Spatial proteomics reveals carboxysome adaptation and mitochondrial proteome in Paulinella chromatophora

The Rhizarian amoeba Paulinella chromatophora represents a unique case of primary endosymbiosis, which has acquired photosynthetic organelles (chromatophores) independently of the endosymbiosis event that gave rise to plastids. Using localization of organelle proteins by isotope tagging following differential centrifugation (LOPIT-DC)-based spatial proteomics, we generated a subcellular protein atlas of P. chromatophora, thus providing a comprehensive view of organelle biology in this lineage. Analysis of chromatophore-targeted proteins revealed that the second part of the bi-partite chromatophore transit peptide (crTP) remains permanently attached post-import. The absence of β-barrel proteins in chromatophore-associated clusters supports the hypothesis of vesicle-mediated protein import as opposed to a translocon-based mechanism. We further identified nucleus-encoded proteins predating chromatophore acquisition in the Paulinella lineage that now associate with chromatophore carboxysomes. Their predicted activities are consistent with an O₂-scavenging system that could locally reduce O₂ levels around the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), thereby enhancing carbon fixation efficiency in the absence of a complete canonical photorespiratory pathway; an example of the repurposing of pre-existing eukaryotic proteins to modify a bacterial compartment. In the mitochondria, we identified both conserved membrane organization alongside highly divergent, lineage-specific proteins. Together, these results demonstrate how spatial proteomics can reveal both conserved organelle features and lineage-specific innovations in a non-model eukaryote.