Utilizing the Split-Ubiquitin Membrane Yeast Two-Hybrid System to Identify Protein-Protein Interactions of Integral Membrane Proteins

Abstract

Various modifications of the conventional yeast two-hybrid system have played an essential role in confirming or detecting protein-protein interactions among nuclear and cytoplasmic proteins. These approaches have permitted the identification of novel interaction partners, as well as provided hints as to their function. However, membrane proteins, such as receptor tyrosine kinases, G protein-coupled receptors, membrane-bound phosphatases, and transporters, which represent important classes of signaling molecules, are difficult to study using classical protein interaction assays because of their hydrophobic nature. Here, we describe a genetic system that allows the identification of integral membrane-interacting proteins. This so-called "split-ubiquitin membrane-based yeast two-hybrid assay" involves fusing the halves of ubiquitin to two interacting proteins, at least one of which is membrane bound. Upon interaction of these two proteins, the halves of ubiquitin are brought together, and the transcription factor that is fused to a membrane protein of interest is cleaved and released. The free transcription factor then enters the nucleus and activates transcription of reporter genes. We also describe how this technology is used to screen complementary DNA libraries to identify novel binding partners of a membrane protein of interest.

Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension

“…Cultures were grown at 30°C. The yeast medium used in iMYTH screening is described in Iyer et al (36).…”

“…However, the highly hydrophobic nature of transmembrane proteins such as Ycf1p complicates the use of these standard methods (36,37). We recently reported the development of iMYTH, a modified version of the split ubiquitin membrane yeast two-hybrid system, showing that it is a powerful method for identifying physiological regulators of membrane proteins such as the ABC transporters (34,37).…”

Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters inArabidopsis

“…The full-length cDNAs of KUP6 and SRK2E/OST1 were cloned in frame with either the C-terminal (Cub) or N-terminal (NubG) subdomain of ubiquitin and then introduced into yeast strain NMY32, as described previously (Iyer et al, 2005). C-terminal regions of KUP6 (1812 to 2348 nucleotides) and KUP8 (1826 to 2346 nucleotides) cloned into pGADT7 were also used to detect interactions with SnRK2s cloned into pGBKT7 in the yeast strain AH109 by the general yeast twohybrid system (Clontech).…”

ABC Transporters in Saccharomyces cerevisiae and Their Interactors: New Technology Advances the Biology of the ABCC (MRP) Subfamily

“…One such approach is the MYTH system, which has recently been developed into a powerful alternative to the traditional YTH system, retaining all of the latter method's advantages yet being suitable for the analysis of proteins localized to a membrane environment (47,71,81,119,138,151,156) (Fig. 7).…”

“…"Baits" are generated by the fusion of C ub -TF to the C terminus of a membrane-anchored protein of interest, which prevents TF from diffusing into the nucleus and activating the transcription of the reporter genes (151). "Preys," in the form of either specifically selected proteins or entire libraries, are generated by the fusion of the N ub G moiety to their N or C termini (71,81). Yeast cells expressing the bait are transformed with prey constructs and plated onto selective medium.…”