FIG. 4.
The carboxyl-terminal domain of Fu tethers Fu to Cos2. (A) Fu-tail associates with Cos2. An immunoblot of Cos2, Fu, Fu-tail, and ΔFu from the various Sf21 lysates is shown (top panel). Fu-tail coimmunoprecipitates with Cos2 from lysates that contain Cos2 and Fu-tail (bottom panel). A lysate containing Cos2 and Fu served as a positive control, while one containing Cos2 and ΔFu served as a negative control for binding in this experiment. A lysate containing Fu was used as an immunoblotting control in the bottom panel (data not shown). (B) Fu-tail quantitatively associates with Cos2. Gel filtration analysis of Sf21 lysates containing Cos2 coexpressed with either Fu-tail (top panel) or ΔFu (bottom panel). Fu-tail elutes at fraction 48 when expressed on its own in Sf21 cells. Cos2 and Fu-tail, from the coinfected Sf21 lysate, comigrate at fraction 32, indicating that Fu-tail can quantitatively associate with Cos2. In a Cos2 and ΔFu coinfection, Cos2 elutes at fraction 34 while ΔFu elutes at fraction 51, verifying that these proteins do not interact significantly, at least not with high affinity. (C) A directed yeast two-hybrid assay demonstrating that Fu-tail directly associates with Cos2. Yeast organisms transformed with various vectors were plated on minimal media without leucine, uracil, or adenine. Yeast transformed with the GAL4 activation domain/Fu-tail and the GAL4 DNA binding domain/Cos2 vectors were able to grow (sector 1). As negative controls, Fu-tail (sectors 2 and 4) and Cos2 (sectors 3 and 4) were also subcloned in their antisense orientation. The reciprocal experiment, in which Fu-tail was subcloned as a fusion protein with the GAL4 DNA binding domain and Cos2 was subcloned as a fusion protein with the GAL4 activation domain (sector 5), was also performed. As before, both Fu-tail (sectors 7 and 8) and Cos2 (sectors 6 and 8) were also subcloned in their antisense orientation.