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• W2018076341 abstract "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. 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. 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 (1Wilson C.B. Remington J.S. Am. J. Obstet. Gynecol. 1980; 138: 357-363Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 2Luft B.J. Remington J.S. Clin. Infect. Dis. 1992; 15: 211-222Crossref PubMed Scopus (1071) Google Scholar). 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 (3Ullman B. Carter D. Infect. Agents Dis. 1995; 4: 29-40PubMed Google Scholar). Different pathogens, however, employ distinct complements of enzymes to scavenge purines from their host environment (4Carter N.S. Rager N. Ullman B. Marr J.J. Nilsen T.W. Komuniecki R.W. Molecular Medical Parasitology. Academic Press, London2003: 197-224Crossref Google Scholar). T. gondii possesses two redundant purine salvage pathways involving the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) 1The 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. and adenosine kinase, both of which have been studied in detail (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 6Schumacher M.A. Carter D. Ross D.S. Ullman B. Brennan R.G. Nat. Struct. Biol. 1996; 3: 881-887Crossref PubMed Scopus (99) Google Scholar, 7Darling J.A. Sullivan Jr., W.J. Carter D. Ullman B. Roos D.S. Mol. Biochem. Parasitol. 1999; 103: 15-23Crossref PubMed Scopus (44) Google Scholar, 8Schumacher M.A. Scott D.M. Matthews I.I. Ealick S.E. Roos D.S. Ullman B. Brennan R.G. J. Mol. Biol. 2000; 296: 549-567Crossref PubMed Scopus (49) Google Scholar). 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 (9Chaudhary K. Darling J.A. Fohl L.M. Sullivan Jr., W.J. Donald R.G. Pfefferkorn E.R. Ullman B. Roos D.S. J. Biol. Chem. 2004; 279: 31221-31227Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In addition to its chemotherapeutic potential, T. gondii HXGPRT has also been exploited as a versatile selectable marker for molecular genetic analysis (10Bohne W. Roos D.S. Mol. Biochem. Parasitol. 1997; 88: 115-126Crossref PubMed Scopus (56) Google Scholar, 11Donald R.G. Roos D.S. Mol. Biochem. Parasitol. 1998; 91: 295-305Crossref PubMed Scopus (118) Google Scholar, 12Knoll L.J. Boothroyd J.C. Mol. Cell. Biol. 1998; 18: 807-814Crossref PubMed Google Scholar). Two isoforms of T. gondii HXGPRT have been identified as the predicted translation products of differentially spliced mRNAs transcribed from a single gene (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). 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) (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 13White E.L. Ross L.J. Davis R.L. Zywno-Van Ginkel S. Vasanthakumar G. Borhani D.W. J. Biol. Chem. 2000; 275: 19218-19223Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The crystal structure of HXGPRT-I is tetrameric (6Schumacher M.A. Carter D. Ross D.S. Ullman B. Brennan R.G. Nat. Struct. Biol. 1996; 3: 881-887Crossref PubMed Scopus (99) Google Scholar, 14Heroux A. White E.L. Ross L.J. Borhani D.W. Biochemistry. 1999; 38: 14485-14494Crossref PubMed Scopus (69) Google Scholar), and the two isozymes form heterotetramers when co-expressed in E. coli (13White E.L. Ross L.J. Davis R.L. Zywno-Van Ginkel S. Vasanthakumar G. Borhani D.W. J. Biol. Chem. 2000; 275: 19218-19223Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). 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. Parasites, Host Cells, Chemicals, and Reagents—RH strain T. gondii tachyzoites and RHΔHXGPRT knock-out mutants (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) were maintained by serial passage in primary human foreskin fibroblasts (HFF cells) (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (511) Google Scholar). Tachyzoite to bradyzoite differentiation was induced in vitro as described previously (10Bohne W. Roos D.S. Mol. Biochem. Parasitol. 1997; 88: 115-126Crossref PubMed Scopus (56) Google Scholar). 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 (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (511) Google Scholar, 16Donald R.G. Roos D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11703-11707Crossref PubMed Scopus (289) Google Scholar, 17Kim K. Soldati D. Boothroyd J.C. Science. 1993; 262: 911-914Crossref PubMed Scopus (234) Google Scholar). 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 (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). 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 (16Donald R.G. Roos D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11703-11707Crossref PubMed Scopus (289) Google Scholar). 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 (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (511) Google Scholar, 18Hu K. Mann T. Striepen B. Beckers C.J. Roos D.S. Murray J.M. Mol. Biol. Cell. 2002; 13: 593-606Crossref PubMed Scopus (139) Google Scholar). 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, 2M. Nishi and D. S. Roos, manuscript in preparation. 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, 2M. Nishi and D. S. Roos, manuscript in preparation. 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 × 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 β-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 × g for 10 min, and the supernatant was subjected to an additional sedimentation at 30,000 × g for 30 min. The 30,000 × 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 (19Pfefferkorn E.R. Borotz S.E. Exp. Parasitol. 1994; 79: 374-382Crossref PubMed Scopus (52) Google Scholar). 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 (15Roos D.S. Donald R.G. Morrissette N.S. Moulton A.L. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (511) Google Scholar), 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 (5Donald R.G. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) 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 × 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 × 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 × 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 (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) 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. 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 ΔHXGPRT knock-out mutants (Fig. 1C). Diffuse hybridization in the ΔHXGPRT 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 ΔHXGPRT 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 (10Bohne W. Roos D.S. Mol. Biochem. Parasitol. 1997; 88: 115-126Crossref PubMed Scopus (56) Google Scholar)), 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 (21Howe D.K. Sibley L.D. J. Infect. Dis. 1995; 172: 1561-1566Crossref PubMed Scopus (1056) Google Scholar): 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 ΔHXGPRT 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 ΔHXGPRT 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”). 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”) (22Morrissette N.S. Murray J.M. Roos D.S. J. Cell Sci. 1997; 110: 35-42Crossref PubMed Google Scholar, 23Morrissette N.S. Sibley L.D. Microbiol. Mol. Biol. Rev. 2002; 66: 21-38Crossref PubMed Scopus (339) Google Scholar). 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 (18Hu K. Mann T. Striepen B. Beckers C.J. Roos D.S. Murray J.M. Mol. Biol. Cell. 2002; 13: 593-606Crossref PubMed Scopus (139) Google Scholar). 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 (24Mann T. Beckers C. Mol. Biochem. Parasitol. 2001; 115: 257-268Crossref PubMed Scopus (189) Google Scholar). 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 (25Stokkermans T.J. Schwartzman J.D. Keenan K. Morrissette N.S. Tilney L.G. Roos D.S. Exp. Parasitol. 1996; 84: 355-370Crossref PubMed Scopus (116) Google Scholar), 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 (26Opitz C. Soldati D. Mol. Microbiol. 2002; 45: 597-604Crossref PubMed Scopus (147) Google Scholar). Hetero-oligomers of HXGPRT Isoforms Form in Vivo—The crystal structure of T. gondii HXGPRT-I is tetrameric (6Schumacher M.A. Carter D. Ross D.S. Ullman B. Brennan R.G. Nat. Struct. Biol. 1996; 3: 881-887Crossref PubMed Scopus (99) Google Scholar, 14Heroux A. White E.L. Ross L.J. Borhani D.W. Biochemistry. 1999; 38: 14485-14494Crossref PubMed Scopus (69) Google Scholar), and the two isoforms form heterotetramers when co-expressed in E. coli (13White E.L. Ross L.J. Davis R.L. Zywno-Van Ginkel S. Vasanthakumar G. Borhani D.W. J. Biol. Chem. 2000; 275: 19218-19223Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). To investigate whether such hetero-oligomers also form in vivo, we transfected parasites stably expressing HXGPRT-I in a ΔHXGPRT 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" @default.
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