Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5-10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.Apicomplexa ͉ plastid ͉ chloroplast

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1 Ap results in loss of apicoplast protein import and subsequent death of the parasite.Apicomplexa are a phylum of obligate parasites that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Recent evidence suggests that apicomplexans evolved from a free-living photosynthetic ancestor (1). This ancestry is reflected in the presence of a chloroplast-like organelle, the apicoplast (2). While no longer engaged in photosynthesis, the apicoplast is essential to parasite survival and home to several critical biosynthetic pathways. Bioinformatic and experimental evidence suggests that the apicoplast is engaged in the synthesis of fatty acids, isoprenoids, and heme (3, 4). Genetic or pharmacological ablation of these pathways blocks parasite growth and the apicoplast, therefore, is currently considered a prime target for antiparasitic drug development (5-7). The organelle was derived by secondary endosymbiosis and reflects the successful union of a red alga and a heterotrophic eukaryote (8). A large number of algal genes were transferred to the host nucleus, and their products must now be routed back into the organelle. Trafficking occurs via the secretory pathway and is guided by an N-terminal bipartite targeting sequence (9). Transport from the ER to the apicoplast is believed to be vesicle mediated and to sidestep the Golgi apparatus (10, 11). Once delivered to the apicoplast, proteins have to cross three additional membranes. Recently, we demonstrated that a homolog of Tic20, a component of the translocon of the inner chloroplast membrane (Tic) 4 complex in plants, is likely required for protein import across the innermost membrane of the apicoplast (12). To date bioinformatics searches have failed to identify a matching translocon of the outer chloroplast membrane in apicomplexans (13). Analysis of the genome of the remnant nucleus of the algal endosymbiont in cryptomonads showed the presence of an ER-associated protein degradation (ERAD) system and offered a candidate for a translocon across the third and potentially the second membrane (14). Classically, ERAD functions in the homeostasis of the secretory ...

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.

Background: Tic22 is a core protein import machinery component of chloroplasts and apicoplasts. Results: Tic22 is a chaperone, required for parasite survival and for protein import in the apicoplast. Conclusion: Tic22 lies between the plastid membranes, maintaining imported proteins unfolded for translocation. Significance: Chaperones which hold proteins in an unfolded state are central to membrane translocation systems and Tic22 plays this role in plastids.

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub-compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.