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 apicoplast--acquired 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.

FIG. 1.

The bipartite apicoplast targeting signal. (a) Diagram representing the constructs used for the experiments shown in panels b, c, and d. The secretory signal is shown in blue, the plastid TP is in green, and YFP is in orange. (b) The fusion of the first 150 aa of FNR to YFP results in its targeting to the apicoplast, seen as oval structures in the apical juxtanuclear region of transfected T. gondii tachyzoites. (c) Deletion constructs containing only the FNR SS fused to YFP (FNR-31, −26, −24, and −22) result in secretion via dense granules (small dots within the parasites) into the parasitophorous vacuole surrounding the parasites. (d) Deletion of the SS (FNR-Δ1-24) results in YFP expression within the parasite cytoplasm only. FNR constructs diagrammed at the top are shown to scale, with summaries of the targeting results shown to the right. AL, apicoplast lumen; AP, apicoplast periphery; C, cytoplasmic; ER, endoplasmic reticulum; R, rhoptry; S, secreted; X, complex localization. Panels B, C, and D are representative images showing direct YFP fluorescence in live parasites. Bars = 5 μm.

FIG. 2.

C-terminal deletions of the TP. (a) Diagram of FNR constructs depicting sequential 5-aa deletions between aa 105 and 150. (b) Western blot of deletion constructs FNR-150 through FNR-110, illustrating mature and processed forms of each fusion construct; note the alternative processing site in constructs shorter than TgFNR-145. Inefficient processing has been previously described (53). (c) Deconvolved fluorescence image of TgFNR-105 (representative of all constructs shown in panel a) illustrating targeting into the apicoplast lumen. The abbreviations are as described in the legend to Fig. 1. For microscopic images, green = YFP (anti-GFP), red = apicoplast (anti-ACP [53]), blue = nuclear and apicoplast DNA (DAPI [42]). A video of the images shown in panel c is available for download (http://roos.bio.upenn.edu/∼oharb/Harb2c.mov).

FIG. 3.

C-terminal deletions of the TP delineate between targeting and translocation. (a) Diagram of FNR constructs depicting sequential 5-aa deletions between aa 40 and 105. (b) Western blot of deletion constructs down to TgFNR-40. (c) Deconvolved fluorescence image of TgFNR-50 (representative of all constructs shown in panel a) illustrating targeting to, but not into, the apicoplast. The abbreviations are as described in the legend to Fig. 1. For microscopic images, green = YFP (anti-GFP), red = apicoplast (anti-ACP [53]), blue = nuclear and apicoplast DNA (DAPI [42]). Bars = 5 μm. A video of the images shown in panel c is available for download (http://roos.bio.upenn.edu/∼oharb/Harb3c.mov).

FIG. 4.

TgFNR-34 targets YFP to multiple compartments. (a) Diagram of TgFNR-34 and -150. (b) Direct YFP fluorescence image of TgFNR-34, showing complex targeting pattern within the parasite and secretion into the parasitophorous vacuole. (c) Fluorescence image of TgFNR-34 (fixed specimen), showing complex targeting pattern within the parasite, including colocalization with rhoptries. The insert (bottom right) is an enlarged view of the apical region of one of the parasites showing green fluorescence at the periphery of the apicoplast and colocalizing with the rhoptries. The abbreviations are as described in the legend to Fig. 1. For microscopic images, green = YFP (anti-GFP or native fluorescence in panel b), red = rhoptries (anti-ROP2/3/4 [3]), blue = nuclear and apicoplast DNA (DAPI [42]). Bars = 5 μm.

FIG. 5.

Redundant motif within apicoplast targeting signal. (a) Diagram depicting deletions that define a redundant domain with the FNR TP. (b) Western blot demonstrating processing of TgFNR-Δ26-82, but not TgFNR-Δ26-86. (c) Despite deletion of one apicoplast targeting signal, TgFNR-Δ26-82 still localizes to the organelle periphery. (d) Deletion of an additional 4 aa results in targeting to both the ER and apicoplast periphery (TgFNR-Δ26-86). The abbreviations are as described in the legend to Fig. 1. For microscopic images, green = YFP (anti-GFP), red = apicoplast (anti-ACP [53]), blue = nuclear and apicoplast DNA (DAPI [42]). Bars = 5 μm.

FIG. 6.

Mapping the second redundant targeting motif within the apicoplast targeting signal. (a) Diagram depicting various deletions generated to map the second redundant targeting motif within the FNR TP. (b) TgFNR-Δ26-50,91-150 is targeted to the apicoplast periphery. (c) TgFNR-Δ26-82,91-150 is secreted into the parasitophorous vacuole. (d and e) Deleting both of the mapped apicoplast targeting signals results in a complex pattern of protein secretion targeting TgFNR-Δ26-50,83-105 to the apicoplast periphery (d), rhoptries (e), and the parasitophorous vacuole (not shown). The abbreviations are as described in the legend to Fig. 1. For microscopic images, green = YFP (anti-GFP or native fluorescence [c]), red = apicoplast (anti-ACP) or rhoptries (anti-ROP2/3/4 [e]). Bars = 5 μm.

FIG. 7.

Scanning the apicoplast TP. (a) Diagram of overlapping sequential 25-aa segments scanning through the TgFNR TP inserted downstream of the SS and upstream of a YFP reporter. Four targeting patterns were observed, labeling the apicoplast (b), ER (c), parasitophorous vacuole (d), or rhoptries and parasitophorous vacuole (e). The abbreviations are as described in the legend to Fig. 1. All micrographs are direct fluorescence images of living parasites. Bars = 5 μm.

FIG. 8.

Summary of signal features within the TgFNR leader. The secretory SS is shown in magenta, and redundant apicoplast targeting domains are shown in green. Computationally predicted and experimentally determined cleavage sites are indicated by black and blue text, respectively. Red highlighting indicates the V-(V/S)-S-F motif that may be associated with release from the ER. The mature FNR gene is indicated, with conserved amino acids shown in red.