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. 2020 Jun 8;9:e55954. doi: 10.7554/eLife.55954

Figure 3. The BBSome recognizes SMO via membrane-embedded residues in SMO helix 8.

(A–B) GST-capture assays were conducted with in vitro translated BBSome subunits tagged with a 6xMyc epitope. Bound material was released by specific cleavage between GST and the fused peptide, and released proteins were detected using a Western blot and anti-Myc antibody (α-Myc WB). The proportions of BBSome subunits recovered in the eluate are plotted; grey circles are individual data points and blue lines are mean values. Even loading of the glutathione beads is demonstrated by staining for the remaining GST-tagged proteins after cleavage elution. (A) Capture of individual BBSome subunits with GST-SMOCtail (aa 543–793) identifies BBS7 as the SMO-binding subunit. (B) Capture assays with truncations of SMOCtail find that SMOH8 is necessary and sufficient for binding to BBS7. (C) Yeast two-hybrid (YTH) assays with SMOCtail against an array of BBS protein fragments identify an interaction between a C-terminal fragment of BBS7 (BBS7C, residues 326–672) and SMOCtail. The composition of the BBS YTH array is shown in Supplementary file 2. (D) YTH assays find that SMOH8 is required for the interaction with BBS7C. Growth controls on diploid-selective medium for panels (C–D) are shown in Figure 3—figure supplement 1C.

Figure 3.

Figure 3—figure supplement 1. Capture assays, controls for YTH and SMOH8 conformation, and BBSome-binding motif in ciliary GPCRs.

Figure 3—figure supplement 1.

(A) Capture of individual BBSome subunits with GST-SSTR3i3 identifies BBS7 as the SSTR3i3-binding subunit. (B) Capture of individual BBSome subunits with GST fused to the first 19 amino acids of SMOCtail (aa 543–559) identifies BBS7 as the SMO-binding subunit. (C). Controls for diploid growth of the yeast array on medium lacking tryptophan and leucine tested in Figure 3C–D. (D) Structural overlay of helix 8 from different human Smoothened proteins (PDB IDs: 4JKV, 4N4W, 4O9R, 4QIM, 4QIN, 5L7D, 5V56, 5V57, 6D32, 6D35, 6OT0). The side chains of the two critical tryptophan residues are shown as sticks. The numbering of the residues is based on mouse Smoothened.