Kiba Takatoshi, Henriques Rossana, Sakakibara Hitoshi, Chua Nam-Hai
Laboratory of Plant Molecular Biology, The Rockefeller University, New York, New York 10065-6399, USA.
Plant Cell. 2007 Aug;19(8):2516-30. doi: 10.1105/tpc.107.053033. Epub 2007 Aug 10.
Circadian clocks comprise several regulatory feedback loops that control gene transcription. However, recent evidence has shown that posttranslational mechanisms are also required for clock function. In Arabidopsis thaliana, members of the PSEUDO-RESPONSE REGULATOR (PRR) family were proposed to be components of the central oscillator. Using a PRR5-specific antibody, we characterized changes in PRR5 protein levels in relation to its mRNA levels under various circadian conditions. Under long-day conditions, PRR5 mRNA levels are undetectable at dusk but PRR5 protein levels remain maximal. Upon dark transition, however, PRR5 levels decrease rapidly, indicating dark-induced, posttranslational regulation. We demonstrated that the Pseudo-Receiver (PR) domain of PRR5 interacts directly with the F box protein ZEITLUPE (ZTL) in vitro and in vivo. Analyses of mutants and transgenic plants revealed an inverse correlation between PRR5 and ZTL levels, which depends on the PR domain. These results indicate that PRR5 is negatively regulated by ZTL, which likely mediates its ubiquitination and degradation. Phenotypic analyses of prr5 ztl double mutants showed that PRR5 is required for ZTL functions. ZTL contains a Light-Oxygen-Voltage domain, and its activity may be directly regulated by blue light. Consistent with this notion, we found that blue light stabilizes PRR5, although it does not alter ZTL levels. Together, our results show that ZTL targets PRR5 for degradation by 26S proteasomes in the circadian clock and in early photomorphogenesis.
生物钟由几个控制基因转录的调节反馈环组成。然而,最近的证据表明,翻译后机制对于生物钟功能也是必需的。在拟南芥中,伪反应调节因子(PRR)家族的成员被认为是中央振荡器的组成部分。我们使用PRR5特异性抗体,表征了在各种昼夜节律条件下PRR5蛋白水平与其mRNA水平相关的变化。在长日照条件下,黄昏时检测不到PRR5 mRNA水平,但PRR5蛋白水平仍保持最高。然而,在黑暗转换时,PRR5水平迅速下降,表明存在黑暗诱导的翻译后调控。我们证明,PRR5的伪受体(PR)结构域在体外和体内都直接与F盒蛋白ZEITLUPE(ZTL)相互作用。对突变体和转基因植物的分析揭示了PRR5和ZTL水平之间的负相关,这取决于PR结构域。这些结果表明,PRR5受ZTL负调控,ZTL可能介导其泛素化和降解。prr5 ztl双突变体的表型分析表明,PRR5是ZTL功能所必需的。ZTL含有一个光-氧-电压结构域,其活性可能直接受蓝光调节。与此观点一致,我们发现蓝光可稳定PRR5,尽管它不会改变ZTL水平。总之,我们的结果表明,在生物钟和早期光形态建成中,ZTL靶向PRR5通过26S蛋白酶体进行降解。