas giram Molbiotec. Adicionar 15 mL de uma suspensão de 50% (gel para carregar solução) a cada fração, e incubar as amostras de 30 &#39;com final mais rotação final. Manter o fluxo para análise. </li><li> Lavar as amostras 3 vezes com solução de carga e uma vez com 500 mL filtrado dH 2 O. </li><li> Peptídeos eluir com 2 - 3 min com 400 mL de 50 mM Na 2 HPO 4 (pH 8,4) buffer. Transferir para um frasco de vidro. </li><li> Acidificar a pH 3 com 5 mL de ácido fórmico 100%, congelamento de flash em nitrogênio líquido, e amostra seca para baixo em uma velocidade vac. </li><li> Fosfopeptídeos ressuspender em 200 mL de TFA 0,1% e C18 limpo como descrito acima. Seco para baixo amostras eluídas em uma velocidade vac. </li><li> Ressuspender peptídeos em 20 l de 0,1% de ácido fórmico. Use 2 mL por análise de espectrometria de massa ou conforme necessário. </li></ol> Notas Durante boa parte deste protocolo que você estará trabalhando &quot;cega&quot;. A lise celular pode ser monitorada por Western Blotting e ensaios Bradford ou BCA, ea digestão com tripsina também pode ser facilmente avaliada. A coluna vai HILIC deu um traço semelhante a esta, ou como pode ser visto por outros [1, 2] dependendo da máquina de cromatografia líquida que é usado. Amostras são monitorados por espectrometria de massa, teste a sua amostras em máquinas de baixo custo, tais como LCQs ou MALDIs antes de ir para as análises de custo alto de uma massa de alta precisão, máquina de alta resolução. Finalmente, se você uma secagem para baixo uma amostra com ácido nele, use vidro.

<ol>
	<li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis.">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li><li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection.">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li><li>Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li><li>Ross, 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=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway.">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li><li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+fission+yeast.">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li><li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCX/IMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry.">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li><li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques.">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li><li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS/MS+and+titanium+oxide+precolumns.">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li><li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry.">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li><li>Zhou, H., Watts, J. D., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+to+the+analysis+of+protein+phosphorylation.">A systematic approach to the analysis of protein phosphorylation.</a> Nat Biotechnol. 19 (4), 375-378 (2001).</li><li>Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+isolation+of+distinct,+overlapping+segments+of+the+phosphoproteome.">Reproducible isolation of distinct, overlapping segments of the phosphoproteome.</a> Nat Methods. 4 (3), 231-237 (2007).</li></ol>

Este método irá produzir um enriquecimento da fosfopeptídeos que podem ser analisadas quantitativamente. Um número de parâmetros podem ser alterados neste protocolo, mas o aspecto mais importante a lembrar é o de preservar a sua fosforilação. Preparação de células para a quantificação pode ser alcançado em um número de diferentes maneiras, por exemplo, se você não pode rotular suas células in vivo, tags, tais como ICAT [3] ou ITRAC [4] pode ser usado extração post para diferencialmente etiqu...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Isotec&trade; L-Arginine-13C6,15N4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>608033</td>
<td>&nbsp;</td>
<tr>
<td>Isotec&trade; L-Lysine-15N2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>609021</td>
<td>&nbsp;</td>
<tr>
<td>GIBCO&#x2122; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>70011044</td>
<td>&nbsp;</td>
<tr>
<td>SigmaFAST Protease Inhibitor Tablets</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Pierce Halt Phosphatase Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>78420</td>
<td>&nbsp;</td>
<tr>
<td>Retsch PM100 Ball Mill Grinder </td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>20.540.0001</td>
<td>&nbsp;</td>
<tr>
<td>Retsch 125 ml Stainless Steel Grinding Jar</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>01.462.0148</td>
<td>&nbsp;</td>
<tr>
<td>Ultramicrospin C18 column</td>
<td>&nbsp;</td>
<td>The Nest Group</td>
<td>SUM SS10</td>
<td>&nbsp;</td>
<tr>
<td>Sep-Pak Vac 500 mg C18</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT043395</td>
<td>&nbsp;</td>
<tr>
<td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>13071</td>
<td>This is the HILIC chromatographic column.</td>
</tr>
<tr>
<td>Guard column 4.6 mm ID x1 cm</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>19021</td>
<td>We recommend this guard column to extend the life of your HILIC column.</td>
</tr>
<tr>
<td>PHOS-Select&trade; Iron Affinity Gel</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P9740</td>
<td>This is the IMAC resin. Aliquot and store.</td>
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quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

Este protocolo descreve o crescimento e estímulo, com o oleato de ácidos graxos, de células de Saccharomyces isotopicamente leves e pesados ​​S.. Células são solo usando um procedimento cryolysis em um moedor de moinho de bolas e os grindate resultando colocada em solução de solubilização da uréia. Este procedimento permite a lise das células em um estado metabolicamente inativas, preservando fosforilação e evitando reorientação da phosphoproteome durante a lise celular. Após a redução, alquilação, digestão com tripsina das proteínas, as amostras são dessalinizada em colunas C18 e da complexidade amostra reduzida por fracionamento através de cromatografia de interação hidrofílica (HILIC). Colunas HILIC preferencialmente reter moléculas hidrofílicas que é bem adequado para phosphoproteomics. Peptídeos fosforilados tendem a eluir mais tarde no perfil cromatográfico do que as contrapartes não fosforilado. Após o fracionamento, fosfopeptídeos são enriquecidos por cromatografia em fase de metal imobilizado, que conta com carga baseada em afinidades de enriquecimento fosfopeptídeos. No final deste procedimento as amostras estão prontas para serem analisadas quantitativamente por espectrometria de massa.

Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Descrição de um processo de fosforilação quantitativa usando cryolysis, solubilziation uréia, fracionamento e enriquecimento HILIC IMAC de peptídeos fosforilados.

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 ...

quantitative-phosphoproteomics-fatty-acid-stimulated-saccharomyces

Research

JoVE Journal

Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K visualizações.  Institute for Systems Biology. Descrição de um processo de fosforilação quantitativa usando cryolysis, solubilziation uréia, fracionamento e enriquecimento HILIC IMAC de peptídeos fosforilados.

Vídeo: Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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 ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Quantificação e análise da formação de HO-endonuclease Stimulated translocações cromossômicas por Strand Único Annealing em Saccharomyces cerevisiae</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

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 ...

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.

Identificação rápida de químicos Genéticos Interações em<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Cicloheximida Análise perseguição de degradação da proteína em<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

A esporulação eficiente dos<em&gt; Saccharomyces cerevisiae</em&gt; Num Multiwell Format 96

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

Phosphoproteomics quantitativa em ácidos graxos estimulada Saccharomyces cerevisiae

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