Targeting and Processing of Nuclear-encoded Apicoplast Proteins in Plastid Segregation Mutants of Toxoplasma gondii

Cynthia Y He,Boris Striepen,Charles H Pletcher,John M Murray,David S Roos

doi:10.1074/jbc.m102000200

Cynthia Y He, Boris Striepen + Show 3 more

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https://doi.org/10.1074/jbc.m102000200

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Abstract

The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.

Highlights

Toxoplasma gondii is an obligate intracellular parasite, a major cause of congenital birth defects in humans and livestock [1, 2], and an important opportunistic infection associated with AIDS [3]
Two previously validated apicoplast targeting signals from nuclear-encoded apicoplast proteins were employed in this study; these signals were derived from the N-terminal domains of the acyl carrier protein (ACP)1 [14] or ferrodoxin NADP-reductase (FNR) [24]
Targeting of Heterologous Proteins into the Apicoplast—We have previously shown that N-terminal fusion of the bipartite targeting sequences from various nuclear-encoded apicoplast proteins permits the targeting of GFP into the apicoplast in stable T. gondii transgenics [14] and that this marker can be exploited to follow organellar replication in living parasites

Summary

Introduction

Toxoplasma gondii is an obligate intracellular parasite, a major cause of congenital birth defects in humans and livestock [1, 2], and an important opportunistic infection associated with AIDS [3] This single-cell eukaryotic pathogen contains an unusual organelle that was acquired by horizontal transfer (secondary endosymbiosis) from a eukaryotic alga [4]. Previous studies have shown that the apicoplast is essential for long term parasite viability (6 – 8). When this organelle is eliminated by either pharmacological or molecular genetic manipulation, parasites are killed with distinctive “delayed death” kinetics. Phenotype remains unexplained, these studies demonstrate that the apicoplast is an essential organelle and a potential target for drug development [9]

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