Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway's enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

Biology Centre CAS Laboratory of Evolutionary Protistology Institute of Parasitology 370 05 České Budějovice Czech Republic

Department of Biochemistry University of Cambridge Cambridge CB2 1TN UK

Department of Pathobiology School of Veterinary Medicine University of Pennsylvania Philadelphia PA 19104 USA

Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Welcome Centre for Integrative Parasitology College of Medical Veterinary and Life Sciences Institute of Infection Immunity and Inflammation University of Glasgow Glasgow G12 8QQ UK

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Complex Endosymbioses I: From Primary to Complex Plastids, Serial Endosymbiotic Events