Toxoplasma gondii, which causes toxoplasmic encephalitis and birth defects, contains an essential chloroplast-related organelle to which proteins are trafficked via the secretory system. This organelle, the apicoplast, is bounded by multiple membranes. In this report we identify a novel apicoplast-associated thioredoxin family protein, ATrx1, which is predominantly soluble or peripherally associated with membranes, and which localizes primarily to the outer compartments of the organelle. As such, it represents the first protein to be identified as residing in the apicoplast intermembrane spaces. ATrx1 lacks the apicoplast targeting sequences typical of luminal proteins. However, sequences near the N terminus are required for proper targeting of ATrx1, which is proteolytically processed from a larger precursor to multiple smaller forms. This protein reveals a population of vesicles, hitherto unrecognized as being highly abundant in the cell, which may serve to transport proteins to the apicoplast.The apicoplast is a relict secondary chloroplast that resides within the cytosol of most apicomplexan parasites, including the intracellular pathogens Toxoplasma gondii and the malaria parasite Plasmodium. It is an essential organelle in these organisms, and since it does not have a counterpart in animal hosts, pathways known to function in the organelle have been considered as potential targets for prophylactic or therapeutic intervention for both toxoplasmosis and malaria (52). Indeed, specific antibiotics that target the transcription and translation systems of the apicoplast, including clindamycin and tetracycline, are known to be toxic to the parasites (9, 14, 23). Other small molecules inhibit key apicoplast metabolic pathways, such as isoprenoid biosynthesis (27) or fatty acid biosynthesis (22). Therefore, identification of additional essential molecules in the apicoplast could yield new drug targets.The apicoplast was acquired by secondary endosymbiosis and is surrounded by four membranes (30); the inner two presumably correspond to the two original chloroplast membranes, while the outer two are thought to be derived from the algal plasma membrane and the endocytic vacuole. Thus, there are eight distinct locations within the apicoplast: one for each of the four membranes, three for the intermembrane spaces, and one for the lumen of the organelle. Little is known about proteins that reside in nonluminal compartments. Since the apicoplast genome has few genes other than those involved in genetic functions (53), the vast majority of apicoplast proteins are encoded by nuclear genes. Many proteins have been predicted to localize to the apicoplast lumen on the basis of a predicted apicoplast targeting sequence (17, 42), as described below.To date, all proteins known to reside in the lumen of the apicoplast are synthesized with an N-terminal bipartite targeting sequence that routes them to the organelle. This sequence is composed of a signal peptide that diverts the nascent protein into the endoplasmic reticulum (ER). A...

SummaryThe apicoplast is a relict plastid essential for viability of the apicomplexan parasites Toxoplasma and Plasmodium. It is surrounded by multiple membranes that proteins, substrates and metabolites must traverse. Little is known about apicoplast membrane proteins, much less their sorting mechanisms. We have identified two sets of apicomplexan proteins that are homologous to plastid membrane proteins that transport phosphosugars or their derivatives. Members of the first set bear N-terminal extensions similar to those that target proteins to the apicoplast lumen. While Toxoplasma gondii lacks this type of translocator, the N-terminal extension from the Plasmodium falciparum sequence was shown to be functional in T. gondii. The second set of translocators lacks an N-terminal targeting sequence. This translocator, TgAPT1, when tagged with HA, localized to multiple apicoplast membranes in T. gondii. Contrasting with the constitutive targeting of luminal proteins, the localization of the translocator varied during the cell cycle. Early-stage parasites showed circumplastid distribution, but as the plastid elongated in preparation for division, vesicles bearing TgAPT1 appeared adjacent to the plastid. After plastid division, the protein resumes a circumplastid colocalization.These studies demonstrate for the first time that vesicular trafficking likely plays a role in the apicoplast biogenesis.