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. Author manuscript; available in PMC: 2014 Jul 21.
Published in final edited form as: Chem Biol. 2012 Oct 26;19(10):1265–1277. doi: 10.1016/j.chembiol.2012.07.023

Figure 3.

Figure 3

Reactivity of the xUB-xE1(A) pair assembled by UB mutant UB10 and Uba1 mutant A5. (A) UB mutants UB9 and UB10 can be activated by Uba1 mutants A4, A5 and A6 to form UB~E1 conjugates. (B) Polyubiquitination of CHIP using UB10 as the xUB, Uba1-A5 as xE1 and UbcH5a as E2. wtUB transfer to CHIP by wtUba1 and UbcH5a was used as a control. (C) xUB (UB10) transfer to the RING domain of Mdm2 catalyzed by xE1 (Uba1-A5) and UbcH5b. (D) Formation of UB~HECT thioester conjugate between xUB (UB10) and the HECT domain of Rsp5. DTT at 150 mM was added to the xUB transfer reaction in the last lane to cleave the thioester bond of the UB~HECT conjugate. (E) Formation of UB~HECT thioester conjugate between xUB (UB10) and the HECT domain of E6AP. DTT at 150 mM was also added to the xUB transfer reaction in the last lane.