Apicoplast Targeting of a Toxoplasma gondii Transmembrane Protein Requires a Cytosolic Tyrosine‐Based Motif

Abstract

Toxoplasma gondii, like most apicomplexan parasites, possesses a nessential relict chloroplast, the apicoplast. Several apicoplast membrane proteins lack the bipartite targeting sequences of luminal proteins. Vesicles bearing these membrane proteins are detected during apicoplast enlargement, but the means of cargo selection remains obscure. We used a combination of deletion mutagenesis, point mutations, and protein chimeras to identify a short motif prior to the first transmembrane domain of the T. gondii apicoplast phosphate transporter 1 (APT1) that is necessary for apicoplast trafficking. Tyrosine 16 was essential for proper localization; any substitution resulted in misdirection of APT1 to the Golgi body. Glycine 17 was also important, with significant Golgi body accumulation in the alanine mutant. Separation of at least eight amino acids from the transmembrane domain was required for full motif function. Similarly placed YG motifs are present in apicomplexan APT1 orthologues and the corresponding N-terminal domain from Plasmodium vivax was able to route T. gondii APT1 to the apicoplast. Differential permeabilization demonstrated that both the N-and C- termini of APT1 are exposed to the cytosol. We propose that this YG motif facilitates APT1 trafficking via interactions that occur on the cytosolic face of nascent vesicles destined for the apicoplast.

A genetic screen in rodent malaria parasites identifies five new apicoplast putative membrane transporters, one of which is essential in human malaria parasites

“…The targeting information required for the correct localisation of this type of membrane protein remains largely unexplored, in part because of a lack of confirmed, leaderless proteins to include in analyses. The only confirmed targeting information for these leaderless proteins is the presence of a tyrosine in the N‐terminal region of the Toxoplasma orthologue of oTPT (DeRocher, Karnataki, Vaney, & Parsons, ), and we recently established that an N‐terminal tyrosine residue is necessary but not sufficient for the apicoplast targeting of Pf oTPT (Lim, Sayers, Goodman, & McFadden, ).…”

Fatty acid metabolism in the Plasmodium apicoplast: Drugs, doubts and knockouts

“…Although the T. gondii genome encodes only one pPT [115], it appears to perform an equivalent function to the Plasmodium transporters. The apicoplast localization of the T. gondii pPT has been confirmed by epitope tagging [115,116], and its ability to transport PEP, DHAP and 3-phosphoglycerate have been demonstrated in vitro [117]. In addition, immuno-electron microscopy has indicated the T. gondii pPT may be targeted to multiple apicoplast membranes [118], although the proximity of the four bilayers prevents its precise localization from being determined.…”

Cryptic organelle homology in apicomplexan parasites: insights from evolutionary cell biology

“…Some proteins target the apicoplast using a tyrosine-based motif [25], but most exploit a classical secretory signal sequence mediating cotranslational translocation across the endoplasmic reticulum; vesicular fusion then mediates traversal of the first apicoplast membrane [21,26]. Phylogenetic and cell biological analyses have shown that the apicoplast has repurposed proteins from the ERAD system (normally used to remove misfolded proteins from the ER) as an apicoplast translocon [27,28].…”

“…Right , apicoplast (membranes numbered sequentially from exterior to interior; Plasmodium shown). Proteins likely cross the outer membrane by vesicle fusion [25], the second membrane using a translocon derived from the endoplasmic reticulum ERAD-system, and a reduced chloroplast import apparatus to cross the third and fourth membranes [27,28]. …”