Figure 2.

OpsDHN1-OpsDHN1 protein interaction using the split-ubiquitin yeast two-hybrid assay. (A) Schematic representation of OpsDHN1 bait and prey constructs. In the pDHB1 construct, the OpsDHN1 gene was fused at its N-terminal to the Ost4 membrane protein, and its C-terminal to the ubiquitin C-terminal moiety. LexA-VP16, the artificial transcription factor, is fused at the C-terminal end of ubiquitin. This construct is under control of the ADH1 promoter region and CYC1 terminator region. In the pPR3-N-prey construct, the ubiquitin N-terminal was fused to the OpsDHN1 gene. This construct is under control of CYC1 promoter region and CYC1 terminator region. (B) Yeast cells carrying the control system interaction LargeT-Cub/Δp53-NubG, and functional assay of OpsDHN1 SK₃ as bait construct using prey control system vectors: NubI, NubG and pPR3-N, and OpsDHN1-Cub/OpsDHN1-NubG interaction. Yeast strains was plated to an OD600 of 0.8, and at serial 10-fold dilutions on semi-selective (SD-LW) and on selective (SD-LWHA) media supplemented with 3-AT (45 and 55 mM). Quantitative β-Galactosidase activity was assayed by hydrolysis of the o-nitrophenyl-b-galactoside (ONPG), as expressed in nmol ONP/min per mg of protein. Data represent the mean ± SD, (n = 3).