FIGURE 4.

Cdc123 binds domain III of Gcd11 to promote eIF2 complex formation and cell viability. A, binding of yeast Cdc123 and the human ortholog hD123 to domain III of Gcd11. Scheme of Gcd11 (top). Indicated are amino acid positions, archaeal homology domains (open boxes) and eukaryotic extensions (gray boxes). Yeast-2-hybrid assays (middle) were performed with DNA-binding domain (BD)-fusions of Cdc123, hD123, Sui2, or Sui3 and transcription activation domain (AD)-fusions of full-length or truncated versions of Gcd11. Expression of the lacZ-reporter was analyzed by assaying β-galactosidase activity. The mean and S.D. are shown (n = 3, for bars marked with an asterisk n = 6). Protein levels of AD-Gcd11 fusions 1–514 and 1–527 were compared by Western blotting and total protein was visualized by Ponceau staining (bottom). B, multiple sequence alignment of the C-terminal region of a/eIF2γ. Indicated are Gcd11 truncations (numbers above the alignment) and the final β-sheet of domain III (arrow) (38). C and D, C-terminal truncations of Gcd11. Flag-Gcd11 and truncated versions were expressed in gcd11Δ/GCD11 heterozygous diploid strains, immunoprecipitated and analyzed for interaction with Cdc123, Sui2, and Sui3. Protein levels in WCE and anti-flag immunoprecipitates (α-flag IP) were determined by Western analysis (C). Strains were sporulated and meiotic progeny was analyzed by tetrad dissection. Δ, gcd11Δ; +, the indicated flag-GCD11-construct (D).