National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, People's Republic of China.
Plant Physiol. 2013 Aug;162(4):1867-80. doi: 10.1104/pp.113.217604. Epub 2013 Jun 26.
The plastidic caseinolytic protease (Clp) of higher plants is an evolutionarily conserved protein degradation apparatus composed of a proteolytic core complex (the P and R rings) and a set of accessory proteins (ClpT, ClpC, and ClpS). The role and molecular composition of Clps in higher plants has just begun to be unraveled, mostly from studies with the model dicotyledonous plant Arabidopsis (Arabidopsis thaliana). In this work, we isolated a virescent yellow leaf (vyl) mutant in rice (Oryza sativa), which produces chlorotic leaves throughout the entire growth period. The young chlorotic leaves turn green in later developmental stages, accompanied by alterations in chlorophyll accumulation, chloroplast ultrastructure, and the expression of chloroplast development- and photosynthesis-related genes. Positional cloning revealed that the VYL gene encodes a protein homologous to the Arabidopsis ClpP6 subunit and that it is targeted to the chloroplast. VYL expression is constitutive in most tissues examined but most abundant in leaf sections containing chloroplasts in early stages of development. The mutation in vyl causes premature termination of the predicted gene product and loss of the conserved catalytic triad (serine-histidine-aspartate) and the polypeptide-binding site of VYL. Using a tandem affinity purification approach and mass spectrometry analysis, we identified OsClpP4 as a VYL-associated protein in vivo. In addition, yeast two-hybrid assays demonstrated that VYL directly interacts with OsClpP3 and OsClpP4. Furthermore, we found that OsClpP3 directly interacts with OsClpT, that OsClpP4 directly interacts with OsClpP5 and OsClpT, and that both OsClpP4 and OsClpT can homodimerize. Together, our data provide new insights into the function, assembly, and regulation of Clps in higher plants.
高等植物的质体天冬氨酸蛋白酶（Clp）是一种进化上保守的蛋白降解装置，由一个蛋白水解核心复合物（P 和 R 环）和一组辅助蛋白（ClpT、ClpC 和 ClpS）组成。Clp 在高等植物中的作用和分子组成才刚刚开始被揭示，主要来自模式双子叶植物拟南芥（Arabidopsis thaliana）的研究。在这项工作中，我们在水稻（Oryza sativa）中分离到一个黄绿叶突变体（vyl），该突变体在整个生长期间产生黄化叶片。幼嫩的黄化叶片在后期发育阶段变绿，伴随着叶绿素积累、叶绿体超微结构和叶绿体发育及光合作用相关基因表达的改变。定位克隆表明，VYL 基因编码一个与拟南芥 ClpP6 亚基同源的蛋白，它靶向叶绿体。VYL 在大多数组织中持续表达，但在发育早期含有叶绿体的叶片部分表达最丰富。vyl 突变导致预测基因产物过早终止，保守的催化三联体（丝氨酸-组氨酸-天冬氨酸）和 VYL 的多肽结合位点丢失。使用串联亲和纯化方法和质谱分析，我们在体内鉴定出 OsClpP4 是 VYL 的一个相关蛋白。此外，酵母双杂交实验表明 VYL 直接与 OsClpP3 和 OsClpP4 相互作用。此外，我们发现 OsClpP3 直接与 OsClpT 相互作用，OsClpP4 直接与 OsClpP5 和 OsClpT 相互作用，并且 OsClpP4 和 OsClpT 都可以形成同源二聚体。总之，我们的数据为 Clp 在高等植物中的功能、组装和调控提供了新的见解。
