Figure 3. Interaction of the CrIFT38 CH domain with a conserved surface patch on CrIFT80 BP1.

(A) Mapping of evolutionarily conserved surface-residues onto the CrIFT80 crystal structure. Two orientations (related to each other by a 180° rotation) are shown. The schematic drawing below the structure indicates the relative positions of individual domains. In the orientation on the left, several highly conserved residues (forming a pronounced patch) are shown at the top of CrIFT80 BP1. (B) GST-pulldown assays showing that the CrIFT80 BP1 domain is sufficient for interaction with CrIFT38CH. (C) The CrIFT80(BP1)/CrIFT38CH complex can be reconstituted by SEC. After mixing CrIFT80(BP1) with a slight excess of CrIFT38CH, a peak for the stable complex is obtained (left), and SDS-PAGE analysis shows that the complex contains stoichiometric levels of the two proteins (right). (D) Isothermal titration calorimetry (ITC) analysis of the interaction of CrIFT38CH with CrIFT80(BP1-BP2). The two proteins form a complex with a dissociation constant of 225 nM.

Figure 3—figure supplement 1. The C-terminus of IFT80 is required for IFT54/20 interaction.

(A) Pull-down of full-length and 1–582 CrIFT80 by CrIFT54-IFT20-GST demonstrating that the C-terminal residues (583 C) of IFT80 are required for the IFT54/20 interaction. (B) Pull-down demonstrating that the N-terminal CH-domain of IFT54 (residues 1–133) is not required for the interaction between IFT80 and IFT54/20. (C) Deletion of the C-terminal 655 C residues of IFT80 significantly reduces the interaction with IFT54/20 in GST pull-downs. (D) In-gel fluorescence measurement of GFP intensity in IMCD3 cells used in the rescue experiments in Ift80 null-mutant IMCD3 cells shown in Figure 9B and C. (E) Quantification of in-gel GFP fluorescence as shown in D), normalized to total protein amount and displayed as -fold intensity compared to IFT80fl. n = 4, mean ±SD, one-sample t-tests compared to 1 (bold) and paired t-test (italic).