Table_3_The Puzzle of Metabolite Exchange and Identification of Putative Octotrico Peptide Repeat Expression Regulators in the Nascent Photosynthetic Organelles of Paulinella chromatophora.XLSX

The endosymbiotic acquisition of mitochondria and plastids more than one billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles—termed chromatophores—that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are continuously exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded solute transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting that mechanisms evolved that coordinate gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, also in P. chromatophora nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Thus, the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, our mass spectrometric analysis revealed an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted expression regulators of the octotrico peptide repeat type in algae and plants. Apparently these chromatophore-targeted proteins evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

逾十亿年前，线粒体（mitochondria）与质体（plastids）的内共生获取事件，是真核生物（eukaryotic life）演化历程中的核心节点。然而，由于二者起源极为久远，这两类细胞器仅能为细胞器发生（organellogenesis）的早期阶段提供有限的研究视角。丝足虫类变形虫（cercozoan amoeba）中的色球藻变胞藻（Paulinella chromatophora）含有一类光合细胞器（photosynthetic organelles）——被称为色素胞（chromatophores）——其起源于约1亿年前的一株蓝细菌（cyanobacterium），与植物和藻类中的质体演化路径完全独立。尽管色素胞的起源时间更近，但其已与宿主细胞（host cell）实现紧密整合：它需导入数百种由细胞核编码的蛋白质（nucleus-encoded proteins），且多种代谢物（metabolites）持续不断地在色素胞的两层包膜（envelope membranes）之间交换。然而，色素胞自身编码的溶质转运蛋白（solute transporters）数量极为有限，不足以维持代谢连通性（metabolic connectivity）与蛋白质导入（protein import）过程。此外，定位于色素胞的生物合成通路（biosynthetic pathways）与多蛋白复合物（multiprotein complexes）中，存在兼具双遗传起源（dual genetic origin）的蛋白质，这表明演化出了协调色素胞与细胞核之间基因表达水平（gene expression levels）的机制。上述研究结果表明，与线粒体和质体中的情况类似，在色球藻变胞藻（Paulinella chromatophora）中，同样演化出了调控色素胞代谢物交换与基因表达的细胞核编码因子（nuclear factors）。本研究通过对富集的不溶性蛋白质组分（insoluble protein fractions）进行质谱分析（mass spectrometric analyses），意外发现：由细胞核编码的转运蛋白并未嵌入色素胞的内膜（inner envelope membrane）中。因此，尽管该膜的屏障功能看似得以维持，但这类经典的代谢物转运蛋白（canonical metabolite transporters）并未存在于该内膜中。与之相反，本研究鉴定出了多组经过扩增的、靶向色素胞的孤儿蛋白（orphan proteins）。其中一类蛋白以单个跨膜螺旋（transmembrane helix）为结构特征，其余类群则含有两亲性螺旋（amphipathic helices）。我们推测，这类蛋白参与调控膜通透性（membrane permeability）。因此，介导色素胞代谢连通性的机制，与线粒体和质体中的对应机制存在根本性差异，但更类似于目前研究尚不明确的、植物与昆虫体内各类细菌内共生体（bacterial endosymbionts）所采用的机制。此外，本次质谱分析还鉴定出了一组经过扩增的、靶向色素胞的螺旋重复蛋白（helical repeat proteins）。这类蛋白的结构域排布（domain architectures），与藻类和植物中已知的、靶向细胞器的八肽重复（octotrico peptide repeat）型表达调控因子（expression regulators）高度相似。显然，这类靶向色素胞的蛋白与靶向质体的表达调控因子经历了趋同演化，且极有可能参与色素胞内的基因表达调控（gene expression control）过程。