Fig. 1. VAB-8 and UNC-51 interactions. (A) Yeast two-hybrid interactions between full-length VAB-8 and various UNC-51 fragments. VAB-8L was fused to the GAL4 DNA binding domain (GAL4BD), and UNC-51 fragments were fused to the GAL4 activation domain (GAL4AD). Binding was determined by ß-galactosidase activity using filter lift assays (Durfee et al., 1993). +, most colonies had turned blue overnight; –, no colony had turned blue overnight. (B) GST and GST-UNC-51(750-856) fusion proteins were expressed and purified from E. coli, and bound to glutathione-conjugated beads. Various protein domains of VAB-8 were transcribed and translated in vitro using reticulocyte lysates (see Materials and methods). The + and – signs represent the strength of binding of each VAB-8 fragment to UNC-51(750-856) as compared to GST alone. (C) Domains of VAB-8 sufficient to bind UNC-51(750-856). (D) Schematic representation of binding interactions between UNC-51 and both VAB-8 and UNC-14. The hatched boxes represent the domains that are sufficient for binding. VAB-8 contains two domains that are sufficient to bind to UNC-51.