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. Author manuscript; available in PMC: 2017 Aug 25.
Published in final edited form as: Cell. 2016 Aug 25;166(5):1198–1214.e24. doi: 10.1016/j.cell.2016.07.027

Figure 6. Distinctive and Specialized Roles of Neddylation, CUL1, and RBX1 in Employing ARIH1 to Target a CRL Substrate.

Figure 6

(A) CUL1 and RBX domains (Zheng et al., 2002), denoting previously known functional interactions.

(B) Pulse-chase assay as in Figure 2A testing role of neddylation in UBCH7-ARIH1-mediated *UB transfer to pCyE with indicated versions of CRL1FBW7ΔD (± neddylation, with NEDD8 Q40E mutant that does not support CUL1-RBX1 conformational change, or with CUL1 ΔWHB lacking domain with neddylation site).

(C) Assay as in (B) except with ARIH1OPEN mutant that is not autoinhibited. ARIH1OPEN overcomes defects from lack of neddylation, indicating role of CRL neddylation in relieving ARIH1 autoinhibition.

(D) Roles of neddylation in promoting ARIH1 “opening” as probed reactivity with *UB-VME, showing defect caused by Q40E and that neddylation is not mimicked by deleting CUL1's WHB domain.

(E) Role of CUL1 N-terminal domain, specifically the CR3 subdomain, in relieving ARIH1 autoinhibition, tested by ARIH1 autoubiquitylation in presence of indicated versions of neddylated CUL1-RBX1 (no SR, no substrate).

(F) Role of CUL1 N-terminal domain, specifically the CR3 subdomain, in stimulating ARIH1 “opening,” probed by ability of indicated versions of neddylated CUL1-RBX1 to stimulate ARIH1 reactivity with *UB-VME.

(G) Effects of RBX1 mutants on ARIH1-mediated ubiquitylation of neddylated CRL substrate, mapped on conventional RING E3-E2 (RBX1-UBC12) structure (Scott et al., 2014). Green, normal; raspberry, marginal defect; red, strong defect (data in Figure S6A).

(H) Role of RBX1 RING on ARIH1-neddylated CRL pathway tested by RBX1/RBX2 domain swaps, assayed as in (B).

(I) Role of RBX1 RING in neddylated CUL1-RBX1-dependent “opening” of ARIH1, assayed as in (D).

(J) Second, the distinctive role of RBX1 RING (directing UB transfer from ARIH1 to neddylated CRL substrate) uncovered by use of ARIH1OPEN mutant. In the absence of autoinhibition, this assay directly tests CRL substrate (pCyE) targeting by the indicated RING mutations in neddylated CRL1FBW7ΔD. Right-most lanes, control confirming ARIH1OPEN autoubiquitylation in absence of neddylated CRL.