Decoys untangle complicated redundancy and reveal targets of circadian clock F-box proteins

First author: Chin-Mei Lee; Affiliations: Yale University (耶鲁大学): New Haven, USA

Corresponding author: Joshua M Gendron

Eukaryotic circadian clocks utilize the ubiquitin proteasome (泛素蛋白酶体) system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering (光周期开花) 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 (泛素化) 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 (免疫沉淀质谱法) 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 degradation is reduced in a ztlmutant plant, showing that CHE is a bona fide (真实的) 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.

真核生物昼夜节律利用泛素蛋白酶体来精确调控周期蛋白的降解。在植物中，F-box类E3泛素连接酶ZTL、FLAVIN-BINDING、KELCH REPEAT、FKF1以及LKP2调控周期途径，并偶联周期在响应昼夜光照条件时作用于植物的光周期开花。为了更好的理解这些基因的生物学功能，作者在拟南芥中表达了一些带有标记但不能使得其靶标泛素化的ZTL、FKF1和LKP2诱饵蛋白。这些诱饵蛋白抑制了其靶蛋白的泛素化进程，能够帮助进一步研究ZTL、FKF1和LKP2依赖和不依赖于泛素化的功能。作者的研究揭示了这些诱饵蛋白对于生物钟和开花时间的作用。此外，E3连接酶诱饵抑制了底物之间的相互作用，利用免疫沉淀质谱法。作者鉴定了一些相互作用伴体。作者对生物钟转录因子CHE进行了进一步的研究，结果显示ZTL会直接与CHE进行互作，并介导CHE的泛素化。本文的研究还显示CHE蛋白在黑暗条件下降解，并且在ztl突变体中，CHE蛋白的降解减缓，说明CHE确实是ZTL的靶向蛋白。本文的工作进一步提升了我们对于ZTL、FKF1和LKP2的分子功能的理解，并提供了一种关于研究众多E3泛素连接酶遗传冗余的有效方法。

通讯：Joshua M Gendron(https://mcdb.yale.edu/people/joshua-gendron)

个人简介：加利福尼亚大学，生物科学学士；斯坦福大学，生物学博士；加利福尼亚大学，博士后。

研究方向：植物中蛋白降解是如何调控生物钟的。

doi: https://doi.org/10.1104/pp.18.00331

Journal: Plant Physiology

Published date: 23 May, 2018

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