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

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

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.

Publication types

  • Validation Study

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Computational Biology
  • DNA Primers / genetics
  • DNA, Recombinant / genetics
  • Genetic Vectors
  • Membrane Proteins / analysis*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Molecular Sequence Data
  • Multiprotein Complexes / analysis
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism
  • Plasmids / genetics
  • Protein Interaction Mapping / methods*
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Transformation, Genetic
  • Two-Hybrid System Techniques* / statistics & numerical data
  • Ubiquitin / metabolism

Substances

  • DNA Primers
  • DNA, Recombinant
  • Membrane Proteins
  • Multiprotein Complexes
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin