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Originally published In Press as doi:10.1074/jbc.M503178200 on April 6, 2005

J. Biol. Chem., Vol. 280, Issue 23, 22053-22059, June 10, 2005
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Differential Localization of Alternatively Spliced Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase Isoforms in Toxoplasma gondii*

Kshitiz Chaudhary{ddagger}§, Robert G. K. Donald{ddagger}§, Manami Nishi{ddagger}, Darrick Carter¶, Buddy Ullman¶, and David S. Roos{ddagger}||

From the {ddagger}Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239-3098

Received for publication, March 23, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A unique feature of the Toxoplasma gondii purine salvage pathway is the expression of two isoforms of the hypoxanthine-xanthine-guanine phosophoribosyltransferase (HXGPRT) of the parasite encoded by a single genetic locus. These isoforms differ in the presence or absence of a 49-amino acid insertion (which is specified by a single differentially spliced exon) but exhibit similar substrate specificity, kinetic characteristics, and temporal expression patterns. To examine possible functional differences between the two HXGPRT isoforms, fluorescent protein fusions were expressed in parasites lacking the endogenous hxgprt gene. Immunoblot analysis of fractionated cell extracts and fluorescence microscopy indicated that HXGPRT-I (which lacks the 49-amino acid insertion) is found in the cytosol, whereas HXGPRT-II (which contains the insertion) localizes to the inner membrane complex (IMC) of the parasite. Simultaneous expression of both isoforms resulted in the formation of hetero-oligomers, which distributed between the cytosol and IMC. Chimeric constructs expressing N-terminal peptides from either isoform I (11 amino acids) or isoform II (60 amino acids) fused to a chloramphenicol acetyl transferase (CAT) reporter demonstrated that the N-terminal domain of isoform II is both necessary and sufficient for membrane association. Metabolic labeling experiments with transgenic parasites showed that isoform II or an isoform II-CAT fusion protein (but not isoform I or isoform I-CAT) incorporate [3H]palmitate. Mutation of three adjacent cysteine residues within the isoform II-targeting domain to serines blocked both palmitate incorporation and IMC attachment without affecting enzyme activity, demonstrating that acylation of N-terminal isoform II cysteine residues is responsible for the association of HXGPRT-II with the IMC.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The obligate intracellular parasite Toxoplasma gondii is a leading cause of congenital birth defects in children and opportunistic infections in immunosuppressed patients, such as those afflicted with AIDS (1, 2). This protozoan pathogen belongs to the phylum Apicomplexa, which also includes many other parasites of medical and veterinary importance. Like many intracellular pathogens, apicomplexans are incapable of de novo purine synthesis, making salvage enzymes essential for survival and therefore an attractive target for chemotherapeutic intervention (3). Different pathogens, however, employ distinct complements of enzymes to scavenge purines from their host environment (4).

T. gondii possesses two redundant purine salvage pathways involving the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT)1 and adenosine kinase, both of which have been studied in detail (5–8). An integrated genetic, biochemical, and genomic approach has shown that these two enzymes are the only physiologically relevant routes of purine acquisition by the parasite, and fitness defects associated with gene knock-outs at either locus demonstrate that both enzymes play important roles in parasite metabolism (9). In addition to its chemotherapeutic potential, T. gondii HXGPRT has also been exploited as a versatile selectable marker for molecular genetic analysis (10–12).

Two isoforms of T. gondii HXGPRT have been identified as the predicted translation products of differentially spliced mRNAs transcribed from a single gene (5). The coding sequence of HXGPRT-II is identical to that of HXGPRT-I, except for the addition of a 147-nucleotide exon encoding 49 amino acids that is inserted seven amino acids downstream of the N terminus (Fig. 1). The presence of this extra exon in HXGPRT-II is also associated with failure to excise an intron in the 5'-untranslated region. Each isoform is able to complement Escherichia coli hpt or gpt mutants and a T. gondii hxgprt knock-out mutant, and they are kinetically similar in vitro (although HXGPRT-II is slightly less efficient in phosphoribosylating guanine) (5, 13). The crystal structure of HXGPRT-I is tetrameric (6, 14), and the two isozymes form heterotetramers when co-expressed in E. coli (13).

To understand the functional significance of these two isoforms, we have determined their subcellular location, temporal expression patterns, and oligomerization status in vivo. The two isoforms form hetero-oligomers in vivo and are differentially localized within the parasite because of acylation of the N-terminal domain unique to isoform II, resulting in targeting to the inner membrane complex (IMC) that forms the structural scaffolding of the parasite.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites, Host Cells, Chemicals, and Reagents—RH strain T. gondii tachyzoites and RH{Delta}HXGPRT knock-out mutants (5) were maintained by serial passage in primary human foreskin fibroblasts (HFF cells) (15). Tachyzoite to bradyzoite differentiation was induced in vitro as described previously (10). ME49 strain T. gondii were obtained from the National Institutes of Health Research and Reference Reagent Program (catalog no. 2858), and unsporulated oocysts and Veg strain tachyzoites were kindly provided by Dr. Michael S. White (Montana State University, Bozeman). DNA-modifying enzymes were purchased from New England Biolabs (Beverly MA), unlabeled substrates of purine salvage enzymes from Sigma, and radiolabeled compounds from Moravek Biochemicals (Brea, CA; nucleotides), PerkinElmer Life Sciences (chloramphenicol, amino acids), and Amersham Biosciences (fatty acids).

Procedures for transient and stable transformation of T. gondii have been described in detail (15–17). T. gondii HXGPRT and E. coli CAT genes were used both as enzyme reporters and as selectable markers to obtain stable transgenic parasites. Selections for HXGPRT+ parasites following transfection with HXGPRT-I or HXGPRT-II expression vectors were carried out in 25 µg/ml mycophenolic acid supplemented with 50 µg/ml xanthine, as described previously (5). Parasites expressing chimeric HXGPRT-CAT enzymes were selected in 20 µM chloramphenicol, and drug-resistant clones were isolated by limiting dilution in 96-well plates (16). Southern blot analysis of several independent drug-resistant parasite clones obtained from transfections with HXGPRT or HXGPRT-CAT expression plasmids indicated 1–3 copies of the transgene in each clone (not shown). No differences in drug resistance were observed among transgenic parasite clones expressing membrane-associated versus cytosolic forms of HXGPRT or CAT reporter enzymes.

Molecular Methods—HXGPRT-I or HXGPRT-II expression vectors and plasmids pdhfrCAT and ptubIMC1-YFP have been described previously (5, 15, 18). For fluorescent protein fusions, the open reading frames of HXGPRT-I and HXGPRT-II were PCR-amplified using sense primer HXBglF, 5'-GAagatctATGGCGTCCAAACCCATTG-3', and antisense primer HXAvrR, 5'-GGCcctaggCTTCTCGAACTTTTTGCGAG-3' (restriction sites indicated in lowercase). The amplified fragments were digested with BglII and AvrII and ligated into appropriately digested ptub-YFP or ptub-mRFP vectors,2 generating plasmids encoding an HXGPRT-I-yellow fluorescent protein (HXGPRT-I-YFP) fusion and an HXGPRT-II-monomeric red fluorescent protein fusion (HXGPRT-II-mRFP), respectively.

To change cysteines 23–25 to serines in the HXGPRT-II expression vector, we used antisense primer 5'-CTTCATTAGGAGTGCTACTACTGAAGATGTCT-3' (altered residues underlined) in a PCR-based site-directed mutagenesis reaction to generate vector pdhfrHXGPRT-II-C(23–25)S. Chimeric HXGPRT-CAT fusion constructs were prepared by swapping DNA fragments from HXGPRT and CAT expression vectors that were modified by the introduction of restriction sites using site-directed mutagenesis to facilitate cloning. Antisense primer 5'-GGGCTCAATACGGCCgtcgacCTTGCCGTAGTC-3' was used to introduce a SalI site in HXGPRT expression vectors at amino acids 12 and 13 of HXGPRT-I (in pdhfrHXGPRT-I) and 61 and 62 of HXGPRT-II (in pdhfrHXGPRT-II and pdhfrHXGPRT-II-C(23–25)S. Antisense primer 5'-CCAGTGATTTTTTTctcgagTTTAGATCTGAC-3' was used to introduce an XhoI site at CAT translational start in vector pdhfrCAT. The dhfr promoter of pdhfrCAT (XhoI derivative) was excised by digestion with XhoI and KpnI (blunted) and replaced with dhfr promoter-HXGPRT N-terminal fragments from pdhfrHXGPRT vectors digested with SalI and HindIII (blunted). The resulting vectors expressed chimeric HXGPRT-CAT fusion proteins with the first 11 amino acids of HXGPRT-I (pdH1CAT) or the first 60 amino acids of HXGPRT-II (pdH2CAT and pdH2-C(23–25)S-CAT). HXGPRT-His6 fusions were generated by PCR amplification of the HXGPRT-I and HXGPRT-II open reading frames using sense primer HXBglF (above) and antisense primer HX6HisAflR, 5'-CGcttaagCGTGATGGTGATGGTGATGCTTCTCGAATGCG-3', digestion with BglII and AflII, and ligation into BglII/AflII-digested ptubACP-YFP-HA,2 replacing the ACP-YFP-HA open reading frames with HXGPRT-I-His6 or HXGPRT-II-His6.

Total RNA was harvested from parasites using the RNeasy RNA extraction kit (Qiagen, Valencia, CA). 3 µg of denatured RNA was loaded onto a formaldehyde-agarose gel, blotted onto Nytran membrane (Amersham Biosciences), and probed with a PCR-amplified HXGPRT cDNA fragment derived from pdhfrHXGPRT-II using HXBglF and HXAvrR. The gel was stained with ethidium bromide before transfer to confirm equal RNA loading.

Subcellular Fractionation and Enzyme Assays—5 x 108 parasites that had recently lysed out of a monolayer of human foreskin fibroblast cells were purified by filtration through 3-micron Nucleopore membranes (Corning, Corning, NY), washed in phosphate-buffered saline (PBS), and resuspended in 0.5 ml of sonication buffer (400 mM sucrose, 100 mM Tris-HCl, pH 7.5, 10 mM KCl, 5 mM MgCl2, 10% glycerol, 10 mM {beta}-mercaptoethanol, 0.1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). Following sonication, crude extracts were fractionated by centrifugation at 3,000 x g for 10 min, and the supernatant was subjected to an additional sedimentation at 30,000 x g for 30 min. The 30,000 x g pellet was washed and resuspended in 50 µl of sonication buffer by vigorous pipetting, and both the supernatant and the pellet fractions were assayed for HXGPRT or CAT enzymatic activity or loaded onto 12% SDS-polyacrylamide gels for Western blot analysis. The concentration of protein in the pellet fraction was ~10-fold higher than an equivalent volume of supernatant.

HXGPRT activities were assayed in transgenic parasites expressing either HXGPRT-I or HXGPRT-II using [8-3H]xanthine as substrate because host HFF cells cannot phosphoribosylate xanthine (19). Crude parasite lysates were prepared by sonication and incubated with [8-3H]xanthine (10 Ci/mmol) and 1.0 mM 5-phosphoribosyl 1-pyrophosphate, and the reaction products were quantified by ascending paper chromatography. CAT activity in the membrane and pellet fractions of parasites expressing either CAT or chimeric HXGPRT-CAT was assayed as described elsewhere (15), by phosphorimaging analysis of thin-layer chromatograms on which acetylated reaction products were separated from the [1,2-14C]chloramphenicol substrate (>50 mCi/mmol).

Immunological Reagents, Immunofluorescence, and Immunoprecipitation—HXGPRT antisera were generated in New Zealand White rabbits by Cocalico Biologicals Inc. (Reamstown, PA) using conventional protocols. The initial inoculation was performed with 100 µg of purified recombinant purified HXGPRT-I (5) in complete Freund's adjuvant. Subsequent boosts with 50 µg of antigen in incomplete Freund's adjuvant were carried out on days 14, 21, 49, 70, and 121. Test bleeds were performed on days 35, 56, and 77, and the rabbits were exsanguinated at day 135. Antibody titer was ascertained by Western blot analysis against whole T. gondii cell lysates. The antisera exhibited good cross-reactivity against recombinant HXGPRT-II, as expected from the high degree of sequence identity between the two isoforms. Polyclonal rabbit antiserum against CAT was obtained from 5 Prime -> 3 Prime, Inc. (Boulder, CO), and monoclonal mouse Tetra-His antibody was purchased from Qiagen. Antisera against TgMLC1 and TgGAP45 were generously provided by Dr. Con Beckers (University of North Carolina, Chapel Hill).

For fluorescence microscopy, confluent monolayers of HFF cells grown on glass coverslips in 6-well plates were infected with 5 x 105 parasites and examined after 18–24 h. Infected monolayers were fixed in 4% paraformaldehyde (in PBS), permeabilized with 0.25% Triton X-100, and treated with blocking buffer (1% BSA, 5% fetal calf serum in PBS) before staining. CAT protein was detected using a specific polyclonal rabbit antisera (diluted 1:200 in blocking buffer) followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma; diluted 1:160). Nuclear DNA was stained with 2.8 µM 4',6'diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR). YFP, mRFP, DAPI, and secondary fluorescent reagents were detected using an Olympus IX70 inverted microscope equipped with a 100-watt mercury vapor lamp with appropriate barrier emission filters (DeltaVision). Images were captured using a Photometrics CoolSNAP Hi Res charge-coupled device camera and DeltaVision softWorx software (Applied Precision, Issaquah, WA).

For metabolic labeling and immunoprecipitation, confluent cultures of HFF cells grown in 175-cm2 T flasks were inoculated with ~5 x 107 parasites and incubated for 36 h (parasites spontaneously lyse the host cell monolayer at ~44 h). Monolayers were then rinsed with labeling medium (serum/methionine/cysteine/pyruvate-free minimal essential medium supplemented with glutamine, Invitrogen) before the addition of radiolabel. For [35S]methionine/cysteine labeling, 150 µCi of 35S-Express (1,200 Ci/mmol) was added to each flask in 25 ml of labeling medium supplemented with 1% dialyzed fetal calf serum. For fatty acid labeling, 0.5 mCi of a [3H]palmitate (5 mCi/ml, 35 Ci/mmol)/BSA suspension (the suspension consisted of 10% ethanol and 6.25 mg/ml fatty acid-free BSA) was added to each flask in 25 ml of serum-free labeling medium. Labeled parasites were harvested upon lysis by filtration through 3-micron Nucleopore membranes and washed in PBS.

For immunoprecipitation, parasites were lysed in PBS containing 0.5% Nonidet P-40, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. All subsequent incubation and washing steps were performed at 4 °C. Lysate was centrifuged for 20 min at 14,000 x g, and antiserum was added to the supernatant (4 µl of anti-T. gondii HXGPRT or 20 µl of anti-CAT antisera for radiolabeling experiments; 4 µl of anti-Tetra His for co-immunoprecipitation). After a 10-min incubation, 50 µl of protein A-agarose beads (Invitrogen) was added and incubated for an additional 60 min with mixing. The beads were then washed three times in 1 ml of 100 mM Tris-HCl, pH 8.3, 0.5 M NaCl, 1 mg/ml BSA, and 0.5% Nonidet P-40, washed twice with 10 mM Tris-HCl, pH 6.8, and boiled in 50 µl of Laemmli buffer (20) before loading onto SDS-polyacrylamide gels. [35S]Methionine/cysteine-labeled gels were dried and analyzed using phosphorimaging, and [3H]palmitate-labeled gels were impregnated with Enhance (PerkinElmer Life Sciences) before drying and exposure to x-ray film (Kodak X-AR) for 10–12 days. For co-immunoprecipitation experiments, SDS-polyacrylamide gels were transferred to a nitrocellulose membrane (Amersham Biosciences) and processed for immunoblot analysis with anti-T. gondii HXGPRT antibody.



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  		FIG. 1.
T. gondii expresses two isoforms of HXGPRT. cDNA library screening, reverse transcriptase-PCR experiments and Northern and Western blotting experiments identify two alternative HXGPRT isoforms. A, cDNA transcripts derived from the HXGPRT locus differ in the presence of an additional intron within the 5'-untranslated region of HXGPRT-I and an additional 147-nucleotide exon in HXGPRT-II; coding regions shaded. B, the predicted N-terminal sequence of HXGPRT-I and HXGPRT-II, including 49 amino acids (aa) encoded by the HXGPRT-II-specific exon noted above (the remaining 223 amino acids extending to the C terminus are identical in both isoforms). Note the three cysteine residues within the HXGPRT-II-specific insertion for which serines were substituted in targeting domain mutant pdH2-C(23–25)S. C, total RNA was harvested from wild-type (RH) and HXGPRT knock-out ({Delta}HX) parasites and hybridized with an HXGPRT-I probe, revealing two distinct bands in the parental strain and a smear attributable to the disrupted gene in the knock-out mutant. Ethidium bromide-stained rRNA (bottom) shows equal loading. Scale (in kb) is based on RNA standards (Invitrogen). D, Western blot analysis of whole parasite lysates from RH and {Delta}HX parasites using anti-T. gondii HXGPRT antiserum shows the presence of two isoforms of the predicted size (26.4 and 31.5 kDa) in wild-type parasites only. Asterisks in C and D indicate isoform I (*) or isoform II (**) transcripts and proteins.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HXGPRT Isoforms Are Differentially Distributed in Transgenic Parasites—The predicted amino acid sequences of the two HXGPRT transcripts expressed in T. gondii parasites (Fig. 1A) differ by the presence or absence of a 49-residue insertion (Fig. 1B). Northern blots of total parasite RNA probed with HXGPRT-I cDNA show the presence of two distinct mRNA transcripts in RH (wild-type) parasites that are missing in {Delta}HXGPRT knock-out mutants (Fig. 1C). Diffuse hybridization in the {Delta}HXGPRT lane is presumed to result from probe binding to aberrant transcripts from the disrupted hxgprt gene. Western blotting of total cell lysates from RH and {Delta}HXGPRT parasites with polyclonal antisera raised against recombinant T. gondii HXGPRT-I confirms the presence of two HXGPRT isoforms of the size predicted for the two differentially spliced transcripts and the absence of any reactive protein in the knock-out mutant (Fig. 1D).

The possibility that the expression of these two isoforms might be developmentally regulated was tested by reverse transcriptase-PCR analysis of various developmental stages throughout the life cycle of the parasite. Similar relative levels of HXGPRT-I and HXGPRT-II expression were observed in the acutely lytic tachyzoite form cultivated in vitro, the latent bradyzoite tissue cyst form (induced in vitro (10)), and the unsporulated oocyst form obtained from a sexual cross in cats (data not shown). Similar levels of both HXGPRT isoforms were also observed in wild-type strains representing all three of the major parasite lineages known from population genetic studies (21): RH (type I), ME49 (type II), and Veg (type III). These results indicate that the expression of differentially spliced isoforms is conserved in all T. gondii parasites but is not stage- or strain-specific.

To further characterize these two isoforms, we examined their distribution within parasite tachyzoites by subcellular fractionation and fluorescence microscopy as shown in Fig. 2. Differential centrifugation was used to isolate a high speed membrane pellet (Fig. 2A, P) and the cytosolic supernatant (S), which were then subjected to SDS-PAGE and immunoblot analysis using anti-HXGPRT antisera (Fig. 2A) and enzyme activity assays (Fig. 2B). As expected, neither immunoreactive material nor enzyme activity was observed in the {Delta}HXGPRT knock-out background. Only isoform I was observed in the HXGPRT-I transgenics and only in the cytosolic fraction (Fig. 2A, lanes 4 and 6). Similarly, activity was observed in the cytosol only (Fig. 2B), and {Delta}HXGPRT parasites expressing a recombinant transgene in which the entire HXGPRT-I protein was fused to a fluorescent protein reporter showed cytosolic localization (Fig. 2C, left panels).

Only isoform II was observed in the HXGPRT-II transgenics, and this protein was predominantly associated with the membrane fraction (Fig. 2A, lanes 7 and 9). Much of the HXGPRT-II enzyme activity was also associated with the pellet (Fig. 2B), and a recombinant fluorescent protein fusion highlighted the surface membranes of the parasite (Fig. 2C, right panels). HXGPRT-II remained associated with the pellet fraction following washes in either 1 M KCl or 6 M urea (data not shown), indicating that membrane association is not simply a consequence of superficial electrostatic interactions. Significant HXGPRT-II protein and enzyme activity was also observed in the cytosol, but cytosolic localization was more prominent in transgenics employing the (relatively strong) pdhfr promoter as compared with the native phpt promoter (compare lanes 8 and 10 in Fig. 2A), suggesting mistargeting due to overexpression. In wild-type parasites both isoforms were observed in both fractions (compare Fig. 2A, lanes 11 and 12; see "Discussion").



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  		FIG. 2.
HXGPRT isoforms are differentially localized in T. gondii tachyzoites. A, immunoblot analysis of soluble (S) and pellet (P) fractions from wild-type parasites (RH, lanes 11 and 12), {Delta}HXGPRT mutants ({Delta}HX, lanes 1 and 2), and {Delta}HXGPRT parasites expressing HXGPRT isoform I (+HXGPRT-I, lanes 3–6) or isoform II (+HXGPRT-II, lanes 7–10). Two independent lines were examined for each transgene, with HXGPRT under the control of either the T. gondii dhfr promoter (pdhfr; lanes 3 and 4 and 7 and 8) or the native hxgprt promoter (phpt lanes 5 and 6 and 9 and 10). B, XPRT activity in soluble (gray bars) and membrane (black bars) fractions of wild-type parasites, {Delta}HXGPRT mutants, and {Delta}HXGPRT parasites expressing isoform I, isoform II, or the isoform II mutant HXGPRT-II-C(23–25)S (+HXGPRT-II*). C, fluorescent protein fusions of HXGPRT isoform I (HXGPRT-I-YFP; left) or isoform II (HGXPRT-II-mRFP; right) were expressed in {Delta}HXGPRT parasites. Panels show differential interference contrast (DIC) and fluorescence images. Note that HXGPRT-I localizes exclusively to the cytosol, whereas HXGPRT-II is predominantly membrane-associated.

 
HXGPRT-II Associates with the Inner Membrane Complex— The association of HXGPRT-II with the membrane fraction in T. gondii prompted efforts to more precisely define its subcellular location. The pellicle of Apicomplexan parasites is composed of three membranes, including the plasma membrane, and a patchwork of underlying flattened vesicles associated with cytoskeletal components (designated the "inner membrane complex") (22, 23). In addition to providing structural stability, the IMC also serves as a scaffold for daughter parasite assembly during cell division; daughters eventually acquire their plasma membrane by budding out of the mother cell (18). The IMC1 protein is a component of the subpellicular network, a membrane skeleton associated with the IMC in both the mother and developing daughter parasites (24). As shown in Fig. 3A, the HXGPRT-II-mRFP fusion associates with the pellicle of both the mother and developing daughter parasites during mitotic division, even before the daughters acquire their plasma membrane. HXGPRT-II-mRFP co-localizes with an IMC1-YFP transgene, confirming association with the IMC rather than the plasma membrane (Fig. 3B). Treatment of these parasites with oryzalin, which disrupts the assembly of new subpellicular microtubules and IMC formation in mother and daughter cells (25), disrupts the localization of both IMC1 and HXGPRT-II (Fig. 3C).

Further confirmation for association of HXGPRT-II with the IMC comes from mass spectrometric analysis of proteins co-precipitated with HXGPRT following cross-linking. Preliminary studies have identified both IMC1 and myosin A in these samples (not shown). Both of these proteins are known to be associated with the IMC (26).

Hetero-oligomers of HXGPRT Isoforms Form in Vivo—The crystal structure of T. gondii HXGPRT-I is tetrameric (6, 14), and the two isoforms form heterotetramers when co-expressed in E. coli (13). To investigate whether such hetero-oligomers also form in vivo, we transfected parasites stably expressing HXGPRT-I in a {Delta}HXGPRT background with HXGPRT-II-His6 fusion constructs. Conversely, parasites stably expressing HXGPRT-II were transfected with HXGPRT-I-His6. As shown in Fig. 4A, immunoprecipitation with a monoclonal anti-His antibody followed by probing with anti-T. gondii HXGPRT antisera demonstrates that untagged HXGPRT-I co-precipitated with HXGPRT-II-His6 (lane 4) and untagged HXGPRT-II co-precipitated with HXGPRT-I-His6 (lane 8). Control immunoprecipitations from parasites expressing only HXGPRT-I or HXGPRT-II verify the specificity of the anti-His antibody (Fig. 4A, lanes 2 and 6). These results provide evidence that the two HXGPRT isoforms form hetero-oligomers in vivo.



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  		FIG. 3.
HXGPRT isoform II localizes to the inner membrane complex of the parasite. A, a transgenic HXGPRT-II-mRFP fusion protein labels the surface of both mother (arrows) and developing daughter cells (arrowheads) in {Delta}HXGPRT parasites, a pattern similar to that observed for the inner membrane complex marker IMC1 (18). B, HXGPRT-II-mRFP co-localizes with IMC1-YFP when both fusion proteins are co-expressed in {Delta}HXGPRT parasites (daughter parasites label more intensely with IMC1) (18). C, HXGPRT-II-mRFP and IMC1-YFP co-localize in untreated controls (–oryzalin) and cells treated with 500 nM oryzalin for 24 h to disrupt daughter parasite assembly. mRFP, red; YFP, green; DAPI, blue.

 
The subcellular location of HXGPRT-I/HXGPRT-II hetero-oligomers was examined by simultaneously expressing both HXGPRT-I-YFP and HXGPRT-II-mRFP in {Delta}HXGPRT parasites. A variety of phenotypes were observed in these transient transfectants, as shown in Fig. 4B. In some parasites, both isoforms were cytosolic (Fig. 4B, panel i). In others, HXGPRT-II was associated with the IMC (Fig. 4B, panels ii and iii); in some of these, HXGPRT-I was predominantly cytosolic (panel ii), whereas in others HXGPRT-I was associated with the membrane (panel iii). HXGPRT-I was never associated with the IMC unless membrane association was observed for HXGPRT-II. These data are consistent with the observation that both isoforms are found in both the cytosolic and membrane fractions of wild-type parasites (Fig. 2A, lanes 11 and 12). In view of the fact that HXGPRT-I is exclusively cytosolic when expressed alone, whereas HXGPRT-II is predominantly associated with the IMC (Fig. 2C), these results strongly suggest that hetero-oligomers form in vivo and are distributed based on the ability of HXGPRT-II to associate with the IMC (see "Discussion").

Palmitoylation of Cysteine Residues in the Isoform II-specific Exon Mediates Targeting to the IMC—Because the coding sequence of HXGPRT-II differs from HXGPRT-I only in the presence of a 49-amino acid insertion near the N terminus (Fig. 1), N-terminal peptides from HXGPRT-I (11 amino acids) or HXGPRT-II (60 amino acids) were fused to the E. coli CAT gene to explore the possibility that this region encodes a membrane association determinant. pdH1-CAT or pdH2-CAT was transiently transfected into {Delta}HXGPRT parasites, and CAT activity was examined in the membrane and pellet fractions, as shown in Fig. 5. In parasites expressing either a nonchimeric CAT control (pdCAT) or HXGPRT-I-CAT, activity was observed only in the cytosolic fraction. In contrast, parasites expressing HXGPRT-II-CAT showed significant levels of CAT activity associated with the membrane pellet (Fig. 5, black bar). Similar results were obtained by immunofluorescence using an anti-CAT antibody (not shown). These results demonstrate that the 49-amino acid insertion characteristic of HXGPRT-II is both necessary and sufficient to target a soluble protein to the parasite IMC.



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  		FIG. 4.
HXGPRT isoforms form hetero-oligomers in vivo. A, {Delta}HXGPRT knock-out parasites stably expressing (untagged) HXGPRT-I or HXGPRT-II were transiently transfected with His-tagged HXGPRT-II or HXGPRT-I constructs, respectively, and parasite lysates separated on an SDS-polyacrylamide gel before (lys) or after (IP) immunoprecipitation with anti-His antibody. Parasite proteins were revealed by blotting and hybridization with anti-T. gondii HXGPRT antisera. When both isoforms were present, immunoprecipitation of HXGPRT-I pulled down HXGPRT-II (lane 8) and vice versa (lane 4). B, {Delta}HXGPRT parasites were co-transfected with HXGPRT-I-YFP (green) and HXGPRT-II-mRFP (red) constructs. The three panels represent the range of localization phenotypes observed (see "Results"). Arrows and arrowheads indicate co-localization of HXGPRT-I and HXGPRT-II in the IMC of mother and daughter parasites, respectively.

 
The HXGPRT-II-specific insertion is relatively hydrophilic and contains no obvious membrane-spanning domain (Fig. 1A) but includes a cluster of three contiguous cysteines, which could potentially mediate membrane targeting via palmitoylation (27). To assess the functional significance of this motif, all three codons were mutated to encode serines, in the context of both a full-length HXGPRT-II construct (pdH2-C(23–25)S) and the chimeric CAT fusion containing only the N-terminal 60 amino acids of HXGPRT-II (pdH2-C(23–25)S-CAT). This modification had no effect on enzyme activity but abolished membrane association of both isoform II (Fig. 2B) and the chimeric CAT enzyme (Fig. 5).



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  		FIG. 5.
The N-terminal domain of HXGPRT-II targets a heterologous reporter to the IMC. CAT activity was assayed in soluble (gray bars) and membrane (black bars) fractions from parasites transiently expressing CAT fused downstream of N-terminal peptides derived from HXGPRT proteins. untransformed, mock-transfected {Delta}HXGPRT parasites; pdCAT, soluble (nonchimeric) CAT; pdH1CAT, chimeric HXGPRT-I-CAT; pdH2CAT, chimeric HXGPRT-II-CAT; pdH2*CAT, mutant HXGPRT-II-C(23–25)S fused to CAT. Diagrams at left show plasmid constructs containing 5'- and 3'-untranslated regions from the dhfr promoter (hatched) flanking CAT cDNA and the N-terminal peptides of HXGPRT-I and HXGPRT-II (solid boxes); asterisk indicates the C(23–25)S mutation.

 
Wild-type parasites, {Delta}HXGPRT knock-outs, and knock-out transgenics expressing HXGPRT-I, HXGPRT-II, or the HXGPRT-II-C(23–25)S mutant were labeled with [3H]palmitate and immunoprecipitated with anti-T. gondii HXGPRT antiserum, as shown in Fig. 6A. The HXGPRT-II isoform was readily labeled with palmitate in both wild-type parasites (Fig. 6A, lane 1) and the HXGPRT-II transgenics (lane 4), but no labeling was observed for HXGPRT-I (lanes 1 and 3). Labeling was abolished in the C(23–25)S mutant (lane 5). Similar results were obtained with transgenic parasites expressing chimeric HXGPRT-CAT (Fig. 6B). Parallel experiments failed to demonstrate any significant incorporation of [3H]myristate or other fatty acyl chains (not shown). These results indicate that the targeting domain cysteines required for membrane association of HXGPRT-II are modified by palmitoylation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A ready supply of nucleotides is critical for all living organisms, and most species exhibit a diverse array of redundant pathways that help to ensure adequate nucleotide availability (28). Many intracellular pathogens have dispensed with de novo purine biosynthesis, perhaps reflecting a high metabolic cost of this process and/or the abundant supply of purine nucleotides available for salvage from the host. Multiple redundant routes of purine salvage are commonly observed, however, underlining the importance of this pathway. For example, in addition to using adenosine kinase to salvage adenosine, T. gondii also salvages hypoxanthine (as well as xanthine and guanine) using HXGPRT and can interconvert adenylate and guanylate nucleotides (9). Neither adenosine kinase nor HXGPRT is essential for parasite survival (5, 29), but both play important roles in T. gondii purine metabolism, as indicated by the significant fitness cost relative to the wild-type parasites when either enzyme is inactivated (9).

Further redundancy may be provided by the expression of multiple enzyme isoforms. Many examples of metabolic isoenzymes are evident in the T. gondii genome, including glucose 6-phosphate isomerase, enolase, lactate dehydrogenase, phosphatidylinositol synthase, etc. (30). All HXGPRT activity in T. gondii is encoded by a single gene, but differential mRNA transcript processing yields two different enzyme isoforms. As far as we are aware, this is the first reported example of differential HXGPRT splicing in any system, providing an unusual basis for diversity.



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  		FIG. 6.
The HXGPRT-II cysteines required for membrane targeting are palmitoylated. A, HXGPRT isoforms were immunoprecipitated from lysates of radiolabeled wild-type parasites (RH), {Delta}HXGPRT knock-outs, and {Delta}HXGPRT parasites expressing transgenic HXGPRT-I, HXGPRT-II, or the HXGPRT-II C(23–25)S mutant (+HXGPRT-II*), using anti T. gondii HXGPRT antisera. B, HXGPRT-CAT proteins were immunoprecipitated from lysates of transgenic parasites expressing chimeric HXGPRT-I-CAT (pdH1CAT), HXGPRT-II-CAT (pdH2CAT), or HXGPRT-II-C(23–25)S-CAT (pdH2*CAT), using anti-CAT antisera. In both panels, control immunoprecipitations from [35S]methionine/cysteine-labeled cultures verify the efficiency of protein recovery. The results demonstrate that HXGPRT-II is palmitoylated on the cysteine residues required for membrane targeting.

 
Applying cell fractionation, immunoblot analysis, and fluorescence microscopy to wild-type parasites, {Delta}HXGPRT knock-out mutants, and a variety of transgenics capable of expressing only HXGPRT-I, HXGPRT-II, or various mutant or chimeric proteins, we have shown that the two HXGPRT isoforms are capable of forming hetero-oligomers in vivo (Fig. 4). This observation extends previous reports indicating heterotetramer formation when HXGPRT-I and HXGPRT-II are co-expressed in E. coli, with the predominant form consisting of one subunit of HXGPRT-I and three of HXGPRT-II (13).

We have also shown that the HXGPRT-I and HXGPRT-II are differentially localized within the parasite (Figs. 2 and 3). HXGPRT-I is predominantly cytosolic, whereas HXGPRT-II localizes to the parasite IMC. When both isoforms are present, distribution appears to be dependent on the relative expression levels; whichever isoform is most abundant determines the subcellular localization of the hetero-oligomer, suggesting that at least two HXGPRT-II subunits are required for membrane localization.

Membrane association of HXGPRT-II is mediated by an isoform-specific 49-amino acid insertion, and this domain is both necessary for membrane localization and sufficient to confer membrane targeting on a heterologous reporter (Fig. 5). Within this insertion, a Cys-Cys-Cys motif is required for membrane targeting (Figs. 2 and 5) and was also found to be palmitoylated (Fig. 6). Mutation of this motif to Ser-Ser-Ser abolishes both lipid labeling and targeting to the IMC in parallel, highlighting the functional significance of these residues. Palmitoylation regulates the membrane association of many eukaryotic proteins and is particularly prominent in signaling molecules (27). Because the membrane targeting domain of HXGPRT-II lacks a transmembrane domain and any obvious prenylation or myristylation motifs (we were unable to label the protein with myristate), its association with the IMC is likely to result from cysteine palmitoylation alone.

The functional significance, if any, of the two HXGPRT isoforms found in T. gondii remains unclear. Kinetic characterization reveals only minimal differences in activity and substrate specificity (5, 13). Moreover, although mycophenolic acid kills HXGPRT knock-out parasites by blocking conversion of AMP and IMP to GMP, parasites can be rescued by either HXGPRT-I or HXGPRT-II (5). This experiment also argues against any significant difference in the ability of cytosolic-versus membrane-associated HXGPRT to salvage intra-versus extracellular purines. Several T. gondii isoenzymes are known to be differentially expressed during the complex life cycle of the parasite (30), but this appears not to be the case for HXGPRT. Differential splicing, resulting in the production of distinct HXGPRT proteins, is conserved in all known strains of T. gondii, however, suggesting conserved function. Multiple HXGPRT isoforms have not been described in Plasmodium spp., although one report suggests that some Plasmodium falciparum HXGPRT may be located within membrane-bound compartments in merozoites and even in the host cell cytoplasm (31).

We are not aware of previous reports describing HXGPRT association with membranes or cytoskeletal elements, but it is intriguing to note the recently reported role of another metabolic enzyme, aldolase, in connecting membrane adhesins with the parasite actin cytoskeleton (32). It is conceivable that HXGPRT-II could also function as a structural enzyme, facilitating stable association of other proteins with the IMC. It is also possible that membrane-associated HXGPRT-II may be required to ensure adequate local pools of nucleotides for proteins associated with IMC (a form of substrate channeling). For example, purine nucleotides may be important for the function of the "glideosome," a protein complex associated with the IMC and containing the molecular motor myosin A, MLC1, and two recently characterized proteins, GAP45 and GAP50 (26, 33). In this context, reversible palmitoylation (34) of HXGPRT-II could play a regulatory role in transiently increasing purine flux at the IMC. It would be interesting to explore the relative contributions of HXGPRT-I and HXGPRT-II to parasite fitness that may shed light on the importance of these isoenzymes in purine salvage versus other nonmetabolic processes within the cell.


   FOOTNOTES
 
* This work was supported by research and training grants from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| An Ellison Medical Foundation Senior Scholar in global infectious diseases. To whom correspondence should be addressed: Dept. of Biology, University of Pennsylvania, 415 S. University Ave., Philadelphia, PA 19104-6018. Tel.: 215-898-2118; Fax: 215-746-6697; E-mail: droos{at}sas.upenn.edu.

1 The abbreviations used are: HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; HFF, human foreskin fibroblast(s); CAT, chloroamphenicol acetyltransferase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; IMC, inner membrane complex; YFP, yellow fluorescent protein; mRFP, monomeric red fluorescent protein; DAPI, 4',6'-diamidino-2-phenylindole. Back

2 M. Nishi and D. S. Roos, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Sarah Shih for help with generating anti-T. gondii HXGPRT antiserum and Dr. Michael Crawford for helpful comments on the manuscript. A plasmid encoding mRFP was kindly provided by Dr. Roger Tsien (University of California, San Diego).



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 DISCUSSION
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