[Systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins]
>>Published online before print August 6, 2004
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0404467101
Genetics
K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions
Petr Obrdlik a,b, Mohamed El-Bakkoury b,c, Tanja Hamacher b,d, Corinna Cappellaro b,d, Cristina Vilarino e, Carola Fleischer f, Heinz Ellerbrok g, Richard Kamuzinzi h, Valérie Ledent h, Damien Blaudez i, Dale Sanders i, Jose L. Revuelta e, Eckhard Boles d, Bruno André c, and Wolf B. Frommer j,k
aZentrum für Molekularbiologie der Pflanzen, Pflanzenphysiologie, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany; cLaboratoire de Physiologie Cellulaire, hUnite de Bioinformatique, Université de Bruxelles, Institut de Biologie et de Médecine Moléculaires, C.P. 300, Rue des Prof. Jeener et Brachet 12, 6041 Charleroi, Belgium; dInstitut für Mikrobiologie, Universität Frankfurt, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany; eDepartmento de Microbiologia y Genetica, Universidad de Salamanca, Edificio Departmental No. 323, 37007 Salamanca, Spain; fGenExpress GmbH, Tempelhofer Weg 11-12, 10829 Berlin, Germany; gRobert Koch Institut, Nordufer 20, 13353 Berlin, Germany; iBiology Department, University of York, York YO10 5YW, United Kingdom; and jCarnegie Institution of Washington, 260 Panama Street, Stanford, CA 94110
Communicated by Christopher R. Somerville, Carnegie Institution of Washington, Stanford, CA, June 25, 2004 (received for review April 3, 2004)
Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a mating-based split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of attachment sites for GATEWAY. Homo- and heteromeric interactions between Arabidopsis K+ channels KAT1, AKT1, and AKT2 were identified. Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K+ channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.<<
Not sure if GenExpress has a financial interest in this work, but it's important if it works on mammalian proteins, which it seems it ought to. I'd have the patents filed and the business development guy ready to go.
Cheers, Tuck |
