NEDD8 links cullin-RING ubiquitin ligase function to the p97 pathway

Abstract

The AAA+ ATPase p97 and its UBA-UBX cofactors are thought to extract ubiquitinated proteins from membranes or protein complexes as a prelude to their degradation. However, for many cofactors ubiquitinated targets have not yet been identified, leaving their biological function unclear. Previous analysis has linked the p97 pathway to cullin-RING ubiquitin ligases (CRLs); here we demonstrate that the human p97 cofactor UBXD7 mediates the p97-CRL interaction through its conserved ubiquitin-interacting motif (UIM). UBXD7 and its yeast ortholog, Ubx5, associate only with the active, NEDD8- or Rub1-modified form of cullins. Disruption of the Ubx5 UIM results in a loss of CRL binding and consequently impedes degradation of a Cul3 substrate. These results uncover an unexpected and conserved role for NEDD8 in linking CRL ubiquitin ligase function to the p97 pathway.

Figure 1. UBXD7 associates with all cullins except CUL5

(a) Flag-tagged UBA-UBX proteins (p47, UBXD8, FAF1, UBXD7 and SAKS1) lacking the UBX domain (ΔUBX) expressed in 293T cells were immunoprecipitated (IP) with anti-Flag antibodies and probed to detect endogenous binding partners CUL2, CUL4, and p97 as indicated. -, no transfection. (b) Same as in a, except cells were transfected with (+) or without (−) vectors encoding Flag-UBXD7 and V5 epitope-tagged CUL 1 through 5. Immunoprecipitations were carried out with antibodies against the Flag or V5 epitope.

Figure 2. The UBXD7-CRL interaction is neddylation dependent

(a–b) 293T cells were transfected with (+) or without (−) the indicated expression constructs and immunoprecipitated (IP) with anti-Flag or anti-HA antibodies prior to being probed with the antibodies indicated. N8-CUL2, NEDD8-conjugated CUL2. (c) Same as in a. HA-CUL1 (K/R) contains the K720R substitution and HA- CUL2 (K/R) contains the K689R substitution. (d) Same as above, except 1 hour prior to cell lysis, cells were treated with the NEDD8 conjugation inhibitor, MLN4924.

Figure 3. UBXD7 directly interacts with neddylated CRLs via its UIM

(a) Wild type UBXD7 protein, indicating its domains. UBA, ubiquitin-associated domain, UAS, UAS/Thioredoxin-like fold domain, UIM, ubiquitin-interacting motif, UBX, ubiquitin regulatory X domain. (b) Recombinant wild-type Flag-UBXD7 or a deletion mutant was incubated with K48-linked polyubiquitin chains prior to immunoprecipitation and western blotting with indicated antibodies. Nx Ub refers to ubiquitin chains of increasing length. (c) A mix (input) of unneddylated and neddylated recombinant full-length CUL2–RBX1 or CUL4a-RBX1 complex was incubated with recombinant WT Flag-tagged UBXD7 or deletion mutants. Following IP with anti-Flag antibodies, recovered proteins were detected by western blotting with indicated antibodies. Nx N8-CUL, cullin modified with 1 or 2 molecules of NEDD8. (d) 293T cells were transfected with full length Flag-tagged UBXD7 or the indicated UBXD7 deletion mutants and treated with MG132. Lysates were immunoprecipitated with anti-Flag antibodies, and co-precipitated endogenous proteins were detected by western blotting with the indicated antibodies. (e) Binding assays were performed as in (c) using a mix of unneddylated and neddylated recombinant CUL2 CTD–RBX1 or CUL2 CTD–RBX2 complex and recombinant WT Flag-tagged UBXD7 or deletion mutants.

Figure 4. The UIM of UBXD7 binds conjugated NEDD8 on CUL2

(a) Modeling of the UBXD7 UIM - NEDD8 interaction using the HRS UIM -ubiquitin crystal structure as a template (PDB code 2D3G, yellow). The UBXD7 UIM in association with NEDD8 is shown in blue. This figure was made in PYMOL. (b) Cells transfected with Flag-UBXD7 (wild type or ΔUBX) with a wild type, deleted (ΔUIM), or substitution mutant UIM were immunoprecipitated with anti-Flag beads and probed with the indicated antibodies. (c) CUL2 was neddylated with wild type (WT) NEDD8 or L8A mutant NEDD8 and binding assays with Flag-UBXD7 were performed as described in Fig. 3c. (d) Same as in (c), except transfections were carried out with wild type Flag-UBXD7 or Flag-UBXD7 in which the UIM was replaced with the UIM of HRS or the first (S5a-1) or second (S5a-2) UIM of S5a. Immunoprecipitations were carried out in either a low (CUL2 (lo)) or medium (CUL2 (me)) stringency binding buffer. (e) Immunoprecipitation of recombinant wild type Flag-tagged UBXD7 or the indicated UBXD7 deletion mutants from a mix (input) containing recombinant full-length CUL2–RBX1 either unmodified or modified with monoubiquitin or NEDD8.

Figure 5. The UIM in yeast Ubx5 promotes UV-dependent degradation of Rpb1

(a) A mix (input) of unmodified SCF and rubylated recombinant SCF (R-SCF) was incubated with recombinant wild type Flag-tagged Ubx5 or the various Ubx5 mutants. Following immunoprecipitation with anti-Flag beads, the recovered proteins were probed with the indicated antibodies. Single and triple mutants are A368Q and E361R, M365E, A368Q respectively. (b) Yeast cultures (wild type, ubx5Δ, ubx5uimΔ) were irradiated with UV and total cell lysates were prepared at the indicated times prior to detection by western blotting using antibodies against Rpb1 and tubulin (tub). (c) Similar as in (b) except western blots were carried out with IR dye-linked secondary antibodies and quantified by LI-COR Odyssey following normalization with tubulin. Error bars show s.e.m., n=3 for each genotype.