Figure 6.

The UND^PUB18 Domain Is Critical for the Ubiquitination of Exo70B1 and Exo70B2 by PUB18 and PUB22, Respectively.

(A) In vitro ubiquitination assays of Exo70B1 and Exo70B2 by PUB18 and ∆UND-PUB18, respectively. Recombinant His-Myc-PUB18 and His-Myc-∆UND-PUB18 E3 Ub ligases were incubated with MBP-Exo70B1 (left panel) or MBP-Exo70B2 (right panel) with or without crude extract (10 μg total proteins) of ABA (100 μM)-treated pub18 pub19 pub22 pub23 quadruple mutant leaves, along with Flag-tagged Ub (Flag-Ub), E1 (UBA1), MG132 (80 μM), and protease inhibitor cocktail. The reaction mixtures were subjected to immunoblot analysis with anti-Flag, anti-MBP, anti-Myc, and anti-α-tubulin antibodies. Ubiquitinated bands were detected by anti-Flag antibody. Equal loading of each lane was confirmed by anti-MBP and anti-Myc antibodies. The level of α-tubulin was used as a loading control of crude extract. Arrows indicate the migration of the unmodified PUB proteins.

(B) In vitro ubiquitination assays of Exo70B2 and Exo70B1 by PUB22 and UND^PUB18-PUB22, respectively. His-Myc-PUB22 and His-Myc-UND^PUB18-PUB22 E3 Ub ligases were incubated with MBP-Exo70B2 (left panel) or MBP-Exo70B1 (right panel) with or without crude extract (10 μg total proteins) of ABA (100 μM)-treated pub18 pub19 pub22 pub23 quadruple mutant leaves, along with Flag-Ub, E1 (UBA1), MG132 (80 μM), and protease inhibitor cocktail. The samples were resolved by SDS-PAGE and immunoblotted with anti-Flag, anti-MBP, anti-Myc, and anti-α-tubulin antibodies. Ubiquitinated bands were detected by anti-Flag antibody. Equal loading of each lane was confirmed by anti-MBP and anti-Myc antibodies. The level of α-tubulin was used as a loading control of crude extract. Arrows indicate the migration of the unmodified PUB proteins.