齐亚飞等《Plant Physiology》2023年

论文题目：Chloroplast SRP54 and FtsH protease coordinate thylakoid membrane associated proteostasis in Arabidopsis

论文作者：Yang Lei (雷洋), Bilang Li (李碧浪), Xiaomin Wang (王晓敏), Junyou Wei (卫俊佑), Peiyi Wang (王佩仪), Jun Zhao (赵军), Fei Yu (郁飞), Yafei Qi (齐亚飞) *

论文摘要：

Thylakoid membrane protein quality control, which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane protein quality control during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.

类囊体膜蛋白质量控制协同膜蛋白易位和未组装蛋白的降解，能够决定去黄化过程中叶绿体的发育。然而，陆地植物中对类囊体膜蛋白质量控制的理解在很大程度上是未知的。我们报道了拟南芥（Arabidopsis thaliana）中浅绿色突变体pga4，在去黄化过程中存在叶绿体发育缺陷。基于图位克隆和互补实验证实PGA4编码叶绿体信号识别颗粒54kDa蛋白（cpSRP54）。我们创建了光捕获叶绿素A/b结合-绿色荧光蛋白融合蛋白（LhcB2-GFP），作为cpSRP54介导的类囊体易位的指示性底物。LhcB2-GFP不能组装成有功能复合体，在去黄化过程中通过类囊体膜上的N-末端降解为短的dLhcB2-GFP。进一步的生物化学和遗传学证据表明，在pga4突变体和类囊体VAR2/AtFtsH2亚基突变引起的var2突变体中，LhcB2-GFP向dLhcB2-GFP的降解被破坏。酵母双杂交分析表明，LhcB2-GFP的N末端与VAR2/AtFtsH2的蛋白酶结构域相互作用。此外，在pga4和var2中过度积累的LhcB2-GFP形成了不溶于温和的非离子洗涤剂的蛋白质聚集体。在遗传学上，cpSRP54是var2“花斑”表型的抑制基因。这些结果证明了cpSRP54和类囊体FtsH在光合复合物组装过程中在维持类囊体膜蛋白质量控制方面的协同作用，并为监测cpSRP54依赖的蛋白易位和FtsH依赖的蛋白降解提供了可跟踪的底物。

论文链接：https://doi.org/10.1093/plphys/kiad199