Mapping the functional yeast ABC transporter interactome.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.

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

The biological function of proteins may be predicted by identification of their interacting partners, and one of the major goals of the postgenomic era is the mapping of protein interaction networks. Membrane proteins are of particular interest because of their role in disease and because of their prevalence as major pharmaceutical targets. Unfortunately, because of their hydrophobic nature, they have long been difficult to study in a high-throughput format. A powerful technology recently developed to facilitate the characterization of membrane protein interactions is the membrane yeast two-hybrid (MYTH) assay. MYTH adapts the principle of split ubiquitin for use as a potent in vivo sensor of protein-protein interactions, allowing large-scale screening for interactors of full-length membrane proteins, from a range of organisms, using Saccharomyces cerevisiae as a host. In this article, we describe a protocol for MYTH bait generation, validation and library screening. The entire MYTH procedure can generally be completed in 4-6 weeks.