Outline of the split-ubiquitin membrane yeast two-hybrid system. (A) A membrane bait protein of interest X is fused to Cub followed by an artificial transcription factor (TF), while another membrane (or cytoplasmic) protein Y is fused to the NubG domain (Y-NubG). On interaction of the X and Y proteins, ubiquitin reconstitution occurs, leading to proteolytic cleavage by UBPs, and the subsequent release of the transcription factor. This factor activates reporter genes to result in HIS3⁺/lacZ⁺ yeast cells. (B) If X and Y do not interact, there is no ubiquitin reconstitution and thus no UBP-mediated cleavage, resulting in HIS^-/lacZ^- yeast cells.

Maps of novel vectors for the expression of Type I transmembrane bait and prey proteins in the split-ubiquitin membrane yeast two-hybrid system. (A) The bait vector pCYC-BAIT-Cub-TF is a LEU2-based low copy number (CEN/ARS) vector bearing a weak yeast CYC1 promoter, the MCS, and the Cub domain followed by the TF. The foreign cDNA sequence encoding a transmembrane bait protein of interest is introduced into the MCS in frame to Cub-TF portion. Also shown is the MCS sequence upstream of the Cub-TF fusion containing the unique XbaI, SpeI, PstI, and HindIII restriction sites. (B) The prey vector pADH-PREY-2HA-NubG is a TRP1-based multicopy (2μ) vector bearing a strong yeast ADH1 promoter, the MCS, and two HA tags followed by the NubG domain. The cDNA or a library of genomic or cDNA fragments is fused in frame to the NubG cassette. Also shown is the MCS sequence upstream of the two HA-NubG cassettes containing the unique restriction sites NdeI, NcoI, SmaI, and BamHI. Both bait and prey vectors were constructed as described in the Methods section.

(A) The structure of the ErbB3-Cub-TF bait protein used in this study. Like other members of the ErbB family, ErbB3 is a type I transmembrane protein consisting of an extracellular ligand binding domain (blue dotted box), a single membrane-spanning region (striped box), and a cytoplasmic protein tyrosine kinase domain (blue open box). The ErbB3 bait was fused to Cub (red box), followed by an artificial transcription factor (TF; green box). The number of amino acids of ErbB3, Cub, and TF portions are indicated. (B) Growth of yeast cells expressing ErbB3-Cub-TF bait with various Nub-fusions on agar plates lacking tryptophan and leucine (left), and tryptophan, leucine, and histidine containing 10 mM 3-aminotriazole (3-AT; middle). The L40 yeast reporter strain was cotransformed with the ErbB3-Cub-TF bait and indicated prey plasmids, and three independent colonies were grown on Leu^-Trp^- and Leu^-Trp^-His^-selective plates prior to assessment of β-galactosidase activity using X-gal filter test (right). (C) ErbB3 is localized within the yeast membrane. Cytosolic (lanes 1 and 3) and membrane (lanes 2 and 4) fractions of yeast cells expressing the ErbB3-Cub-TF bait were subjected to SDS-PAGE. The insoluble fraction (lane 2) was treated with 1% Triton X-100 to solubilize the proteins, and centrifuged to separate the soluble (lane 3) from insoluble proteins (lane 4). The ErbB3-Cub-TF bait and a control endogenous yeast membrane protein Sec61p were detected by immunoblot analysis using a mouse monoclonal anti-ErbB3 antibody (upper panel), and an anti-Sec61 polyclonal antibody (lower panel). The positions of molecular markers are indicated.

ErbB3 and RGS4 form a complex in human cells. Cell lysates of HEK293T cells coexpressing ErbB3 and RGS4 were immunoprecipitated with either IgG antibody (lanes 2 and 3) or goat polyclonal anti-RGS4 antibody (lanes 4 and 5) using either 100 mM (lanes 2 and 4) or 150 mM NaCl (lanes 3 and 5), and analyzed by immunoblotting with either rabbit polyclonal anti-RGS4 antibody (upper panel) or mouse monoclonal anti-ErbB3 antibody (lower panel). One-tenth of the same extract was used as the input control (lane 1).

Mapping of the ErbB3 interaction domain within RGS4. (A) RGS4-NubG deletion constructs. RGS4 contains an N-terminal amphipathic region important for membrane localization (amino acid 1–33, striped box) followed by the RGS box (amino acid 58–178, grey box) and the C-terminal tail (amino acid 179–205). The NubG domain of ubiquitin is shown as a dotted ellipse. (B) Quantitative β-galactosidase assay showing the binding of different RGS4-NubG deletion mutants to the ErbB3-Cub-TF bait. Values given are the mean of four transformants, each assayed at least twice. The asterisk indicates the original RGS4 clone isolated in the split-ubiquitin membrane yeast two-hybrid screen. (C) Expression and localization of the RGS4–2HA-NubG deletion mutants. The RGS4–2HA-NubG deletion mutants were generated by PCR followed by in vivo recombination into the prey plasmid pADH-PREY-2HA-NubG previously digested with NdeI. Western blot analyses were performed using membrane (left panels) and cytosolic (right panels) fractions of the yeast L40 reporter strain expressing ErbB3-Cub-TF and the prey NubG (lane 1), RGS4-NubG (1–178; lane 2), RGS4-NubG (1–205; lane 3), RGS4-NubG (34–205; lane 4), RGS4-NubG (1–148; lane 5), RGS4-NubG (58–178; lane 6), and RGS4-NubG (58–148; lane 7). The blots were incubated with anti-RGS4 antibody. The positions of molecular markers are indicated. The bands that are not marked with an asterisk are considered as degradation products of RGS4. The RGS4-NubG (1–178) protein found in the split-ubiquitin membrane yeast two-hybrid screen (lane 2) migrates more slowly than the RGS4-NubG (1–205; lane 3) because it contains an additional 45 amino acids upstream of the initial RGS4 start codon.