Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii

Abstract

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1’s involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites.

Dually localized proteins found in both the apicoplast and mitochondrion utilize the Golgi-dependent pathway for apicoplast targeting inToxoplasma gondii

“…Previously published data has indicated that the proteins TgDer1AP, TgATrx1, TgATrx2, TgPPP1 and TgTic22 are apicoplast resident proteins (DeRocher et al, 2008;Agrawal et al, 2009;Sheiner et al, 2011;Glaser et al, 2012;Biddau et al, 2018). Consistent with this data, C-terminal HA fusions of these proteins were localised to the apicoplast, as seen by colocalisation with the apicoplast marker ACP (Fig.1).…”

“…Table 1 can also be categorised based on their membrane association. Apicoplast proteins are either luminal proteins, membrane proteins or have been observed by immuno-EM to be present in the intermembrane space, but closely associated with the membrane, for example TgATrx1 and TgPPP1 (DeRocher et al, 2008;Sheiner et al, 2011;Biddau et al, 2018). Among the dually localised proteins, only…”

A subcellular atlas ofToxoplasmareveals the functional context of the proteome

“…The abundance distribution profiles of ER 2 proteins are more similar to those of the apicoplast rather than ER 1 (Figure S1), suggesting some degree of association between these two. Given that most apicoplast proteins traffic through the ER, this association might reflect a role of these proteins in folding and redox processes during sorting of proteins to the apicoplast (Biddau et al, 2018). Indeed, BiP was recently found among proteins pulleddown by an apicoplast-residing thioredoxin TgATrx2 (Biddau et al, 2018).…”

A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

“…ATG8's novel apicoplast function was discovered serendipitously by its unexpected localization on the apicoplast instead of autophagosomes (Kitamura et al, 2012). Though candidate approaches have yielded new molecular insight (Biddau et al, 2018;Fellows et al, 2017;Sheiner et al, 2015), in general they are indirect and may bias against novel pathways.…”