Figure 4. Detailed mapping of the interaction of Tif4631p with Snu71P and Prp11p.

(A) Schematic representation of the deletion mutants of Tif4631p. Pull-down experiments were performed by incubation of immobilized GST-Pp11p or GST-Snu71P with ³⁵S-labeled full-length or truncated Tif4631p mutants and analysed as described in Figure 3A. Densitometry was used to quantify the respective autoradiographs and the quantification results are presented as % percentages of the input protein used for the pull-down experiments. (B) Immobilized GST-Prp11p (lanes 1–7) was incubated with ³⁵S-labeled Tif4631p (wt) or the Tif4632p-mutant proteins indicated, as described in Figure 3. The pulled-down proteins were resolved by SDS-PAGE, and visualized by autoradiography. 1/5 of input ³⁵S-labeled proteins were also analysed (lanes 8–15). (c) Immobilized GST-Prp11p (lanes 4–6) was incubated with ³⁵S-labeled Tif4631p (wt) or the Tif4632p mutants indicated. The pulled-down proteins were resolved by SDS-PAGE, and visualized by autoradiography. GST alone immobilized on Glutathione-Sepharose beads was used as negative control (lanes 7–9). 1/5 of input ³⁵S-labeled proteins were also analysed (lanes 1–3). The full length ³⁵S-labeled proteins are indicated by arrows. (D) Immobilized GST-Snu71p (lanes 4–6) were incubated with ³⁵S-labeled Tif4631p (wt) or the Tif4632p mutants indicated. The pulled-down proteins were resolved by SDS-PAGE, and visualized by autoradiography. GST alone immobilized on Glutathione-Sepharose beads was used as negative control (lanes 7–9). 1/5 of input ³⁵S-labeled proteins were also analysed (lanes 1–3). The full length ³⁵S-labeled proteins are indicated by arrows. (E) Immobilized GST-Prp11p (lanes 1–3) or TAP-Mud13p (lanes 4–6) were incubated with ³⁵S-labeled wild-type Tif4631p (wt) or Tif4631p mutants lacking amino acid residues 491–501 (Δ491-501), or 534–544 (Δ534-544). The pulled-down proteins were resolved by sDs-PAGe and visualized by autoradiography. 1/5 of the input ³⁵s-labeled proteins was also analysed for comparison. (F) Schematic representation of the domain of Tif4631p required for interaction with Prp11p, Snu71p and CBC. Other characterized domains of Tif4631p are also indicated (see text for details).