A bipartite chromatophore transit peptide and N-terminal protein processing in the Paulinella chromatophore.

The amoeba Paulinella chromatophora contains photosynthetic organelles, termed chromatophores, which evolved independently from plastids in plants and algae. At least one-third of the chromatophore proteome consists of nucleus-encoded (NE) proteins that are imported across the chromatophore double envelope membranes. Chromatophore-targeted proteins exceeding 250 amino acids (aa) carry a conserved N-terminal extension presumably involved in protein targeting, termed the chromatophore transit peptide (crTP). Short imported proteins do not carry discernable targeting signals. To explore whether the import of proteins is accompanied by their N-terminal processing, here we identified N-termini of 208 chromatophore-localized proteins by a mass spectrometry-based approach. Our study revealed extensive N-terminal acetylation and proteolytic processing in both NE and chromatophore-encoded (CE) fractions of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Surprisingly, only the N-terminal ∼50 aa (part 1) become cleaved upon import. This part contains a conserved adaptor protein-1 complex-binding motif known to mediate protein sorting at the trans-Golgi network followed by a predicted transmembrane helix, implying that part 1 anchors the protein co-translationally in the endoplasmic reticulum and mediates trafficking to the chromatophore via the Golgi. The C-terminal part 2 contains conserved secondary structural elements, remains attached to the mature proteins, and might mediate translocation across the chromatophore inner membrane. Short imported proteins remain largely unprocessed. Finally, this work illuminates N-terminal processing of proteins encoded in an evolutionary-early-stage organelle and suggests host-derived posttranslationally acting factors involved in regulation of the CE chromatophore proteome. © American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.