Figure 6.
CPB-1 and FBF proteins physically interact. (A) CPB-1, FBF (amino acids 121–614), GLD-1, TAT (the first exon), and MS2 coat protein (amino acids 1–131) were tested in the yeast two-hybrid system using fusions to either the LexA DNA-binding domain or the Gal4 transcriptional activation domain. β-Galactosidase activity per μg of protein is shown (Zhang et al. 1999). (B) The N-terminal 80 amino acids of CPB-1 domain are required for interaction with FBF. C-terminal truncations of the CPB-1 protein were fused to the LexA DNA-binding domain and tested in the yeast two-hybrid system with FBF (amino acids 121–614) fused to the Gal4 transcriptional activation domain. (C) Interaction between a GST–FBF fusion protein and CPB-1. A fusion protein consisting of GST and FBF (GST–FBF) or GST alone was coupled to Sepharose-conjugated glutathione beads and incubated with radiolabeled CPB-1 protein. Bound proteins were eluted from the beads and analyzed by SDS-PAGE. (D) The interaction between FBF and CPB-1 appears to be RNA independent. Radiolabeled CPB-1 protein was made by in vitro translation and the lysate was either treated with RNAse A or mock-incubated without the nuclease. After treatment the lysates were incubated with GST–FBF beads and the bound proteins were eluted and analyzed by SDS-PAGE. RNA was isolated from aliquots of the RNAse or mock-treated lysates and was analyzed by electrophoresis and ethidium bromide staining.