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. 2014 Nov 14;26(11):4532–4546. doi: 10.1105/tpc.114.131540

Figure 4.

Figure 4.

ATL1 Possesses E3 Ubiquitin Ligase Activity.

ATL1 autoubiquitinates in vitro. Recombinant ATL1 protein was used for in vitro ubiquitination assays in the presence of the E1 protein Uba2, the E2 protein UbcH5a, and Mg-ATP. Multiple ubiquitinated protein forms were detected when wild-type ATL1 was used (lane 1). No autoubiquitination was detected when the ATL1 RING mutant, ATL1C153A/H155A (lane 2), or His-MBP tag (lane 3) was used. No autoubiquitination was detected when ATL1 was incubated in a reaction lacking the E1 protein (lane 4), the E2 protein (lane 5), or ubiquitin (lane 6). The presence of recombinant protein was detected using antihistidine antibody.