The Plant Endoplasmic Reticulum pp 191-203 | Cite as
ER Membrane Protein Interactions Using the Split-Ubiquitin System (SUS)
Abstract
Protein–protein interactions (PPIs) play fundamental roles in all cellular processes. Especially membrane proteins facilitate a range of important biological functions in stimuli perception, signaling, and transport. Here we describe a detailed protocol for the yeast mating-based Split-Ubiquitin System (mbSUS) to study PPIs of ER membrane proteins in vivo. In contrast to the prominent Yeast Two-Hybrid, mbSUS enables analysis of full-length membrane proteins in their native cellular context. The system is based on the ubiquitin proteasome pathway leading to the release of an artificial transcription factor followed by activation of reporter genes to visualize PPIs. The mating-based approach is suitable for both small- and large-scale interaction studies. Additionally, we describe protocols to apply the recently established SUS Bridge assay (SUB) which is optimized for the detection of ternary protein interactions.
Key words
Protein–protein interaction Ternary interaction Yeast Split Ubiquitin mbSUS SUB Membrane proteins GatewayNotes
Acknowledgements
We are grateful to Eva Schwörzer and Laure Grefen for excellent technical support. Work in our lab is supported through seed funding of the SFB1101 and an Emmy Noether fellowship of the German Research Foundation (DFG) to C.G. (GR 4251/1-1).
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