FIGURE 3.

Analyses of amino acid sequence of C-tail necessary for its binding to 9^ΔC-1-1. A, schematic illustration of human C-tail and its deletions used in this study. C-tail, C-N, and C-C contain aa 272–391, aa 272–332, and aa 333–391, respectively. Constructs Δ336, Δ341, Δ351^d5, Δ351, Δ356, Δ361, and Δ371 harbor internal deletions of aa 336–345, aa 341–350, aa 351–355, aa 351–360, aa 356–365, aa 361–370, and aa 371–380 from C-C, respectively; C-tail^Δ351d5 and C-tail^Δ351 are the same deletions as Δ351^d5 and Δ351, respectively, from the C-tail. C-tail^FFAA harbors Ala substitutions at both Phe-365 and Phe-366, which were expressed as fusions with GST/FLAG tags at their N-terminal portion. Below is the sequence of the C-tail, including the 15-aa stretch (aa 351–375) required for interaction with CRS. The NLS and FF motif (Phe-365/Phe-366) are boxed in white and black, respectively. B, glutathione-Sepharose beads pre-bound with GST only (mock), GST/FLAG-tagged C-tail (C-tail), C-tail mutant (C-tail^FFAA), or its deletions (C-N, C-C, Δ336, Δ341, Δ351^d5, Δ351, Δ356, Δ361, Δ371, C-tail^Δ351d5, and C-tail^Δ351) were mixed with 10 pmol alone (left panel) or 5 and 10 pmol (+, and ++, respectively) (right panel) of purified 9^ΔC-1-1, and incubated at 4 °C for 1 h. After washing the beads, 50 fmol (left panel) and 100 fmol (right panel) of purified 9^ΔC-1-1 and 10% (left panel) or 20% (right panel) of the bound fractions were analyzed by immunoblotting using anti-Rad1 antibody (upper panels) and staining with Ponceau S for the C-tail and its deletions (lower panels). Asterisks indicate degraded C-tail fragments.