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

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium, 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 exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded 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 coordination of gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, 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. Furthermore, we identified an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted octotrico peptide repeat expression regulators in algae and plants suggesting their convergent evolution as nuclear regulators of gene expression levels in the chromatophore. Importance The endosymbiotic acquisition of mitochondria and plastids >1 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 chromatophore in Paulinella evolved ~100 million years ago and thus, offers the possibility to gain valuable insights into early stages and common rules in organelle evolution. Critical to organellogenesis appears to be the establishment of nuclear control over metabolite exchange and gene expression in the endosymbiont. Here we show that 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, we describe a novel class of chromatophore-targeted helical repeat proteins which evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.