K ⁺ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions

Abstract

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

Roles of ion channels and transporters in guard cell signal transduction

“…For proteins known to be involved in GC signaling, FRET-based studies could be helpful in analyzing subcellular co-localization and interaction [154]. Recent development of mass spectrometry-based proteomic approaches coupled with purification of protein complexes, as well as study of membrane protein interactions using the split ubiquitin based system [155,156], should also prove useful in identification of new protein partners of known components. Large scale genomic and proteomic analysis of guard cells could also help to identify novel regulators of ion channels and transporters.…”

Arabidopsis extra‐large G proteins (XLGs) regulate root morphogenesis

“…Nuclear localization is also indicated by the fact that Arabidopsis XLGs themselves, if cloned into the Cub vector of the split ubiquitin system (Obrdlik et al, 2004), can bring the artificial transcription factor to the yeast nucleus, therefore causing self-activation in this assay (data not shown).…”

RGF1 INSENSITIVE 1 to 5, a group of LRR receptor-like kinases, are essential for the perception of root meristem growth factor 1 in Arabidopsis thaliana

“…To identify this putative LRR-RLK which can physically interact with BAK1, we carried out yeast two-hybrid analysis to test the interaction of BAK1 with all other 161 LRR-RLKs, whose cDNAs were cloned previously [44]. Using BAK1 as bait, we totally identified 83 LRR-RLKs which can interact with BAK1 in an mbSUS yeast two-hybrid system [45]. Interestingly, a clade of 5 LRR-RLKs in the LRR XI subfamily, At3G24240, At5G48940, At4G26540, At5G56040, and At1G34110, all show some degree of interaction with BAK1 (data not shown).…”