Decoys provide a scalable platform for identification of plant E3 ubiquitin ligases that regulate circadian function
- Yale University, United States
- South China University of Technology, China
Abstract
The circadian clock relies on regulated degradation of clock proteins to maintain rhythmicity. Despite this, we know few components that mediate protein degradation. This is due to high levels of functional redundancy within plant E3 ubiquitin ligase families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian function, we generated a library of transgenic Arabidopsis plants expressing dominant-negative 'decoy' E3 ubiquitin ligases. We determined their effects on the circadian clock and identified dozens of new potential regulators of circadian function. To demonstrate the potency of the decoy screening methodology to overcome redundancy and identify bona fide clock regulators, we performed follow-up studies on MAC3A (PUB59) and MAC3B (PUB60). We show that they redundantly control circadian period by regulating splicing. This work demonstrates the viability of ubiquitin ligase decoys as a screening platform to overcome genetic challenges and discover E3 ubiquitin ligases that regulate plant development.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data is provided for Figures 2,3,4,6, S1 and S2.
Article and author information
Author details
Funding
National Science Foundation (EAGER #1548538)
- Joshua M Gendron
National Institutes of Health (R35 GM128670)
- Joshua M Gendron
Gruber Foundation
- Ann Feke
Rudolph J. Anderson Fund
- Chin-Mei Lee
Forest B.H. and Elizabeth D.W. Brown Fund
- Wei Liu
- Chin-Mei Lee
National Science Foundation (GRFP DGE-1122492)
- Ann Feke
National Institutes of Health (T32 GM007499)
- Ann Feke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jürgen Kleine-Vehn, University of Natural Resources and Life Sciences, Austria
Version history
- Received: December 20, 2018
- Accepted: April 4, 2019
- Accepted Manuscript published: April 5, 2019 (version 1)
- Version of Record published: April 25, 2019 (version 2)
Copyright
© 2019, Feke et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Decoys provide a scalable platform for identification of plant E3 ubiquitin ligases that regulate circadian function
eLife 8:e44558.
https://doi.org/10.7554/eLife.44558
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Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.
