Identification of Novel ErbB3-Interacting Factors Using the Split-Ubiquitin Membrane Yeast Two-Hybrid System

Abstract

Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.

Bone Microenvironment and Androgen Status Modulate Subcellular Localization of ErbB3 in Prostate Cancer Cells

“…Interestingly, several transcription factors have been found to associate with ErbB-3 in yeast two-hybrid approaches, including p23/p198 protein (also known as Ebp1; ref. 33), early growth response-1 (34), and the zinc finger protein ZNF207 (34). Yoo, Zhang, and others showed that treating AU565 breast cancer cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3 and subsequent translocation of Ebp1 to the nucleus, where it suppressed androgen receptor-mediated gene transcription (35)(36)(37).…”

Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex

“…PCR products of the ERG28 bait and the 14 preys (14 ergosterol biosynthetic enzyme open reading frames) were digested and cloned into the bait (pCub) and prey (pNub) vectors, respectively (14). The bait construct (pERG28-Cub-TF) and all of the prey constructs (pERGX-HA-Nub) were transformed into the L40 yeast reporter strain and grown on CSM-Leu and CSM-Trp, respectively.…”

“…The bait vector, pCYC-BAIT-Cub-TF (pCub), and prey vector, pADH-PREY-HA-Nub (pNub), were kindly provided by Dr. Igor Stagljar (14). pCub is a CEN plasmid containing LEU2 and a weak yeast CYC1 promoter, multiple cloning sites followed by the C-terminal Cub sequence, and a TF consisting of protein A, LexA, and VP16.…”

“…Although various modifications of the YTH system have been described (18)(19)(20), the analysis of interactions between membrane proteins has remained a significant challenge because of the hydrophobic nature of membrane proteins as well as the fact that integral and membrane-associated proteins often undergo posttranslational modifications or oligomerizations via interactions between transmembrane domains (14). A strategy called the split-ubiquitin membrane YTH system was recently described (14) that takes advantage of the split-ubiquitin approach first described by Johnsson and Varshavsky (21). The N-and C-terminal halves of ubiquitin (22) [split-ubiquitin: N-terminal ubiquitin (Nub) and C-terminal ubiquitin (Cub)] were fused to bait and prey proteins, respectively.…”

“…However, because of the limitations of each of these biochemical methods, it is difficult to obtain reliable information regarding partners in membrane-protein interaction assays (14). For example, Gavin et al (15) used tandem affinity copurification and mass spectrometry in a large-scale approach to characterize multiprotein complexes in S. cerevisiae , and among 1,799 encoded proteins they chose to study, only 40 proteins that were integrally or peripherally associated with membranes were purified, and none was involved in ergosterol biosynthesis.…”