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. Author manuscript; available in PMC: 2010 Apr 3.
Published in final edited form as: Cell. 2009 Apr 3;137(1):133–145. doi: 10.1016/j.cell.2009.01.041

Figure 6.

Figure 6

Ubc6 and Doa10 contribute to the synthesis of K11 linkages.

(A) Purified GST-Ubc6 and a catalytically inactive mutant (GST-Ubc6m, C87S). When incubated with E1, and Ub for 2 h, Ubc6 was self-ubiquitinated. No free Ub polymers (e.g. dimers, trimers, and etc.) were observed.

(B) The Cys87 residue in Ubc6 is essential for its activity (immunoblotting with GST Ab).

(C) Time course of Ubc6 in vitro ubiquitination reaction.

(D) Comparison of Ubc6 self-ubiquitination (30 min) by WT or R11 Ub.

(E) MS measurement of polyUb linkages on Ubc6 (2 h reaction). The polyUb-Ubc6 was resolved on a SDS gel, excised and analyzed by MS using synthetic peptides as internal standards. Total amount of polyUb linkages was normalized to 100%. ND: not determined.

(F) MS analysis of Ub-R11-Ubc6 conjugates.

(G) Deletion of UBC6, DOA10, or both genes in yeast reduced the global level of K11 linkages. Data in panel E–G are represented as mean and SEM.