Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

Abstract

Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle-the apicoplastacquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP ؉ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.

Organellar dynamics during the cell cycle of Toxoplasma gondii

“…HDEL) (Hager et al, 1999). (4) Construction methods have previously been described for the basic GFP-expressing vector pdhfrΔCAT-GFP, and the secretion vectors ptubP30 L -GFP and ptubP30 L -YFP Matrajt et al, 2002), as well as constructs that label the ER (ptubP30 L -GFP-HDEL) (Hager et al, 1999), Golgi (ptubGRASP-YFP) (Pelletier et al, 2002), IMC (ptubIMC1-YFP) , micronemes (ptubMIC3-GFP) , rhoptries (ptubROP1-CAT-YFP) (Dzierszinski et al, 2004) and apicoplast [ptubACP L -EGFP (Waller et al, 1998); ptubFNR L -YFP (Striepen et al, 2000;Harb et al, 2004); ptubFNR L -DsRed (He et al, 2001)]. For labeling the mitochondrion, sequences encoding the 55 N-terminal residues of T. gondii heatshock protein 60 (Toursel et al, 2000) were amplified from tachyzoite cDNA using primers 5Ј-atgcagatctaaaATGcttgcccgcgcttca-3Ј and 5Ј-cagtcctagggccgagagtgactccgac-3Ј, and introduced as a BglII-AvrII fragment into ptubIMC1-YFP and ptubFNR L -DsRed, yielding ptubHSP60 L -YFP and ptubHSP60 L -DsRed, respectively.…”

Analysis of Euglena gracilis Plastid-Targeted Proteins Reveals Different Classes of Transit Sequences

“…Though not involving the Golgi dictyosomes, a vesicular transfer between the CER and the third membrane has been proposed (23), and a recent report supports such a mechanism (37). Apicomplexans, in particular, provide a valuable model system for dissecting the targeting process in complex plastids with four membranes, with several studies indicating not only that there is partially redundant targeting information in the presequences of apicoplast-targeted proteins (26,81,82) but also that there is a distinction between the information for targeting and that for import into the apicoplast (26). Recent work has even identified proteins that interact with the TP and that may be involved in sorting from the ER to the apicoplast (82).…”

DNA topoisomerases in apicomplexan parasites: promising targets for drug discovery

“…The PfGyrB monomer contains a unique 45 amino acid insertion region within the Toprim domain that is essential for its in vitro and in vivo functions (Dar et al 2007). Both subunits are larger than their bacterial counterparts, and the N-terminal domain contains the elements driving the enzyme to the apicoplast (Harb et al 2004;Khor et al 2005) (figure 1c). DNA gyrases are the only Top that introduce negative supercoils into relaxed or positively supercoiled DNA.…”