The N-Terminal UND Motif of the Arabidopsis U-Box E3 Ligase PUB18 Is Critical for the Negative Regulation of ABA-Mediated Stomatal Movement and Determines Its Ubiquitination Specificity for Exocyst Subunit Exo70B1.

The Arabidopsis thaliana U-box E3 ligases PUB18/PUB19 and PUB22/PUB23 are negative regulators of drought stress responses. PUB18/PUB19 regulate the drought stress response in an abscisic acid (ABA)-dependent manner, whereas PUB22/PUB23 regulate this response in an ABA-independent manner. A major structural difference between PUB18/PUB19 and PUB22/PUB23 is the presence of the UND (U-box N-terminal domain). Here, we focused on elucidating the molecular mechanism that mediates the functional difference between PUB18 and PUB22 and found that the UND ^PUB18 was critically involved in the negative regulation of ABA-mediated stomatal movements. Exo70B1, a subunit of the exocyst complex, was identified as a target of PUB18, whereas Exo70B2 was a substrate of PUB22. However, the ∆UND-PUB18 derivative failed to ubiquitinate Exo70B1, but ubiquitinated Exo70B2. By contrast, the UND ^PUB18 -PUB22 chimeric protein ubiquitinated Exo70B1 instead of Exo70B2, suggesting that the ubiquitination specificities of PUB18 and PUB22 to Exo70B1 and Exo70B2, respectively, are dependent on the presence or absence of the UND ^PUB18 motif. The ABA-insensitive phenotypes of the pub18 pub19 exo70b1 triple mutant were reminiscent of those of exo70b1 rather than pub18 pub19, indicating that Exo70B1 functions downstream of PUB18. Overall, our results suggest that the UND ^PUB18 motif is crucial for the negative regulation of ABA-dependent stomatal movement and for determination of its ubiquitination specificity to Exo70B1.
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