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• W2076804827 abstract "The apicoplast is a distinctive organelle associated with apicomplexan parasites, includingPlasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant “poison” that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity. The apicoplast is a distinctive organelle associated with apicomplexan parasites, includingPlasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant “poison” that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity. acyl carrier protein ferrodoxin NADP-reductase green fluorescent protein red fluorescent protein fluorescence-activated cell sorting chloramphenicol acetyltransferase Toxoplasma gondii is an obligate intracellular parasite, a major cause of congenital birth defects in humans and livestock (1Dubey J.P. Welcome F.L. J. Am. Vet. Med. Assoc. 1988; 193: 697-700PubMed Google Scholar, 2Roizen N. Swisher C.N. Stein M.A. Hopkins J. Boyer K.M. Holfels E. Mets M.B. Stein L. Patel D. Meier P. Pediatrics. 1995; 95: 11-20PubMed Google Scholar), and an important opportunistic infection associated with AIDS (3Luft B.J. Hafner R. Korzun A.H. Leport C. Antoniskis D. Bosler E.M. Bourland III, D.D. Uttamchandani R. Fuhrer J. Jacobson J. N. Engl. J. Med. 1993; 329: 995-1000Crossref PubMed Scopus (371) Google Scholar). This single-cell eukaryotic pathogen contains an unusual organelle that was acquired by horizontal transfer (secondary endosymbiosis) from a eukaryotic alga (4Köhler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Crossref PubMed Scopus (611) Google Scholar). The apicoplast has been identified in many apicomplexan parasites, including Toxoplasma, Plasmodium, Eimeria, Babesia, Theileria, etc. (5Wilson R.J. Williamson D.H. Microbiol. Mol. Biol. Rev. 1997; 61: 1-16Crossref PubMed Scopus (186) Google Scholar). Previous studies have shown that the apicoplast is essential for long term parasite viability (6Fichera M.E. Roos D.S. Nature. 1997; 390: 407-409Crossref PubMed Scopus (500) Google Scholar, 7Sullivan M. Li J. Kumar S. Rogers M.J. McCutchan T.F. Mol. Biochem. Parasitol. 2000; 109: 17-23Crossref PubMed Scopus (55) Google Scholar, 8He C.Y. Shaw M.K. Pletcher C.H. Striepen B. Tilney L.G. Roos D.S. EMBO J. 2001; 20: 330-339Crossref PubMed Scopus (152) Google Scholar). When this organelle is eliminated by either pharmacological or molecular genetic manipulation, parasites are killed with distinctive “delayed death” kinetics. Plastid-deficient parasites are capable of normal growth within and escape from the first host cell, but their replication is inhibited immediately upon invasion into a new host cell. Although the mechanistic basis of the delayed death phenotype remains unexplained, these studies demonstrate that the apicoplast is an essential organelle and therefore a potential target for drug development (9McFadden G.I. Roos D.S. Trends Microbiol. 1999; 7: 328-333Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The apicoplast possesses a 35-kilobase pair circular organellar genome containing rRNA and tRNA genes and 28 open reading frames (10Wilson R.J. Denny P.W. Preiser P.R. Rangachari K. Roberts K. Roy A. Whyte A. Strath M. Moore D.J. Moore P.W. Williamson D.H. J. Mol. Biol. 1996; 261: 155-172Crossref PubMed Scopus (464) Google Scholar). All identified genes in the apicoplast genome are predicted to encode housekeeping proteins (RNA polymerase, ribosomal proteins, etc.). However, most apicoplast proteins are thought to be encoded in the nuclear genome, as is well known for other endosymbiotic organelles (mitochondria, chloroplasts, etc.). These nuclear-encoded apicoplast proteins must be imported across the four membranes surrounding this organelle (9McFadden G.I. Roos D.S. Trends Microbiol. 1999; 7: 328-333Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 11Roos D.S. Crawford M.J. Donald R.G.K. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Crossref PubMed Scopus (129) Google Scholar). Apicomplexan parasite genome and EST sequence databases (12Ajioka J.W. Int. J. Parasitol. 1998; 28: 1025-1031Crossref PubMed Scopus (31) Google Scholar, 13The Plasmodium Genome Consortium Nucleic Acids Res. 2001; 29: 66-69Crossref PubMed Google Scholar) provide a useful resource for identifying nuclear-encoded genes destined for the apicoplast. Candidate nuclear-encoded apicoplast proteins include enzymes associated with the biosynthesis of fatty acids (14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar, 15Zuther E. Johnson J.J. Haselkorn R. McLeod R. Gornicki P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13387-13392Crossref PubMed Scopus (116) Google Scholar) and terpenoids (16Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Turbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1022) Google Scholar). Several of these proteins have been localized to the apicoplast by immunostaining and/or fusion with fluorescent protein reporters (14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar, 16Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Turbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1022) Google Scholar, 17Striepen B. He C.Y. Matrajt M. Soldati D. Roos D.S. Mol. Biochem. Parasitol. 1998; 92: 325-338Crossref PubMed Scopus (164) Google Scholar, 18Jelenska J. Crawford M.J. Harb O.S. Zuther E. Haselkorn R. Roos D.S. Gornicki P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2723-2728Crossref PubMed Scopus (93) Google Scholar). Pharmacological experiments using inhibitors of type II fatty acid biosynthesis (cerulenin, triclosan, and thiolactomycin), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (fosmidomycin), or acetyl-CoA carboxylase (aryloxyphenoxypropionates) also support the presence of these enzymes (14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar, 15Zuther E. Johnson J.J. Haselkorn R. McLeod R. Gornicki P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13387-13392Crossref PubMed Scopus (116) Google Scholar, 16Jomaa H. Wiesner J. Sanderbrand S. Altincicek B. Weidemeyer C. Hintz M. Turbachova I. Eberl M. Zeidler J. Lichtenthaler H.K. Soldati D. Beck E. Science. 1999; 285: 1573-1576Crossref PubMed Scopus (1022) Google Scholar, 19Zhu G. Marchewka M.J. Woods K.M. Upton S.J. Keithly J.S. Mol. Biochem. Parasitol. 2000; 105: 253-260Crossref PubMed Scopus (74) Google Scholar, 20Surolia N. Surolia A. Nat. Med. 2001; 7: 167-173Crossref PubMed Scopus (402) Google Scholar), although formal proof that these drugs target the apicoplast itself is lacking. To substantiate the association of predicted metabolic pathways with the apicoplast and to further characterize this organelle, we have attempted to purify the apicoplast using various cell fractionation methods. Nuclear-encoded apicoplast proteins are characterized by a bipartite targeting sequence consisting of a signal peptide at the extreme N terminus followed by a plastid transit domain (11Roos D.S. Crawford M.J. Donald R.G.K. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Crossref PubMed Scopus (129) Google Scholar). Mutational studies (21DeRocher A. Hagen C.B. Froehlich J.E. Feagin J.E. Parsons M. J. Cell Sci. 2000; 113: 3969-3977PubMed Google Scholar, 22Waller R.F. Reed M.B. Cowman A.F. McFadden G.I. EMBO J. 2000; 19: 1794-1802Crossref PubMed Scopus (424) Google Scholar) indicate that this bipartite structure is both necessary and sufficient for targeting proteins into the apicoplast. Targeting appears to proceed via the secretory pathway with sequential removal of the signal peptide and the plastid transit peptide (11Roos D.S. Crawford M.J. Donald R.G.K. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Crossref PubMed Scopus (129) Google Scholar). Although removal of the secretory signal sequence (in the endoplasmic reticulum) is difficult to detect by Western blotting, the processing of the plastid-targeting domain is easily observed and presumed to occur within the plastid lumen (22Waller R.F. Reed M.B. Cowman A.F. McFadden G.I. EMBO J. 2000; 19: 1794-1802Crossref PubMed Scopus (424) Google Scholar). We have exploited the ability to target heterologous reporter proteins into the apicoplast to develop fluorescent and enzymatic markers for organellar purification. One recombinant fusion protein was found to disrupt apicoplast segregation (8He C.Y. Shaw M.K. Pletcher C.H. Striepen B. Tilney L.G. Roos D.S. EMBO J. 2001; 20: 330-339Crossref PubMed Scopus (152) Google Scholar), yielding plastid-deficient and “super-apicoplast”-containing parasites by fluorescence-activated cell sorting. These mutants allow the examination of apicoplast protein synthesis, targeting, and processing in the presence or absence of the organelle. T. gondii tachyzoites were maintained by serial passage in human foreskin fibroblast cell monolayers cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with heat-inactivated fetal bovine serum at 37 °C in a humidified CO2 incubator as described previously (23Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (506) Google Scholar). Several nuclear-encoded apicoplast proteins have been shown to contain a bipartite leader sequence consisting of a secretory signal sequence thought to mediate translocation across one membrane (into the endoplasmic reticulum) followed by a plastid transit domain thought to mediate translocation across the remaining three membranes surrounding the apicoplast (11Roos D.S. Crawford M.J. Donald R.G.K. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Crossref PubMed Scopus (129) Google Scholar,21DeRocher A. Hagen C.B. Froehlich J.E. Feagin J.E. Parsons M. J. Cell Sci. 2000; 113: 3969-3977PubMed Google Scholar, 22Waller R.F. Reed M.B. Cowman A.F. McFadden G.I. EMBO J. 2000; 19: 1794-1802Crossref PubMed Scopus (424) Google Scholar) (Fig. 1). Two previously validated apicoplast targeting signals from nuclear-encoded apicoplast proteins were employed in this study; these signals were derived from the N-terminal domains of the acyl carrier protein (ACP)1(14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar) or ferrodoxin NADP-reductase (FNR) (24Vollmer M. Thomsen N. Wiek S. Seeber F. J. Biol. Chem. 2001; 276: 5483-5490Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Plasmids ACP-GFP, ACPL-GFP, and ACP-GFP-mROP1 have been described previously (8He C.Y. Shaw M.K. Pletcher C.H. Striepen B. Tilney L.G. Roos D.S. EMBO J. 2001; 20: 330-339Crossref PubMed Scopus (152) Google Scholar, 14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar). Plasmid FNRL-RFP (kindly provided by Dr. Boris Striepen) contains 150 amino acids from the N terminus of ferrodoxin NADP-reductase (FNRL) fused to a red fluorescent protein marker (DsRed,CLONTECH). Plasmid ACPL-CAT (kindly provided by Dr. Robert G. K. Donald) is a fusion between the ACP leader sequence (ACPL) (14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar) and the chloramphenicol acetyltransferase (CAT). 50 µg of plasmid DNA (Qiagen Maxi-preps) was transfected into 107 freshly lysed-out tachyzoites by electroporation as described previously (23Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (506) Google Scholar). In transient assays, protein expression was typically assayed at 24 h post-transfection. Stable transformants were selected in the presence of 20 µm chloramphenicol. GFP, RFP, and fluorescein isothiocyanate fluorescence was detected using a Zeiss Axiovert 35 inverted microscope equipped with a 100-watt Hg-vapor lamp and fluorescein and rhodamine filter sets. Confocal microscopy was performed on a Zeiss Laser Scanning Microscope (LSM510). For immunofluorescence assays, parasites were fixed with 3.7% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 1% bovine serum albumin in phosphate-buffered saline (pH 7.4) at room temperature. The expression of CAT was detected using a polyclonal rabbit antiserum (1:1000) (5 Prime → 3 Prime, Inc., Boulder, CO) followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:160; Sigma). Several protocols were evaluated for apicoplast purification. The Stansted cell disrupter (25Kelemen M.V. Sharpe J.E. J. Cell Sci. 1979; 35: 431-441PubMed Google Scholar) was found to be the most effective, as discussed under “Results” (cf. Fig. 2). ACPL-CAT parasites were harvested from five heavily infected 175-cm2 tissue culture flasks (∼2 × 109 tachyzoites) by filtering through 3-µm pore size polycarbonate filters (Nucleopore) and centrifuged at 1500 × g for 15 min. Parasites were washed twice with homogenization buffer (250 mm sucrose, 5 mmtriethanolamine-HCl, 1 mm EDTA, pH 7.6), resuspended in 10 ml of the same buffer supplemented with protease inhibitors (0.1 µg/ml each of aprotinin, pepstatin A, and leupeptin and 0.5 µm phenylmethylsulfonyl fluoride) and DNase I (1 µg/ml), mechanically disrupted in the Stansted biological cell disrupter at ∼2000 p.s.i., and centrifuged at 1500 ×g at 4 °C for 15 min to remove unbroken parasites. The supernatant containing various organelles, vesicles, and other subcellular structures was centrifuged at 30,000 × gat 4 °C for 30 min onto a 2.2 m sucrose cushion (buffered with 5 mm triethanolamine-HCl and 1 mm EDTA, pH 7.6). The interface (a crude apicoplast fraction) was collected, mixed with 2.2 m sucrose to 0.6 ml, and loaded at the bottom of a sucrose step gradient (0.3 ml each of 1.6, 1.5, 1.4, 1.35, 1.3, 1.25, 1.2, and 1.0 m sucrose buffered with 5 mm triethanolamine-HCl and 1 mmEDTA, pH 7.6). Ultracentrifugation was carried out in a Beckman L8 55 Ultracentrifuge for 1 h at 112,000 × g using an SW55 rotor, and 0.2-ml fractions were collected from the top of the gradient. Sucrose gradient fractions were assayed as follows. The total amount of protein in each fraction was measured by Bradford assay (Bio-Rad). As high sucrose concentrations may interfere with colorimetry, fractions were centrifuged at 200,000 × g for 1 h in a Beckman TL-100 tabletop Ultracentrifuge to remove the sucrose before the assay. Protein gel electrophoresis was performed on a 12% SDS-polyacrylamide gel electrophoresis gel. For Western blotting, proteins were transferred onto nitrocellulose membranes (Schleicher and Schuell) using standard protocol (26Maniatis T. Sambrook J. Fritsch E.F. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 18:64-66Google Scholar), and antigens were detected using the ECL system (Amersham Pharmacia Biotech). After exposure to x-ray film (Eastman Kodak Co.), densitometry was performed using the ImageQuant software (Molecular Dynamics). CAT assays were performed using 14[C]chloramphenicol and thin layer chromatography on Silica Gel G-coated plates (Fisher) essentially as described previously (23Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Methods Cell Biol. 1994; 45: 27-63Crossref PubMed Scopus (506) Google Scholar). Quantification was carried out using a Storm PhosphorImager (Molecular Dynamics) equipped with the ImageQuant software. Specific activity in each fraction was calculated as the amount of ACPL-CAT, ROP7, or CAT activity (arbitrary units) divided by the total protein in each fraction. Enrichment ratios were defined as the specific activity in each fraction divided by the specific activity of the whole parasite control. For extraction in sodium carbonate (27Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1381) Google Scholar), freshly prepared membranes were incubated with 0.2 mNa2CO3 (pH 11.5) on ice for 45 min followed by centrifugation at 100,000 × g at 4 °C for 1 h to pellet membranes and insoluble structures. For extraction in Triton X-114 (28Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar, 29Brusca J.S. Radolf J.D. Methods Enzymol. 1994; 228: 182-193Crossref PubMed Scopus (132) Google Scholar), a freshly prepared 10% (v/v) stock solution of Triton X-114 (Sigma) in phosphate-buffered saline was added to membranes to a final concentration of 2%. Extraction was carried out on ice for 16 h followed by centrifugation at 15,000 g × 4 °C for 10 min in a tabletop microcentrifuge or at 100,000g × 4 °C for 1 h in a Beckman L8 55 Ultracentrifuge to remove Triton-insoluble material. To examine phase separation, the supernatant was incubated for 10 min at 37 °C and centrifuged at 15,000 × g for 10 min at room temperature, and the resulting extracts were washed several times by repeating the above procedure. Proteins in the detergent phase were precipitated for 1 h in acetone at −20 °C and solubilized in SDS loading buffer for polyacrylamide gel electrophoresis in parallel with the aqueous phase samples. FACS was performed on a Becton Dickinson FACS Vantage SE dual laser flow cytometer equipped with Coherent Innova 305 argon and Coherent Spectrum argon-krypton water-cooled lasers. GFP was excited using the primary argon laser tuned to 488 nm at 200 milliwatts, and GFP fluorescence was detected using a standard 560SP dichroic mirror along with a 530DF30 band-pass filter in FL1. RFP was excited using the secondary argon-krypton laser tuned to 568 nm at 150 milliwatts, and RFP fluorescence was detected using a 710DRLP dichroic mirror along with a 610DF30 band-pass filter in FL5. All filters were purchased from Omega Optical. Light scatter and fluorescence were collected using logarithmic amplification with forward scatter light as the threshold parameter. T. gondii tachyzoites were harvested as above and resuspended in culture medium to 5 × 106parasites/ml. Extracellular parasites were sorted using standard high speed conditions on the FACS Vantage (70-µm nozzle tip, 45-p.s.i. sheath pressure). This resulted in a drop drive frequency of 71,000 drops/s, a drive level of 6.5 volts, and a drop breakoff of 33.4 drops. At these settings, parasites flow through the cell sorter at a rate of 12,000/s with abort rates typically less than 10%. Pulse Processing Plus was used to generate a forward scatter (pulse width) parameter for aggregate detection. Parasites were sorted into standard 12 × 75-mm polystyrene collection tubes containing 0.5 ml of phosphate-buffered saline (pH 7.4). We have previously shown that N-terminal fusion of the bipartite targeting sequences from various nuclear-encoded apicoplast proteins permits the targeting of GFP into the apicoplast in stable T. gondiitransgenics (14Waller R.F. Keeling P.J. Donald R.G.K. Striepen B. Handman E. Lang-Unnasch N. Cowman A.F. Besra G.S. Roos D.S. McFadden G.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12352-12357Crossref PubMed Scopus (636) Google Scholar) and that this marker can be exploited to follow organellar replication in living parasites (see Ref. 30Striepen B. Crawford M.J. Shaw M.K. Tilney L.G. Seeber F. Roos D.S. J. Cell Biol. 2000; 151: 1423-1434Crossref PubMed Scopus (187) Google Scholar and Fig.1 A). To facilitate two-color fluorescence studies, a red fluorescent protein reporter was also targeted into the apicoplast, as shown in Fig. 1 B. To provide an enzymatic marker for subcellular fractionation, the CAT enzyme was targeted to the apicoplast as well (Fig. 1 C). All of these fusion proteins target specifically to the apicoplast, which is visible as a small dot in the apical juxtanuclear region of each tachyzoite (4Köhler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Crossref PubMed Scopus (611) Google Scholar). The four tachyzoites shown in each panel are the progeny of a single clonal parasite infection; T. gondiitachyzoites replicate synchronously within the intracellular parasitophorous vacuole until host cell lysis (producing 2, 4, 8, 16, … daughters/vacuole). Transgenic parasites stably expressing ACPL-CAT were disrupted by various methods (Dounce homogenizer, French press, Stansted cell disrupter, or homogenization with silicon carbide in a mortar and pestle). Cell fractionation was then carried out through a series of differential and equilibrium density gradient centrifugation steps as described under “Experimental Procedures.” Fractions were assayed for protein concentration, CAT activity, and the abundance of particular proteins by Western blotting with specific antibodies. The Stansted cell disrupter yielded the cleanest results for several subcellular organelles including the apicoplast. Results from a representative experiment are shown in Fig.2. Although we consistently observed a high enrichment ratio for rhoptries at ∼1.4/1.5 m sucrose (∼38-fold in this experiment; see panels B andD) and micronemes (data not shown), the apicoplast was widely distributed across the gradient with no fraction exhibiting >6-fold enrichment (maximum at the 1.25/1.3 m sucrose interface; see panels A and C). Similar distribution of the apicoplast and rhoptries has been observed in many different experiments using sucrose or Percoll gradients. Electron microscopic analysis (not shown) suggests that the distinctive four-membrane architecture that characterizes the apicoplast (4Köhler S. Delwiche C.F. Denny P.W. Tilney L.G. Webster P. Wilson R.J. Palmer J.D. Roos D.S. Science. 1997; 275: 1485-1489Crossref PubMed Scopus (611) Google Scholar, 9McFadden G.I. Roos D.S. Trends Microbiol. 1999; 7: 328-333Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) is very fragile, making the isolation of intact organelles with a uniform density very difficult. Frustrated by the apparent heterogeneity in apicoplast density, we attempted to develop an alternative strategy based on affinity purification. Such approaches have been successfully applied to chloroplast purification in plant systems (31Kausch A.P. Owen Jr., T.P. Narayanswami S. Bruce B.D. BioTechniques. 1999; 26: 336-343Crossref PubMed Scopus (31) Google Scholar). As no apicoplast surface antigen has been identified to date (and the outer membrane of the apicoplast may be virtually indistinguishable from the endoplasmic reticulum/Golgi apparatus (11Roos D.S. Crawford M.J. Donald R.G.K. Kissinger J.C. Klimczak L.J. Striepen B. Curr. Opin. Microbiol. 1999; 2: 426-432Crossref PubMed Scopus (129) Google Scholar, 32Schwartzbach S.D. Osafune T. Loffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar)), we engineered a series of constructs in which proteins containing a bipartite apicoplast-targeting signal were fused to a transmembrane anchor and cytoplasmic epitope tag (33Sulli C. Fang Z. Muchhal U. Schwartzbach S.D. J. Biol. Chem. 1999; 274: 457-463Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Unfortunately, however, none of these constructs exhibited efficient targeting to the apicoplast (data not shown). Interestingly, one construct engineered in the course of these studies (ACP-GFP-mROP1) was found to target specifically to the apicoplast, alter its morphology, and block organelle segregation during parasite mitosis (8He C.Y. Shaw M.K. Pletcher C.H. Striepen B. Tilney L.G. Roos D.S. EMBO J. 2001; 20: 330-339Crossref PubMed Scopus (152) Google Scholar). To determine the localization of the ACP-GFP-mROP1 (a fusion of the ACP targeting signal to GFP and a fragment of the rhoptry protein ROP1 (34Soldati D. Lassen A. Dubremetz J.F. Boothroyd J.C. Mol. Biochem. Parasitol. 1998; 96: 37-48Crossref PubMed Scopus (89) Google Scholar)), stable transgenic parasites expressing FNRL-RFP within the apicoplast were transiently transfected with ACP-GFP-mROP1, as shown in Fig. 3. In untransfected parasites, each tachyzoite in every parasitophorous vacuole exhibits a red apicoplast attributable to the FNRL-RFP reporter (Fig. 1 B). Parasites expressing the ACP-GFP-mROP1 fusion, however, produce only one apicoplast per vacuole (Fig. 3 A, compare with Fig. 1). This single apicoplast contains both GFP and RFP, whereas other parasites within the same vacuole exhibit only weak, punctate GFP fluorescence in the apical region and very little RFP fluorescence (despite the continued expression of FNRL-RFP from the stably integrated transgene; see below). Confocal microscopy demonstrates that the ACP-GFP-mROP1 labels only the periphery of the apicoplast, whereas FNRL-RFP labels the organellar lumen (Fig. 3 B). RFP and GFP labeling are mutually exclusive in these parasites, as shown by the quantitative fluorescence profile, suggesting the localization of these markers to different compartments within the same organelle. This observation is consistent with previous immunoelectron microscopy showing ACP-GFP-mROP1 staining near the periphery of the apicoplast (8He C.Y. Shaw M.K. Pletcher C.H. Striepen B. Tilney L.G. Roos D.S. EMBO J. 2001; 20: 330-339Crossref PubMed Scopus (152) Google Scholar). Integral membrane proteins and proteins that are tightly associated with membranes (including glycosylphosphatidylinositol-anchored proteins such as the major T. gondii surface antigen P30) remain associated with membrane fractions after extraction with either carbonate or Triton X-114 (27Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1381) Google Scholar, 28Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar). To determine whether the ACP-GFP-mROP1 fusion protein is associated with the complex four-membrane structure surrounding the apicoplast, we extracted crude apicoplast fractions with either Na2CO3 or Triton X-114, as shown in Fig. 4. Both the processed (∼41 kDa) and unprocessed (∼46 kDa) forms of ACP-GFP are properly targeted to the apicoplast (22Waller R.F. Reed M.B. Cowman A.F. McFadden G.I. EMBO J. 2000; 19: 1794-1802Crossref PubMed Scopus (424) Google Scholar). The mature (processed) form was efficiently extracted by carbonate, consistent with the interpretation that this is a soluble protein (left panel,lane 3). The unprocessed form was only partially extracted, perhaps reflecting the association with apicoplast membranes during the processing event (35Sulli C. Schwartzbach S.D. Plant Cell. 1996; 8: 43-53Crossref PubMed Scopus (49) Google Scholar). In contrast, whereas the ACP-GFP-mROP1 protein was partially processed, neither the mature nor the processed forms was extracted by carbonate treatment (left panel, topof lane 4). After solubilization in Triton X-114, both the processed and unprocessed forms of ACP-GFP and ACP-GFP-mROP1 partitioned into the aqueous phase (right panel, lane 6), consistent with the lack of any stable membrane association. Protease protection assays were employed to further probe the subcellula" @default.
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• W2076804827 title "Targeting and Processing of Nuclear-encoded Apicoplast Proteins in Plastid Segregation Mutants of Toxoplasma gondii" @default.
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