Early Steps in Organelle Evolution – How Unicellular Hosts Tame their Bacterial Endosymbionts

10多亿年前线粒体和质体的内共生获得深刻影响了真核生物的起源和多样化。为了解释细菌内共生体转化为遗传整合细胞器的分子机制，本项目旨在研究细胞器发生的早期阶段：绒毛虫阿米巴（Paulinella chroophora）中的光合蓝藻内共生体（色素体）和锥虫Angomonas deanei中的β -变形细菌内共生体。前5年的投资产生了以下见解。(i)在P. chroophora中，细胞核和染色质编码的蛋白质是高度互补的。（ii）许多补偿染色质基因损失的核基因是通过细菌供体的水平基因转移获得的。（iii）对染色质的遗传控制基本上转移到细胞核，大约三分之一的染色质蛋白质组由核编码的染色质靶向蛋白组成。（iv）一个长达200个氨基酸的n端前序列似乎可以靶向蛋白质进入染色质，但标记短输入蛋白（<90个氨基酸）进入染色质的信号尚不清楚。(v)在A. deanei中，仅鉴定出8个核编码的内共生体靶向蛋白（ETPs），表明A. deanei与P. chroophora相比，代表了更早的内共生体整合状态。鉴定出的etp可能是获得宿主对细胞内细菌控制的关键角色。（vi）通过开发deanei的遗传工具，我们建立了这种生物作为一种高效的、遗传上可处理的内共生模型，在这种模型中，可以产生靶向基因敲除并从核基因组中表达转基因。这些工具有助于鉴定etp在内共生体包膜、分裂部位或细胞质上的不同亚细胞定位。迄今为止分析的三个etp无法获得纯合基因敲除，这表明这些蛋白质具有基本功能。在这项延长Emmy Noether项目一年的提案中，我们将重点探索a . deanei中etp的进化、靶向机制和功能特征。为此，我们的目标是：(i)开发一种条件基因敲除系统；（ii）应用这个条件系统来研究etp的细胞功能；(3)探索寄主蛋白靶向deanei内共生体的分子机制演化。这项工作对我们理解真核细胞控制内共生细菌的分子机制至关重要，这可以导致进化时间将两个共生伙伴完全合并为一个高效的单一生物体。

The endosymbiotic acquisition of mitochondria and plastids more than a billion years ago profoundly impacted origin and diversification of eukaryotes. To decipher the molecular mechanisms that lead to the transformation of a bacterial endosymbiont into a genetically integrated cell organelle, this projects aims at studying early stages in organellogenesis: the photosynthetic cyanobacterial endosymbionts (chromatophores) in the cercozoan amoeba Paulinella chromatophora and the beta-proteobacterial endosymbionts in the trypanosomatid Angomonas deanei.The first five years of funding resulted in the following insights. (i) In P. chromatophora, nucleus and chromatophore-encoded proteins are highly complementary. (ii) Many nuclear genes that compensate for gene losses from the chromatophore were acquired through horizontal gene transfers from bacterial donors. (iii) Genetic control over the chromatophore shifted substantially to the nucleus with around one third of the chromatophore proteome being composed of nucleus-encoded, chromatophore-targeted proteins. (iv) A ~200 amino acid-long N-terminal pre-sequence seems to target proteins into the chromatophore, however the signal marking short imported proteins (<90 amino acids) for import into the chromatophore remains unknown. (v) In A. deanei, only 8 nucleus-encoded endosymbiont-targeted proteins (ETPs) were identified, suggesting that A. deanei represents an earlier state of endosymbiont integration compared to P. chromatophora. The identified ETPs likely represent key players in gaining host control over the intracellular bacterium. (vi) Through the development of genetic tools for A. deanei we established this organism as efficient, genetically tractable endosymbiosis model in which targeted gene knock-outs can be generated and transgenes expressed from the nuclear genome. These tools were instrumental for the identification of distinct subcellular localizations of the ETPs over the endosymbiont envelope, division site, or cytoplasm. The inability to obtain homozygous gene knock-outs for three ETPs analyzed so far, suggests an essential function for these proteins.In this proposal for a one-year extension of the Emmy Noether program, we focus on exploring evolution, targeting mechanisms, and functional characterization of the ETPs in A. deanei. For this purpose, we aim (i) to develop a conditional gene knock-out system for A. deanei; (ii) to apply this conditional system to investigate the cellular functions of the ETPs; and (iii) to explore the evolution of molecular mechanism underlying the targeting of host proteins to the endosymbiont in A. deanei.This work is critical to our understanding of the molecular mechanisms through which a eukaryotic cell can gain control over an endosymbiotic bacterium and which can lead over evolutionary time to the complete merger of the two symbiotic partners into a highly efficient single organism.