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. Author manuscript; available in PMC: 2024 Apr 24.
Published in final edited form as: Dev Cell. 2023 Apr 4;58(8):677–693.e9. doi: 10.1016/j.devcel.2023.03.003

Figure 1. Absence of detectable binding of the BBSome and IFT complexes to UbK63 chains.

Figure 1.

A. Diagram of the biochemical strategy. Recombinant GST-NEDD4 bound to glutathione (GSH) resin was incubated with E1 (UBA1), E2 (UBCH5), ATP, and ubiquitin to grow UbK63 polyubiquitin chains onto NEDD4. UbK63 chains grown on immobilized NEDD4 capture ubiquitin-binding proteins (UBP) from cell or tissue extracts, and proteins bound to UbK63 chains are specifically eluted by cleaving ubiquitin chains with the deubiquitinase USP2. B-D. Top panel: Coomassie-stained gel of bead-bound GST-NEDD4 after completion of autoubiquitination reaction. Ub was omitted from the ubiquitination reaction for the control. Middle and lower panels show the products of the treatment with USP2. Middle panel: Coomassie-stained gel of bead-bound GST-NEDD4 ± (Ub)n shows that Ub chains are fully digested by USP2. Lower panel: Coomassie-stained gel of material released from beads by USP2 treatment. Ubiquitin is only present when chains have been assembled on NEDD4. Bottom panels: western blots of known UBP and ciliary trafficking components. 0.1% input and 42% of cleavage eluates were loaded for the western analyses. In B and C, bovine retinal extracts were applied onto GST-NEDD4 ± (Ub)n beads. Myosin VI (MYO6) and TOM1/TOM1L2 are known UbK63 readers and efficient binding is detected. No binding to UbK63 is detected for the IFT-A complex subunits IFT122 and IFT139, the IFT-B complex subunits IFT172 and IFT38, the BBSome subunits BBS9, BBS4 and BBS5, or the BBSome-associated protein LZTFL1. In D, BBSome purified from bovine retina was applied onto GST-NEDD4 ± (Ub)n beads. E. IMCD3 cells stably expressing GFP-tagged TOM1L2, BBS5, BBS4, or BBS1 were lysed, and extracts were passed over GST-NEDD4 ± (Ub)n beads. Lower panels: Coomassie-stained gel showing Ub release by USP2. Upper panels: Immunoblotting for GFP reveals specific binding of TOM1L2 to UbK63 chains, but not of the BBSome subunits BBS1, BBS4, or BBS5. GST-NEDD4 without Ub in the chain building reaction served as a control.