Figure 2.
The Insertion domain of MavC is essential for UBE2N ubiquitination but not for ubiquitin deamidation and MavC self‐ubiquitination. A) Deletion of the Insertion domain of MavC (MavC∆mid) abolished UBE2N ubiquitination but had no effect on ubiquitin deamidation and MavC self‐ubiquitination. Ubiquitination reactions containing MavC, MavCC74A, or MavC∆mid were resolved by SDS‐PAGE and visualized by Coomassie staining (left panel) or immunoblotting with MavC‐specific antibodies (right panel). B) The MavC∆mid mutant did not catalyze UBE2N ubiquitination in cells infected by L. pneumophila. U937 cells were infected with the indicated L. pneumophila strains for 1 h at a MOI of 10, after which the cell lysates separated by SDS‐PAGE were probed by immunoblotting with the indicated antibodies. Note that the MavC∆mid mutant was translocated into host cells at levels higher than wild‐type bacteria. C) MavC mutant lacking the Insertion domain is capable of ubiquitin deamidation. Proteins in reactions containing ubiquitin and MavC or its mutants were separated by native PAGE and visualized by Coomassie staining.