FIG. 5.

Analysis of E4orf4 binding to the Met30 fragment and an amino-terminal B55α polypeptide. (A) Expression of the yeast Met30 fragment blocks killing by E4orf4. Yeast strain W303-1A previously transformed with pYES2-HA-E4orf4 and pEG202-CDC55 was transformed by vector DNA expressing the 37-residue Met30 fragment. Transformed yeast cells were streaked onto glucose plates or raffinose (Raf) and galactose (Gal) plates and allowed to grow at 30°C for 5 days. Sectors are indicated. (B) Bacterial two-hybrid analysis of E4orf4 interactions with fragments of Met30 and B55α and full-length B55α. Bacterial two-hybrid reporter strain XL1-Blue MRF′KAN was cotransformed with DNAs expressing a pTRG RNAP-α fusion product with the Met30 fragment, B55α (1-124), or full-length B55α, with E4orf4 fused to lambda-cl DNA-binding protein. Transformed cells were first grown on non-ampicillin-containing plates and then streaked onto plates supplemented with 125 μg/ml ampicillin. Cells were allowed to grow at 37°C for 24 h. LGF2, pBT-LGF2 interaction control plasmid encoding the dimerization domain (40 amino acids) of the Gal4 transcriptional activator protein; Gal11, pTRG-GAL11 interaction control plasmid encoding a domain (90 amino acids) of the mutant form of the Gal11 protein; Orf4, pBT-E4orf4; Met30, pTRG-MET30-37 expressing the yeast MET30 fragment; B55 (1-124), pTRG-B55α (1-124) expressing amino acids 1 to 124 of mammalian PP2A B55α; B55, pTRG-B55α expressing full-length mammalian PP2A B55α. No vector, contains just the reporter strain without plasmid transformation.