An optimized
split ubiquitin system for systematic identification and characterization of
membrane protein interactions.
Petr Obrdlik (1),
Mohamed El Bakkoury (2), Tanja Hamacher (3), Corinna Cappellaro (3), Binghua Wu
(1), Eckhard Boles (3), Bruno André (2), Wolf Bernd Frommer (1)
(1) ZMBP, Pfanzenphysiologie, Universitaet Tuebingen, Auf der Morgenstelle,
Tuebingen, 72076, Germany (petr.obrdlik@zmbp.uni-tuebingen.de); (2) Laboratoire
de Physiologie Cellulaire, Universite Libre de Bruxelles, IBMM, Rue des Prof.
Jeener et Brachet 10, 6041 Gosseiles, Belgium; (3) Institut für Mikrobiologie,
Goethe-Universitaet Frankfurt, Marie-Curie-Str. 9, D-60439 Frankfurt am Main,
Germany
Protein-protein interactions and the organization of proteins into functional complexes are crucial for most, if not all, cellular functions. Yet the proteomic approaches applied so far are effective only for soluble protein complexes, and are much less suitable for studying membrane-associated complexes. We have developed a new split ubiquitin system for systematic identification of membrane protein interactions. It consists of a set of vectors, which allow cloning of a PCR product by in vivo cloning and two novel yeast strains of different mating types, which allow testing of interactions via mating approach. In addition, the vectors contain attB1 and attB2 sites compatible with the Gateway cloning system. Thus the new system can be also used as an entry point for further functional, cellular and biochemical studies of membrane proteins. The system was tested with membrane transporters of the model plant Arabidopsis thaliana. The screen revealed novel, not yet described oligomerization of plant ammonium transporters. Furthermore the system also allowed detection of interactions among plant shaker-like potassium channels.