Chloroplast SRP54 and FtsH protease coordinate thylakoid membrane-associated proteostasis in Arabidopsis
Chloroplast SRP54 and FtsH protease coordinate thylakoid membrane-associated proteostasis in Arabidopsis
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DOI：
10.1093/plphys/kiad199
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发表时间：
2023-03-30
期刊：
影响因子：
7.4
通讯作者：
Qi，Yafei
中科院分区：
文献类型：
--
作者：
Lei，Yang;Li，Bilang;Qi，Yafei
类囊体膜蛋白质量控制（PQC）需要协调膜蛋白易位和未组装蛋白的降解，决定去黄化过程中叶绿体的发育。尽管付出了很多努力，但陆地植物中这一过程的调控在很大程度上还是未知的。在这里，我们报道了拟南芥（Arabidopsis thaliana）中淡绿色拟南芥4（pga4）突变体的分离和表征，该突变体在去黄化过程中叶绿体发育存在缺陷。基于图谱的克隆和互补测定证实 PGA4 编码叶绿体信号识别颗粒 54 kDa (cpSRP54) 蛋白。生成异质光捕获叶绿素/b结合绿色荧光蛋白 (LhcB2-GFP) 融合蛋白作为 cpSRP54 介导的类囊体易位的指示性报告基因。 LhcB2-GFP 功能失调，并在去黄化过程中通过类囊体膜上引发的 N 端降解降解为短形式 dLhcB2-GFP。进一步的生化和遗传学证据表明，在类囊体 FtsH 的丝状温度敏感 H2 (VAR2/AtFtsH2) 亚基突变引起的 pga4 和黄色杂色 2 (var2) 突变体中，LhcB2-GFP 降解为 dLhcB2-GFP 受到破坏。酵母双杂交实验表明 LhcB2-GFP 的 N 末端与 VAR2/AtFtsH2 的蛋白酶结构域相互作用。此外，pga4和var2中过度积累的LhcB2-GFP形成蛋白质聚集体，不溶于温和的非离子洗涤剂。从遗传学上来说，cpSRP54是var2叶片杂色表型的抑制基因座。总之，这些结果证明了 cpSRP54 和类囊体 FtsH 在光合复合物组装过程中维持类囊体膜 PQC 的协调作用，并为监测 cpSRP54 依赖性蛋白质易位和 FtsH 依赖性蛋白质降解提供了可追踪的底物和产物。
Thylakoid membrane protein quality control (PQC), 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 ofpale green Arabidopsis4(pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed thatPGA4encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophylla/bBinding-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 inpga4andyellow 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 inpga4andvar2formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically,cpSRP54is a suppressor locus for the leaf variegation phenotype ofvar2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.