Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Understanding protein interactions is crucial for predicting biological functions and mapping cellular networks.
• Membrane proteins are significant pharmaceutical targets but challenging to study due to their hydrophobic nature.
• High-throughput methods are needed to characterize membrane protein interactions.

Purpose of the Study:

• To describe a protocol for the membrane yeast two-hybrid (MYTH) assay.
• To facilitate the large-scale screening of membrane protein interactors.
• To enable the study of full-length membrane proteins from various organisms.

Main Methods:

• Adaptation of the split ubiquitin principle for in vivo protein-protein interaction sensing.
• Utilizing Saccharomyces cerevisiae as a host for membrane protein interaction studies.
• Detailed protocol for MYTH bait generation, validation, and library screening.

Main Results:

• The MYTH assay provides a potent in vivo sensor for protein-protein interactions.
• The protocol allows for large-scale screening of interactors for full-length membrane proteins.
• The entire MYTH procedure can be completed within 4-6 weeks.

Conclusions:

• The MYTH assay is a powerful technology for characterizing membrane protein interactions.
• This method overcomes challenges associated with studying hydrophobic membrane proteins.
• The described protocol enables efficient and large-scale analysis of membrane protein interactomes.