Abstract
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.
Highlights
Apicomplexans are eukaryotic pathogens and responsible for important human and animal diseases including malaria and toxoplasmosis
The apicoplast endoplasmic reticulum associated degradation (ERAD) system is broadly conserved between most apicomplexans and surprisingly retains the ubiquitylation machine typically associated with destruction
By genetic analysis in T. gondii we demonstrate that loss of the apicoplastlocalized ubiquitin-conjugating enzyme leads to loss of apicoplast protein import and parasite demise
Summary
Apicomplexans are eukaryotic pathogens and responsible for important human and animal diseases including malaria and toxoplasmosis. Targeting depends on a bipartite leader peptide, the first section of which mediates co-translational import into the endoplasmic reticulum, and the second part mediates delivery to the apicoplast, likely through fusion of endosomal vesicles with the outermost membrane of the organelle [4]. The two inner membranes of the apicoplast are homologous to the membranes of the primary chloroplast and protein transport depends on systems derived from the chloroplast TIC and TOC machinery [6,7,8,9]. Insight into the third translocon emerged first in cryptomonads, an algal group that like Apicomplexa harbors a secondary plastid. The secondary plastids of cryptomonads retained a remnant of the algal nucleus, the nucleomorph. Analysis of the gene content of the nucleomorph led to the discovery of plastid proteins that resembled components of the endoplasmic reticulum associated degradation (ERAD) machinery [10].
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