FIG. 7.

FF domain repeats interact with multimeric ligand. (A) Sequence of recombinant His-tagged protein containing five copies of the FF domain binding motif YEWDLD, each separated by a five-residue linker (GAGAG). (B) Recombinant YEWDLD oligomer and CA150 FF domains interact in vitro. Constructs expressing the His-tagged YEWDLD motifs were expressed in bacteria. Either 1 or 5 μM GST alone or GST-tagged FF domains 1 to 3 were incubated with the immobilized, multimeric binding sites. One micromolar inputs were loaded in the first two lanes on the left. Immunoblotting with antibodies against GST allowed detection of bound proteins. (C) Oligomerized motifs can compete for Tat-SF1 interaction. Beads carrying either GST alone or recombinant GST-tagged proteins containing FF domains 1 to 3 with or without the tethered binding motifs were mixed with HEK 293 whole-cell lysate. Interacting protein was detected with antibodies against endogenous Tat-SF1. α-Tat-SF1, anti-Tat-SF1. (D) Domain architecture of Tat-SF1, which has two RRM domains. Several regions of the protein, including parts of the N terminus, RRM domain 1 or 2, and C terminus were used to map the interaction with the CA150 FF domains. A point mutation abolishing binding to the FF domain target site YEWDLD (YEAALD) was also made. Fragments are shown at the bottom, with open boxes denoting RRM domains. The number of potential FF domain binding motifs in each segment is shown in parentheses. (E) The FF domains of CA150 are able to associate with several regions of Tat-SF1. Flag-tagged fragments of Tat-SF1 were transfected into HEK 293 cells, and expression was monitored by immunoblotting with anti-Flag (α-Flag) antibodies (top panel). Lysate was incubated with recombinant GST-tagged FF1 to 3 bound to glutathione beads. Proteins retained after washing were detected by immunoblotting (bottom panel). WB, Western blotting.