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 matingbased 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.split ubiquitin ͉ proteomics ͉ KAT1 ͉ Arabidopsis ͉ GATEWAY