Dual Targeting of Antioxidant and Metabolic Enzymes to the Mitochondrion and the Apicoplast of Toxoplasma gondii

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Abstract
Toxoplasma gondii is an aerobic protozoan parasite that possesses mitochondrial antioxidant enzymes to safely dispose of oxygen radicals generated by cellular respiration and metabolism. As with most Apicomplexans, it also harbors a chloroplast-like organelle, the apicoplast, which hosts various biosynthetic pathways and requires antioxidant protection. Most apicoplast-resident proteins are encoded in the nuclear genome and are targeted to the organelle via a bipartite N-terminal targeting sequence. We show here that two antioxidant enzymes—a superoxide dismutase (TgSOD2) and a thioredoxin-dependent peroxidase (TgTPX1/2)—and an aconitase are dually targeted to both the apicoplast and the mitochondrion of T. gondii. In the case of TgSOD2, our results indicate that a single gene product is bimodally targeted due to an inconspicuous variation within the putative signal peptide of the organellar protein, which significantly alters its subcellular localization. Dual organellar targeting of proteins might occur frequently in Apicomplexans to serve important biological functions such as antioxidant protection and carbon metabolism. Toxoplasma gondii is a human and animal pathogen representative of the large group of Apicomplexa. Most members of this phylum contain, in addition to a tubular mitochondrion, a second endosymbiotic organelle indispensable for parasite survival, called the apicoplast. This non-photosynthetic plastid is the site of several anabolic pathways, including the biosynthesis of fatty acids, isoprenoids, iron-sulphur cluster, and heme. Virtually all enzymes active inside the apicoplast are encoded by the nuclear genome and targeted to the organelle via the endoplasmic reticulum courtesy of a bipartite amino terminal recognition sequence. The metabolic activities of the apicoplast impose a high demand for antioxidant protection. We show here that T. gondii possesses a superoxide dismutase and a peroxidase that are shared between the two organelles by an unusual mechanism of bimodal targeting whereby the nature of the signal peptide influences the destination of the protein to both organelles. Dual targeting also extends to other classical metabolic enzymes such as aconitase, uncovering unexpected metabolic pathways occurring in these organelles. In consequence, the bioinformatic predictions for plastidic or mitochondrial targeting on the basis of the characteristics of N-terminal presequences are insufficient in the absence of an experimental confirmation.