Euglena spp. are phototrophic flagellates with considerable ecological presence and impact. Euglena gracilis harbours secondary green plastids, but an incompletely characterised proteome precludes accurate understanding of both plastid function and evolutionary history. Using subcellular fractionation, an improved sequence database and MS we determined the composition, evolutionary relationships and hence predicted functions of the E. gracilis plastid proteome. We confidently identified 1345 distinct plastid protein groups and found that at least 100 proteins represent horizontal acquisitions from organisms other than green algae or prokaryotes. Metabolic reconstruction confirmed previously studied/predicted enzymes/pathways and provided evidence for multiple unusual features, including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism, and dual sets of the SUF pathway for FeS cluster assembly, one of which was acquired by lateral gene transfer from Chlamydiae. Plastid paralogues of trafficking-associated proteins potentially mediating fusion of transport vesicles with the outermost plastid membrane were identified, together with derlin-related proteins, potential translocases across the middle membrane, and an extremely simplified TIC complex. The Euglena plastid, as the product of many genomes, combines novel and conserved features of metabolism and transport.

Department of Biochemistry University of Cambridge Cambridge CB2 1QW UK

Department of Plant Sciences University of Oxford Oxford OX1 3RB UK

Faculty of Science Charles University BIOCEV Vestec 252 50 Czechia

Faculty of Science University of Ostrava Ostrava 710 00 Czechia

Faculty of Science University of South Bohemia České Budějovice 370 05 Czechia

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice 370 05 Czechia

School of Life Sciences University of Dundee Dundee DD1 5EH UK

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