Name: split-ubiquitin based membrane yeast two-hybrid (myth) system: a powerful tool for identifying protein-protein interactions
Uploaded: 2010-02-01T16:25:00
Description: Watch this Scientific Journal Video about Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions at JoVE.com

We willen graag Dawn Edmonds bedanken voor een kritische lezing van dit manuscript. De Stagljar lab wordt ondersteund door middelen van de Canadese Stichting voor Innovatie (CFI), het Canadese Instituut voor Gezondheid Onderzoek (CIHR), het Hart en Stroke Foundation, de Canadese Cancer Society, en Novartis.

1. Achtergrond informatie Eiwit-eiwit interacties (PPI) zijn de fundamentele bouwstenen die betrokken zijn in het bestuur van alle cellulaire processen. Bijgevolg is het essentieel dat alle interacties goed zijn gereguleerd om cellulaire homeostase te behouden, zoals een verschuiving in deze biologische evenwicht vaak een rol speelt bij de ziekte en kanker cel transformatie. Membraan geassocieerde eiwitten behoren tot de meest biologisch belangrijke klasse van eiwitten als zij kunnen starten c...

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	<li>Stagljar, I., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+membrane+protein+interactions+using+yeast-based+technologies.">Analysis of membrane protein interactions using yeast-based technologies.</a> Trends Biochem Sci. 27 (11), 559-563 (2002).</li><li>Iyer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+the+split-ubiquitin+membrane+yeast+two-hybrid+system+to+identify+protein-protein+interactions+of+integral+membrane+proteins.">Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.</a> Sci STKE. 275, pl3-pl3 (2005).</li><li>Paumi, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+protein-protein+interactions+for+the+yeast+ABC+transporter+Ycf1p+by+integrated+split-ubiquitin+membrane+yeast+two-hybrid+analysis.">Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.</a> Mol Cell. 26 (1), 15-25 (2007).</li><li>Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+system+based+on+split-ubiquitin+for+the+analysis+of+interactions+between+membrane+proteins+in+vivo.">A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo.</a> Proc Natl Acad Sci U S A. 95 (9), 5187-5192 (1998).</li><li>Gisler, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein-protein+interactions+between+the+mammalian+integral+membrane+transporters+and+PDZ-interacting+partners+using+a+modified+split-ubiquitin+membrane+yeast+two-hybrid+system.">Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.</a> Mol Cell Proteomics. 7 (7), 1362-1377 (2008).</li><li>Scheper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+N-glycosylation+and+protein+translocation+across+the+endoplasmic+reticulum+membrane+by+Sss1+protein.">Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein.</a> J Biol Chem. 278 (39), 37998-38003 (2003).</li><li>Thaminy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+ErbB3-interacting+factors+using+the+split-ubiquitin+membrane+yeast+two-hybrid+system.">Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.</a> Genome Res. 13 (7), 1744-1753 (2003).</li><li>Johnsson, N., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+ubiquitin+as+a+sensor+of+protein+interactions+in+vivo.">Split ubiquitin as a sensor of protein interactions in vivo.</a> Proc Natl Acad Sci U S A. 91 (22), 10340-10344 (1994).</li><li>Kelleher, D. J., Gilmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Saccharomyces+cerevisiae+oligosaccharyltransferase+is+a+protein+complex+composed+of+Wbp1p,+Swp1p,+and+four+additional+polypeptides.">The Saccharomyces cerevisiae oligosaccharyltransferase is a protein complex composed of Wbp1p, Swp1p, and four additional polypeptides.</a> J Biol Chem. 269 (17), 12908-12917 (1994).</li><li>Chevallier, 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=Cloning+and+transcriptional+control+of+a+eucaryotic+permease+gene.">Cloning and transcriptional control of a eucaryotic permease gene.</a> Mol Cell Biol. 2 (8), 977-984 (1982).</li></ol>

MYTHE is de eerste hoge doorvoer systeem dat de identificatie van interacties tussen full-length membraaneiwitten en cytosolische of membraan-gebonden partners mogelijk maakt. Het is gebruikt om membraan eiwit studeren uit een reeks van organismen [3-7]. Er zijn echter specifieke details die mogelijk moeten worden onderzocht om ervoor te zorgen het eiwit-of-interest vatbaar is om te studeren met een mythe. Veel membraan-gebonden eiwitten worden gericht aan het plasmamembraan via een signaals...

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split-ubiquitin based membrane yeast two-hybrid (myth) system: a powerful tool for identifying protein-protein interactions

De fundamentele biologische en klinische belang van integrale membraaneiwitten geleid tot de ontwikkeling van een gist-gebaseerd systeem voor de high-throughput identificatie van eiwit-eiwit interacties (PPI) voor de volledige lengte transmembraan eiwitten. Daartoe ons lab ontwikkelde de split-ubiquitine op basis van Membraan Yeast Two-Hybrid (mythe) systeem. Deze technologie maakt het mogelijk voor de gevoelige detectie van voorbijgaande aard en stabiele eiwit interacties met behulp van<em&gt; Saccharomyces cerevisiae</em&gt; Als een gastheerorganisme. MYTHE maakt gebruik van de observatie dat ubiquitine kunnen worden gescheiden in twee stabiele groepen: de C-terminale helft van gist ubiquitine (C<sub&gt; Ub</sub&gt;) En de N-terminale helft van de ubiquitine-groep (N<sub&gt; Ub</sub&gt;). In de mythe, is dit principe aangepast voor gebruik als een &#39;sensor&#39; van eiwit-eiwit interacties. Kortom, is het integraal membraan aas eiwit gefuseerd met C<sub&gt; Ub</sub&gt; Die gekoppeld is aan een kunstmatige transcriptiefactor. Prey eiwitten, hetzij in individuele of bibliotheek-formaat, zijn gefuseerd aan het N<sub&gt; Ub</sub&gt; Groep. Eiwit interactie tussen het aas en prooi leidt tot een reconstructie van de ubiquitine-groepen, de vorming van een full-length &#39;pseudo-ubiquitine&#39; molecuul. Dit molecuul wordt op zijn beurt erkend door cytosolische enzymen ubiquitinerings, wat resulteert in een splitsing van de transcriptie factor, en daaropvolgende inductie van reporter-gen expressie. Het systeem is zeer flexibel, en is bijzonder goed geschikt voor high-throughput screening. Het is met succes gebruikt om interacties met behulp van integrale membraaneiwitten van zowel gist en andere organismen te onderzoeken.

Watch this Scientific Journal Video about Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions at JoVE.com

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTHE kan de gevoelige detectie van voorbijgaande aard en stabiele interacties tussen eiwitten die worden uitgedrukt in het model organisme Saccharomyces cerevisiae. Het is met succes toegepast op exogene en gist integrale membraaneiwitten bestuderen om hun interagerende partners in een high throughput manier te identificeren.

Split-Ubiquitine membraanfilters op Yeast Two-Hybrid (mythe) Systeem: een krachtig hulpmiddel voor het identificeren van eiwit-eiwit interacties

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

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