Determination of a Chloroplast Degron in the Regulatory Domain of Chlorophyllide a Oxygenase
Determination of a Chloroplast Degron in the Regulatory Domain of Chlorophyllide a Oxygenase
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DOI：
10.1074/jbc.m109.008144
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发表时间：
2009-12-25
影响因子：
4.8
通讯作者：
Tanaka, Ayumi
中科院分区：
文献类型：
--
作者：
Sakuraba, Yasuhito;Tanaka, Ryouichi;Tanaka, Ayumi
叶绿素b是植物的主要光合色素之一。叶绿素b的生物合成调控对植物适应变化的环境条件具有重要意义。在叶绿体中，叶绿素b由叶绿素a通过叶绿素内酯加氧酶（CAO）合成，这是一种rieske型单加氧酶。该酶的活性通过叶绿素b的反馈机制在蛋白质稳定性水平上受到调节。Clp蛋白酶和CAO的n端结构域（称为a结构域）对调节机制至关重要。在这项研究中，我们的目的是确定特定的氨基酸残基或序列内的A域是必不可少的这种调节。为了实现这一目标，我们在拟南芥的A结构域中随机引入碱基替换，并通过分析1000个转化子来寻找潜在的重要残基。然而，单氨基酸取代都没有显著地稳定CAO。因此，我们在A结构域产生了一系列缺失，并在缺乏cao的拟南芥突变体背景下表达了这些缺失。我们发现氨基酸序列（97）QDLLTIMILH（106）对蛋白质稳定性的调节至关重要。我们进一步确定该序列诱导绿色荧光蛋白的不稳定。这些结果表明，该序列作为一种降解信号，可被叶绿体中功能的蛋白酶识别。
Chlorophyll b is one of the major photosynthetic pigments of plants. The regulation of chlorophyll b biosynthesis is important for plants in order to acclimate to changing environmental conditions. In the chloroplast, chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), a Rieske-type monooxygenase. The activity of this enzyme is regulated at the level of protein stability via a feedback mechanism through chlorophyll b. The Clp protease and the N-terminal domain (designated the A domain) of CAO are essential for the regulatory mechanism. In this study, we aimed to identify the specific amino acid residue or the sequence within the A domain that is essential for this regulation. To accomplish this goal, we randomly introduced base substitutions into the A domain and searched for potentially important residues by analyzing 1,000 transformants of Arabidopsis thaliana. However, none of the single amino acid substitutions significantly stabilized CAO. Therefore, we generated serial deletions in the A domain and expressed these deletions in the background of CAO-deficient Arabidopsis mutant. We found that the amino acid sequence (97)QDLLTIMILH(106) is essential for the regulation of the protein stability. We furthermore determined that this sequence induces the destabilization of green fluorescent protein. These results suggest that this sequence serves as a degradation signal that is recognized by proteases functioning in the chloroplast.