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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00750

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Mutation of an unusual mitochondrial targeting sequence of SODB2 produces multiple targeting fates in Toxoplasma gondii

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA

View larger version (81K): [in a new window]   Fig. 1. Structural properties of TgSODB2. (A) Comparison of amino acid sequences of TgSODB2 (accession no. AY176062), Perkinsus marinus FeSOD (PmSOD1, AY095212), TgSODB (AAC63943), Plasmodium falciparum FeSOD (PfFeSODB, CAA89971) and Vibrio cholerae FeSOD (VcFeSOD, NP_231679). Protein sequences were aligned using the ClustalW program (DNAStar-Megalign) and identical residues among sequences are shaded. TgSODB2 contains all residues deemed important for enzyme function (marked with #) and metal ion binding (marked with *) and shows extensive similarity with TgSODB and other FeSODs. The N-terminal hydrophobic sequence of TgSODB2 is boxed and the single basic residue (arg12) within this region is indicated with an arrow. (B) Schematic representation of the TgSODB2 N-terminal extension containing a hydrophobic sequence (HS) and presequence (PS). The HS is predicted by SignalP to be a signal peptide for targeting to the secretory pathway, the PS is predicted by iPSORT to be a mitochondrial or chloroplast targeting sequence, and the entire N-terminal extension is predicted to be an apicoplast targeting sequence. (C) Helical wheel projections (Webgenetics; http://webgenetics.com/cgibin/wg?form=wheel&ID=0) of TgSODB2 amino acids 1 to 30 (TgSODB21-30) and 31 to 60 (TgSODB231-60) and TgHSP70 amino acids 1-30 (TgHSP701-30). Arrows denote charged residues, all of which are basic amino acids. Note that TgSODB21-30 is predicted to form a hydrophobic helix with only one charged residue (arg12) whereas TgSODB231-60 exhibits the properties of an amphipathic helix that is similar to the mitochondrial import presequence of TgHSP70.

View larger version (49K): [in a new window]   Fig. 2. Expression and analysis of recombinant TgSODB2. (A) Schematic of constructs used in this study including two E. coli N-terminally histidine-tagged (His) expression constructs: hisrSOD26-287 contains the presequence (PS) and the SOD domain, and his-rSOD85-287 consists of the SOD domain alone. An arrow denotes the approximate position of cleavage of his-rSOD26-287 by an endogenous E. coli protease. (B) Coomassie Blue-stained SDS-PAGE gels showing insoluble (insol) and soluble (sol) fractions of E. coli expressing his-rSOD26-287 and his-rSOD85-287 and final purification of his-rSOD85-287 (pure). The open arrowhead denotes the position of his-rSOD26-287 in the insoluble fraction; closed arrows denote the position of his-rSOD85-287 in the soluble fraction of induced cells. (C) SOD activity assay using native gel electrophoresis. A 12.5% polyacrylamide gel was first stained for SOD activity (see Materials and Methods) and then stained with Coomassie Blue. Light areas indicate regions of SOD activity: 10 µg E. coli FeSOD (positive control), 10 µg purified rSOD85-287 showing strong dismutase activity, and 15 µg purified recombinant T. gondii MIC5 (negative control).

View larger version (68K): [in a new window]   Fig. 3. Comparison of GFP fluorescence in intracellular parasites transiently expressing TgSODB21-84/GFP, TgSODB21-287/GFP, or TgACP/GFP. The apicoplast is delineated as a single, discrete dot of GFP fluorescence in pTgACP/GFP transfected cells (third row, four-cell stage), while parasites transfected with the SOD constructs show a twisting pattern of GFP fluorescence characteristic of the parasite mitochondrion (first and second rows, four- and two-cell stages, respectively). Mock transfected parasites showed no fluorescence when photographed with the same exposure time. Scale bar: 5 µm.

View larger version (81K): [in a new window]   Fig. 4. TgSODB2 localizes to the parasite mitochondrion. Parasites were transiently transfected with pTgSODB21-84/GFP and pTgSODB21-287/GFP and imaged for GFP and Mitotracker-redTM fluorescence. (A) Deconvolved images of intracellular parasites (two-cell stage), showing extensive MitotrackerTM staining of host mitochondria, as well as colocalization of GFP and MitotrackerTM in the parasite mitochondrion. (B) Extracellular parasites, showing colocalization of GFP and MitotrackerTM. Scale bar, 5 µm.

View larger version (53K): [in a new window]   Fig. 5. TgSODB2 is imported into the mitochondrion. A western blot of lysate from parasites stably expressing TgSODB21-287/GFP shows two bands (grey arrows) in addition to the endogenous TgSODB21-287 and TgSODB285-287 bands (black arrows) seen in 2F lysate: a faint band at about 60 kDa corresponding in size to TgSODB21-287/GFP and a much more abundant product at about 50 kDa corresponding to TgSODB285-287/GFP. mrSODB2 recognized all four bands, whereas GFP recognized only the 50 and 60 kDa, GFP-containing products. HFF cell lysate is included as a negative control.

View larger version (55K): [in a new window]   Fig. 6. Mutating the N-terminal extension results in mistargeting phenotypes. Parasites transiently transfected with pTgSODB226-84/GFP, which contains a deletion of the presequence (PS), showed GFP fluorescence in a punctate pattern within the parasites (four-cell stage). GFP signal was also detected in the PV, where it colocalized with GRA4. Transfection with pTgSODB21-25/GFP bearing a deletion of the hydrophobic sequence (HS) resulted in diffuse localization within the parasite cytoplasm in a pattern distinct from the mitochondrion, visualized by MitotrackerredTM staining. Transfection with pTgSODB2R12A/GFP, in which arg12 was replaced by an ala (asterisk), showed a discrete localization pattern that colocalized with an apicoplast targeted protein, FNR(l)-dsRed. Scale bar, 5 µm.

View larger version (68K): [in a new window]   Fig. 7. TgSODB2 does not target to the mitochondrion via the ER. Adding an ER retention signal (HDEL) to dense granule and apicoplast targeting constructs to create pTgSODB226-84/GFP-HDEL and pTgSODB2R12A/GFP-HDEL, respectively, caused ER retention of GFP in a cup-shaped pattern. Retention was complete for parasites expressing TgSODB226-84/GFP-HDEL, with no vacuolar staining overlapping with GRA4. Some apicoplast staining that colocalized with FNR(l)-dsRED was seen for pTgSODB2R12A/GFP-HDEL transfected parasites, with extensive smearing and ER involvement. Addition of HDEL had no effect on targeting of cytoplasmic fusion protein TgSODB21-25/GFP, indicating that the HDEL signal cannot misdirect proteins not destined for the secretory system. No ER signal was seen for TgSODB21-287/GFP-HDEL, which colocalized perfectly with MitotrackerTM, implying that TgSODB2 does not enter the ER and probably traffics from the cytosol. Scale bar: 5 µm.

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