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

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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.