Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom.
Plant Physiol. 2011 Sep;157(1):147-59. doi: 10.1104/pp.111.181891. Epub 2011 Jul 14.
Two different, essential Omp85 (Outer membrane protein, 85 kD)-related proteins exist in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD), encoded by atTOC75-III; and OEP80 (Outer Envelope Protein, 80 kD), encoded by AtOEP80/atTOC75-V. The atToc75-III protein is closely related to the originally identified pea (Pisum sativum) Toc75 protein, and it forms a preprotein translocation channel during chloroplast import; the AtOEP80 protein is considerably more divergent from pea Toc75, and its role is unknown. As knockout mutations for atTOC75-III and AtOEP80 are embryo lethal, we employed a dexamethasone-inducible RNA interference strategy (using the pOpOff2 vector) to conduct in vivo studies on the roles of these two proteins in older, postembryonic plants. We conducted comparative studies on plants silenced for atToc75-III (atToc75-III↓) or AtOEP80 (AtOEP80↓), as well as additional studies on a stable, atToc75-III missense allele (toc75-III-3/modifier of altered response to gravity1), and our results indicated that both proteins are important for chloroplast biogenesis at postembryonic stages of development. Moreover, both are important for photosynthetic and nonphotosynthetic development, albeit to different degrees: atToc75-III↓ phenotypes were considerably more severe than those of AtOEP80↓. Qualitative similarity between the atToc75-III↓ and AtOEP80↓ phenotypes may be linked to deficiencies in atToc75-III and other TOC proteins in AtOEP80↓ plants. Detailed analysis of atToc75-III↓ plants, by electron microscopy, immunoblotting, quantitative proteomics, and protein import assays, indicated that these plants are defective in relation to the biogenesis of both photosynthetic and nonphotosynthetic plastids and preproteins, confirming the earlier hypothesis that atToc75-III functions promiscuously in different substrate-specific import pathways.
两种不同的、基本的 Omp85（外膜蛋白，85kDa）相关蛋白存在于拟南芥（Arabidopsis thaliana）叶绿体的外被膜中：Toc75（叶绿体外被膜转位体，75kDa），由 atTOC75-III 编码；和 OEP80（外被膜蛋白，80kDa），由 AtOEP80/atTOC75-V 编码。AtToc75-III 蛋白与最初鉴定的豌豆（Pisum sativum）Toc75 蛋白密切相关，在叶绿体导入过程中形成前体蛋白转运通道；AtOEP80 蛋白与豌豆 Toc75 的差异较大，其作用尚不清楚。由于 atTOC75-III 和 AtOEP80 的敲除突变是胚胎致死的，我们采用了地塞米松诱导的 RNA 干扰策略（使用 pOpOff2 载体），在体内研究这两种蛋白在较老的、胚胎后植物中的作用。我们对沉默 atToc75-III（atToc75-III↓）或 AtOEP80（AtOEP80↓）的植物进行了比较研究，以及对一个稳定的、atToc75-III 错义等位基因（toc75-III-3/altered response to gravity1 的修饰因子）的额外研究，结果表明，这两种蛋白在胚胎后发育阶段对叶绿体的生物发生都很重要。此外，两者对光合和非光合发育都很重要，尽管程度不同：atToc75-III↓表型比 AtOEP80↓表型严重得多。AtToc75-III↓和 AtOEP80↓表型之间的定性相似性可能与 AtOEP80↓植物中 atToc75-III 和其他 TOC 蛋白的缺陷有关。通过电子显微镜、免疫印迹、定量蛋白质组学和蛋白导入分析对 atToc75-III↓植物进行详细分析，表明这些植物在光合和非光合质体和前体蛋白的生物发生方面都有缺陷，证实了之前的假设，即 atToc75-III 在不同的底物特异性导入途径中具有混杂功能。
