Figure 7. TEB4 facilitates a K48R-specific ubiquitination ligation reaction.
(A) The TEB4 RING finger facilitates the in vitro Lys48-specific linkage of ubiquitin to ubiquitin. The indicated protein components were incubated at 30 °C for 10 min in a reaction buffer containing 25 mM Tris (pH 7.6), 10 mM MgCl2 and 1 mM ATP. Where indicated, the reaction contains 10 nM E1, 10 μM UBC7, 10 μM GST–TEB4 RING and 50 mM poly-His-tagged ubiquitin or the ubiquitin mutant Ub-K48R. Reactions were stopped by the addition of non-reducing SDS sample buffer, and protein components were then separated by SDS/PAGE and visualized by staining with Coomassie Brilliant Blue. The migration of purified HsUbc7 and GST–TEB4 RING are shown in lanes 1 and 2 respectively. A ubiquitin dimer is formed in the reaction containing E1, GST–TEB4 RING, UBC7 and ubiquitin (lane 4) but not when ubiquitin was replaced by the mutant Ub-K48R (lane 5) or in the absence of GST–TEB4 RING (lane 3). (B) Schematic representation of the transfer of thioester-linked ubiquitin on UBC7 to a free ubiquitin. (C) A ubiquitin dimer is formed by the transfer of thioester-linked ubiquitin on UBC7 to a free ubiquitin. A reaction mixture containing 25 mM Tris (pH 7.6), 1 mM MgCl2, 0.5 mM ATP, 1 nM E1, 1 μM UBC7 and 2 μM Oregon Green-labelled ubiquitin [34] was incubated at 30 °C for 1 min to generate UBC7-ubiquitin thioester (UBC7-Ub). The reaction was stopped by the addition of EDTA to a final concentration of 10 mM. The mixture was then incubated at 30 °C for 15 min with the addition of 40 μM of a truncated ubiquitin that lacks the C-terminal Gly-Gly residues and 10 μM of RING proteins as indicated. At the end of the incubation, the reaction was stopped by the addition of non-reducing SDS sample buffer. Proteins were separated by SDS/PAGE and proteins bearing the Oregon Green fluorescence (UBC7-Ub thioester and ubiquitin dimer) were visualized on a fluorescence gel scanner. Free ubiquitin migrated at the dye front and its fluorescence is masked by the presence of Bromophenol Blue in the SDS sample buffer and is not shown here.