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. 2009:548:247-71.
doi: 10.1007/978-1-59745-540-4_14.

Analysis of membrane protein complexes using the split-ubiquitin membrane yeast two-hybrid (MYTH) system

Saranya Kittanakom  1 , Matthew Chuk, Victoria Wong, Jamie Snyder, Dawn Edmonds, Apostolos Lydakis, Zhaolei Zhang, Daniel Auerbach, Igor Stagljar
Affiliations

Analysis of membrane protein complexes using the split-ubiquitin membrane yeast two-hybrid (MYTH) system

Saranya Kittanakom et al. Methods Mol Biol. 2009.

Abstract

Recent research has begun to elucidate the global network of cytosolic and membrane protein interactions. The resulting interactome map facilitates numerous biological studies, including those for cell signalling, protein trafficking and protein regulation. Due to the hydrophobic nature of membrane proteins such as tyrosine kinases, G-protein coupled receptors, membrane bound phosphatases and transporters it is notoriously difficult to study their relationship to signaling molecules, the cytoskeleton, or any other interacting partners. Although conventional yeast-two hybrid is a simple and robust technique that is effective in the identification of specific protein-protein interactions, it is limited in its use for membrane proteins. However, the split-ubiquitin membrane based yeast two-hybrid assay (MYTH) has been described as a tool that allows for the identification of membrane protein interactions. In the MYTH system, ubiquitin has been split into two halves, each of which is fused to a protein, at least one of which is membrane bound. Upon interaction of these two proteins, the two halves of ubiquitin are reconstituted and a transcription factor that is fused to the membrane protein is released. The transcription factor then enters the nucleus and activates transcription of reporter genes. Currently, large-scale MYTH screens using cDNA or gDNA libraries are performed to identify and map the binding partners of various membrane proteins. Thus, the MYTH system is proving to be a powerful tool for the elucidation of specific protein-protein interactions, contributing greatly to the mapping of the membrane protein interactome.

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