Figure 4

UBXN7 directly interacts with NEDD8 and cullins in vitro. (A) The UIM motif of UBXN7 directly recognizes NEDD8. The deletion of the UIM motif exclusively reduces UBXN7 binding to NEDD8 while deletion of the UBA domain abolishes the interaction with ubiquitin. NEDD8 or ubiquitin-agarose beads were incubated with the indicated recombinant variants of UBXN7. (B) Wild-type UBXN7 efficiently pulls-down CUL2 irrespective of its modification status (left panel). Bacterially expressed Flag-UBXN7 was pre-incubated with full-length CUL2 either unmodified or partially neddylated and then immunoprecipitated using anti-Flag beads. (C) The in vitro interaction of UBXN7 with full-length CUL2 is not affected by UIM deletion. Wild-type or UIM-deleted Flag-UBXN7 was incubated with a mixture of neddylated and non-neddylated CUL2 and then immunoprecipitated as in (B). (D) Cullin-neddylation increases the binding of full-length and UBA-deleted UBXN7, but not of the UIM-deleted mutant. GSH-beads coated with either non-neddylated or a mixture of neddylated and non-neddylated CUL1(342-776)/GST-RBX1 were incubated with the indicated UBXN7 variants. Naked GSH-beads were used as a control. The Flag western blot in the right panel shows similar input levels for the three UBXN7 variants.