Table_4_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.

超过十亿年前，真核生物通过内共生作用获取线粒体和质体对其生命进化起到了核心作用。然而，由于这些细胞器的古老起源，它们仅能为我们提供有限的信息来揭示细胞器起源的初期阶段。围口目变形虫保罗宁氏色素体含有光合细胞器，即所谓的色素体，这些色素体约在一亿年前从蓝藻中进化而来，与植物和藻类中的质体独立演化。尽管色素体的起源较为近期，但它与宿主细胞的整合却非常紧密。色素体能够导入数百个由细胞核编码的蛋白质，并且两种色素体包膜之间的代谢物持续进行交换。然而，色素体编码的溶质转运蛋白的有限集合似乎不足以支持代谢连通性或蛋白质的导入。此外，色素体定位的合成途径以及多蛋白复合体中包含双遗传起源的蛋白质，这表明进化出了协调色素体与细胞核之间基因表达水平的机制。这些发现表明，类似于线粒体和质体的情况，在保罗宁氏色素体中，细胞核因子也进化出来，以控制色素体中的代谢物交换和基因表达。在本研究中，通过富集不溶性蛋白组分的质谱分析，我们意外地发现，细胞核编码的转运蛋白并未插入到色素体内部包膜。因此，尽管表面上维持了其屏障功能，但经典的代谢物转运蛋白却缺失于该膜中。相反，我们鉴定出几组扩大的短色素体靶向孤儿蛋白。其中一组蛋白的特征是具有单个跨膜螺旋，而其他蛋白则含有两亲性螺旋。我们假设这些蛋白参与调节膜通透性。因此，产生色素体代谢连通性的机制与线粒体和质体的机制根本不同，但可能更类似于植物和昆虫中各种细菌内共生体中尚不甚明了的机制。此外，我们的质谱分析揭示了扩大的色素体靶向螺旋重复蛋白家族。这些蛋白显示出与已知藻类和植物中八聚三肽重复型已知细胞器靶向表达调控因子相似的域结构。显然，这些色素体靶向蛋白与质体靶向表达调控因子趋同进化，并且很可能参与色素体中的基因表达调控。