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Plant Physiol. 2018 Jul;177(3):1170-1186. doi: 10.1104/pp.18.00331. Epub 2018 May 23.

Decoys Untangle Complicated Redundancy and Reveal Targets of Circadian Clock F-Box Proteins.

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Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511.
Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093.
Department of Neurology, Keck School of Medicine, University of Southern California, Los Angeles, California 90089.
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511


Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis (Arabidopsis thaliana). These dominant-negative forms of the proteins inhibit the ubiquitylation of target proteins and allow for the study of ubiquitylation-independent and -dependent functions of ZTL, FKF1, and LKP2. We demonstrate the effects of expressing ZTL, FKF1, and LKP2 decoys on the circadian clock and flowering time. Furthermore, the decoy E3 ligases trap substrate interactions, and using immunoprecipitation-mass spectrometry, we identify interacting partners. We focus studies on the clock transcription factor CCA1 HIKING EXPEDITION (CHE) and show that ZTL interacts directly with CHE and can mediate CHE ubiquitylation. We also demonstrate that CHE protein is degraded in the dark and that degradation is reduced in a ztl mutant plant, showing that CHE is a bona fide ZTL target protein. This work increases our understanding of the genetic and biochemical roles for ZTL, FKF1, and LKP2 and also demonstrates an effective methodology for studying complicated genetic redundancy among E3 ubiquitin ligases.

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