Figure 3.
AtAIRP2 is a cytosolic RING E3 Ub ligase. A, In vitro E3 Ub ligase assay. Left panel, bacterially expressed MBP-AtAIRP2 was incubated with ATP in the presence or absence of Ub, Arabidopsis E1 (His-UBA1), and Arabidopsis E2 (His-UBC8) at 30°C for 2 h. Reaction mixtures were separated by SDS-PAGE and subjected to immunoblot analysis using either anti-MBP antibody or anti-Ub antibody. Right panel, MBP-AtAIRP2 and single-amino acid substitution mutant MBP-AtAIRP1H163A were incubated at 30°C for 2 h in the presence of ATP, Ub, E1, and E2. Ubiquitinated proteins were detected by either anti-MBP or anti-Ub antibody. WT, Wild type. B, Cytosolic localization of AtAIRP2. 35S:sGFP, 35S:AtAIRP2-sGFP, 35S:AtAIRP1-sGFP, and 35S:AREB1-sGFP gene constructs were transformed into onion epidermal cells using particle bombardment. Localization of the expressed proteins was visualized by fluorescence microscopy (dark and bright fields) in both unplasmolyzed and plasmolyzed onion cells. Arabidopsis AREB1 and RING E3 Ub ligase AtAIRP1 were used as specificity controls for nuclear and cytosolic proteins, respectively. DAPI, 4′,6-Diamino-phenylindole. Bars = 100 μm. [See online article for color version of this figure.]