Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Department of Biology, Duke University, Durham, NC 27708, USA.
Mol Plant. 2020 Mar 2;13(3):446-458. doi: 10.1016/j.molp.2019.12.013. Epub 2019 Dec 31.
Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O-fucosyltransferase SPINDLY (SPY) and the O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDO-RESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.
翻译后修饰在精细调控真核生物昼夜节律系统中发挥着至关重要的作用。在植物中，O-糖基化对昼夜节律的影响及其潜在机制仍 largely 未知。O-岩藻糖基转移酶 SPINDLY（SPY）和 O-GlcNAc 转移酶 SECRET AGENT（SEC）是高等植物中两种突出的 O-糖基化酶，在植物生长发育中具有重叠和独特的功能。与 O-GlcNAc 在调节动物昼夜节律中的关键作用不同，我们在此报告，核定位的 SPY 而非 SEC 特异性调节拟南芥昼夜节律的节奏。通过鉴定 SPY 的相互作用组，我们确定了核心昼夜节律组件之一的伪响应调节因子 5（PRR5）作为一种新的与 SPY 相互作用的蛋白质。PRR5 在植物中可被 SPY 进行 O-岩藻糖基化，而 SPY 催化结构域中的点突变消除了 PRR5 的 O-岩藻糖基化。PRR5 的蛋白质丰度在 spy 突变体中显著增加，而 PRR5 的降解速率大大降低，这表明 PRR5 的蛋白水解由 SPY 介导的 O-岩藻糖基化促进。此外，多条遗传证据表明 PRR5 是 SPY 的主要下游靶点，以特异性介导其对昼夜节律的调节。总体而言，我们的发现为 SPY 的 O-岩藻糖基转移酶活性在调节昼夜节律中的特定作用提供了新的见解，并暗示 O-糖基化可能在调节昼夜节律系统中发挥进化保守的作用，在哺乳动物中通过 O-GlcNAc 化，但在高等植物中通过 O-岩藻糖基化。
