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

Abstract

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.

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

“…To determine the actual membrane topology of human Tmem65, we employed a membrane yeast two-hybrid (MYTH) system, as previously described (Fig. 4c,d) [26][27][28] . To determine protein topography, both N-terminal (TF-Cub-Tmem65) and C-terminal (Tmem65-Cub-TF) 'bait' Tmem65 proteins were tested for protein-protein interactions (PPIs) in the presence of a non-interacting yeast integral membrane protein Ost1p (ref.…”

Interactome mapping suggests new mechanistic details underlying Alzheimer's disease

“…It would be tempting to attribute this limited coverage to the intrinsic properties of many AD-related proteins which, due to their transmembrane or secreted nature, are experimentally difficult to handle. To check whether this was the case, we compared the number of interactions detected for each of the nine seed proteins employed (three secreted, three transmembrane, and three intracellular), and found no significant difference (10.3, 11.7, and 10.3 interactions per protein on average, respectively), although the use of different Y2H setups specially designed to deal with transmembrane proteins might indeed increase the coverage (Snider et al 2010). Thus, in this particular study, failure to detect interactions is likely to be the result of the high stringency applied to our Y2H assays, particularly designed to minimize false-positives, although this criterion might penalize detection of some weak or transient interactions.…”

Conserved Motifs in the Msn2-Activating Domain are Important for Msn2-Mediated Yeast Stress Response

“…To examine whether motif A or motif B is essential for the physical interaction of Msn2 with Gal11, we have utilized the Split Ubiquitin yeast two-hybrid (Y2H) assay. The split ubiquitin is a Y2H assay that is based on conditional proteolysis of ubiquitinated Ura3-conjugated proteins (Snider et al, 2010). This assay was extensively used to monitor interactions between membrane proteins and between transcriptional-activating domains, such as Gal4 and Gcn4, with other cellular proteins (Lim et al, 2007;Snider et al, 2010).…”