Trypanosoma brucei Plasma Membrane-Type Ca2+-ATPase 1 (TbPMC1) and 2 (TbPMC2) Genes Encode Functional Ca2+-ATPases Localized to the Acidocalcisomes and Plasma Membrane, and Essential for Ca2+ Homeostasis and Growth
2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês
10.1074/jbc.m309978200
ISSN1083-351X
AutoresShuhong Luo, Peter Rohloff, Joanna M. Cox, Sérgio Akira Uyemura, Roberto Docampo,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoTrypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca2+ pumps (Ca2+-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca2+-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H+-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. T. brucei Ca2+-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca2+-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA (RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca2+ and decreased mobilization of Ca2+ from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca2+-ATPases are essential for parasite viability and validate them as targets for drug development. Trypanosoma brucei adaptation and survival in its host involve integrated regulation of Ca2+ pumps (Ca2+-ATPases), which are essential in calcium ion homeostasis. Here we report the cloning and sequencing of two genes (TbPMC1 and TbPMC2) encoding plasma membrane-type Ca2+-ATPases (PMCAs) of T. brucei, an agent of African trypanosomiasis. Indirect immunofluorescence analysis using antibodies against the proteins and against epitope tags introduced into each protein showed that TbPMC1 co-localized with the vacuolar H+-pyrophosphatase to the acidocalcisomes while TbPMC2 localized to the plasma membrane. Northern and Western blot analyses revealed that TbPMC1 and TbPMC2 are up-regulated during blood stages. TbPMC1 and TbPMC2 suppressed the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. T. brucei Ca2+-ATPase genes were functionally characterized by using double-stranded RNA interference (RNAi) methodology to produce inducible Ca2+-ATPase-deficient procyclic forms. Similar results were obtained with bloodstream form trypomastigotes, except that the RNAi system was leaky and mRNA and protein levels recovered with time. The induction of dsRNA (RNAi) caused gross morphological alterations, and growth inhibition of procyclic forms. Induction of RNAi against TbPMC1 but not against TbPMC2 caused elevated levels of cytosolic Ca2+ and decreased mobilization of Ca2+ from intracellular stores following ionophore addition. These results establish that T. brucei PMCA-Ca2+-ATPases are essential for parasite viability and validate them as targets for drug development. P-type ATPases are proteins involved in the active pumping of charged substrates across biological membranes and the formation of a phosphorylated intermediate during their reaction cycle is their distinguishing feature (1Carafoli E. Annu. Rev. Biochem. 1987; 56: 395-433Google Scholar). Ca2+-ATPases, within the P-type ATPase family, are involved in the unidirectional removal of Ca2+ from the cytosol. These pumps have been found in the plasma membrane and intracellular organelles of different organisms. A sequence analysis of conserved core sequences of all P-type ATPases has grouped them in five subfamilies designated type I-V (2Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Google Scholar). Type II covers Ca2+-ATPases (types IIA and IIB), Na+,K+- and H+,K+-ATPases (type IIC), a small family of fungal ATPases (type IID), and the solitary P-type ATPases of unknown function of some bacteria (2Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Google Scholar). Mammalian plasma membrane Ca2+ pumps (PMCA), which are responsive to calmodulin and extrude Ca2+ out of the cells, comprise one subcluster within type IIB. Sequences found in Saccharomyces cerevisiae (3Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Google Scholar), Dictyostelium discoideum (4Moniakis J. Coukell M.B. Forer A. J. Biol. Chem. 1995; 270: 28276-28281Google Scholar), Trypanosoma cruzi (5Lu H.-G. Zhong L. Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cel. Biol. 1998; 18: 2309-2323Google Scholar), Toxoplasma gondii (6Luo S. Vieira M. Graves J Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Google Scholar), and Entamoeba histolytica (7Ghosh S.K. Rosenthal B. Rogers R. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 125-130Google Scholar) make up another subcluster. These enzymes are characterized by the absence of C-terminal calmodulin-binding sequences and their intracellular localization in compartments different from the sarco/endoplasmic reticulum or the Golgi complex. The S. cerevisiae (3Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Google Scholar) and E. histolytica (7Ghosh S.K. Rosenthal B. Rogers R. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 125-130Google Scholar) enzymes are localized in vacuoles, while the D. discoideum (8Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Google Scholar), T. cruzi (5Lu H.-G. Zhong L. Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cel. Biol. 1998; 18: 2309-2323Google Scholar), and T. gondii (6Luo S. Vieira M. Graves J Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Google Scholar) enzymes are localized in very acidic compartments known as acidocalcisomes. Acidocalcisomes are characterized by their acidic nature, their high electron density, and their high concentration of calcium, magnesium, and other elements in addition to pyrophosphate (PPi) and polyphosphate (polyP) (9Docampo R. Moreno S.N.J. Mol. Biochem. Parasitol. 2001; 33: 151-159Google Scholar). Sleeping sickness, also known as African trypanosomiasis, is caused by protozoan parasites within the T. brucei complex. These parasites have a digenetic life cycle, with two main stages: the bloodstream form that lives in the bloodstream of its mammalian host, and the procyclic form that lives in the insect vector (tsetse fly). The parasite is spread to humans through the bite of the tsetse fly and is believed to infect 300,000-500,000 Africans, most in the sub-Saharan regions of the continent, and causes about 55,000 deaths each year. Chemotherapy against this disease, however, is unsatisfactory (10Docampo R. Moreno S.N.J. Parasitol. Res. 2003; 90: S10-S30Google Scholar). It is therefore important to identify enzymes and metabolic processes in T. brucei that might be potential targets for drug development. Trypanosoma brucei procyclic and bloodstream trypomastigotes have been demonstrated to accumulate Ca2+ in acidocalcisomes via a vanadate-sensitive Ca2+-ATPase (11Vercesi A.E. Moreno S.N.J. Docampo R. Biochem. J. 1994; 304: 227-233Google Scholar). Although a sarco/endoplasmic reticulum-type Ca2+-ATPase (SERCA) has been characterized in T. brucei (12Nolan D. Revelard P. Pays E. J. Biol. Chem. 1994; 269: 26045-26051Google Scholar), the information available on the presence of a PMCA-type Ca2+-ATPase is however limited to a few contradictory reports. Although a Ca2+-ATPase activity was initially reported in membrane-rich fractions of T. brucei (13Bababunmi E. Emech J.K. Bolaji O.M. Ann. N. Y. Acad. Sci. 1982; 402: 349-367Google Scholar), a further communication by the same authors described its absence (14Bababunmi E. Microbiol. Letters. 1987; 36: 71-76Google Scholar). In addition, a high affinity Ca2+-ATPase that does not show any demonstrable dependence on Mg2+ was also reported (15McLaughlin J. Mol. Biochem. Parasitol. 1985; 15: 189-201Google Scholar) in the plasma membrane of T. brucei and it was suggested that this enzyme might have a role in the regulation of intracellular calcium homeostasis in this trypanosomatid. The relative importance of this protein, however, has not been determined nor has it been demonstrated that this protein functions as a Ca2+ pump. A more recent report (16Benaim G. Lopez-Estraño C. Docampo R. Moreno S.N.J. Biochem. J. 1993; 296: 759-763Google Scholar) showed the presence of a calmodulin-stimulated Ca2+ pump in plasma membrane vesicles from T. brucei bloodstream forms. It is evident that a more thorough molecular characterization of the Ca2+-ATPases present in T. brucei will be essential for structure-function analysis and further development of potential chemotherapeutic agents. The feasibility of targeting this type of ion pumps for drug development is demonstrated by the current use of anti-ulcer drugs, surface-active glycosides, and artemisinin derivatives, against typical P-type ATPases such as the H+/K+-ATPase of the gastric mucosa (17Mattsson J.P. Väänänen K. Wallmark B. Lorentzon P. Biochim. Biophys. Acta. 1991; 1065: 261-268Google Scholar), the Na+,K+-ATPase of the heart (18Schatzmann H.-J. Helv. Physiol. Acta. 1953; 11: 346-354Google Scholar), and the SERCA-type Ca2+-ATPase of the malaria parasites (19Eckstein-Ludwig U. Webb R.J. van Goethem I.D.A. East J. Lee A.G. Kimura M. O'Neill P.M. Bray P.G. Ward S.A. Krishna S. Nature. 2003; 424: 957-961Google Scholar), respectively. In the present study, we report the identification in T. brucei of the TbPMC1 and TbPMC2 genes, which encode proteins with homology to the PMCA-type Ca2+-ATPases of vacuolar and acidocalcisome localizations (3Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Google Scholar, 4Moniakis J. Coukell M.B. Forer A. J. Biol. Chem. 1995; 270: 28276-28281Google Scholar, 5Lu H.-G. Zhong L. Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cel. Biol. 1998; 18: 2309-2323Google Scholar, 6Luo S. Vieira M. Graves J Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Google Scholar, 7Ghosh S.K. Rosenthal B. Rogers R. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 125-130Google Scholar, 8Marchesini N. Ruiz F.A. Vieira M. Docampo R. J. Biol. Chem. 2002; 277: 8146-8153Google Scholar). Indirect immunofluorescence analysis using antibodies against a conserved region present in both isoforms showed their localization in the plasma membrane, and co-localization with the vacuolar H+-pyrophosphatase (V-H+-PPase) 1The abbreviations used are: V-H+-PPase, vacuolar H+-pyrophosphatase; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; RACE, rapid amplification of cDNA ends; ORF, open reading frame; RNAi, RNA interference; PMCA, plasma membrane-type Ca2+-ATPase. to the acidocalcisomes. Using antibodies against epitope tags introduced into each protein it was possible to demonstrate an acidocalcisome localization of TbPMC1 and a plasma membrane localization of TbPMC2. TbPMC1 and TbPMC2 were able to functionally complement the PMC1 gene, encoding the vacuolar PMCA-type Ca2+-ATPase of S. cerevisiae, providing genetic evidence for their function as calcium pumps. RNA interference (RNAi) studies demonstrated that these genes are essential cellular components for Ca2+ homeostasis, growth, and survival of T. brucei, especially in the presence of high extracellular Ca2+ concentrations, validating them as targets for chemotherapy. Culture Methods—Procyclic forms cell line 29-13 and bloodstream form BF cell line (114hyg5′-328, also known as the single-marker cell line), co-expressing T7 RNA polymerase and Tet repressor were gifts from G. A. M. Cross (Rockefeller University) (20Wirtz E. Leal S. Ochatt C. Cross G.A.M. Mol. Biochem. Parasitol. 1999; 99: 89-101Google Scholar). Procyclic forms were grown in SDM-79 (21Brun R. Schönenberger M. Acta Trop. 1979; 36: 289-292Google Scholar) supplemented with 10% fetal bovine serum in the presence of G418 (15 μg/ml) and hygromycin (50 μg/ml) to maintain the integrated genes for T7 RNA polymerase and tetracycline repressor, respectively. Bloodstream forms were grown in HMI-9 medium (22Hirumi H. Hirumi K. J. Parasitol. 1989; 75: 985-989Google Scholar) supplemented with 10% fetal bovine serum with 10% Serum Plus (JRH Biosciences), and 2.5 μg/ml G418. Cell densities were determined using a Neubauer chamber. Procyclic forms were diluted to 1 × 106 cell/ml and bloodstream forms to 0.5 × 105 cell/ml and cultured in appropriate media. Growth curves were plotted by using the product of the cell density and the dilution factor. Chemicals and Reagents—DMEM, Dulbecco's phosphate-buffered saline (PBS), Fetal bovine serum, Tween 20, Triton X-100, proteinase K, RNase A, Hepes, CNBr-activated Sepharose 4B, EGTA, anti-α-tubulin antibodies, and poly-l-lysine (mol. wt 70,000) were purchased from Sigma Chemical Co. Trizol reagent, Taq polymerase, SuperScript PCR buffer, Superscript II reverse transcriptase, and pCR 2.1-TOPO cloning kit were from Invitrogen (Carlsbad, CA). The Poly(A)Tract mRNA isolation system and Prime-a-Gene Labeling System were from Promega (Madison, WI). [α-32P]dCTP (3,000 Ci/mmol) and the enhanced chemiluminescence (ECL) detection kit were from Amersham Biosciences. Zeta Probe GT nylon membranes and the protein assay were from Bio-Rad (Hercules, CA). The primers were purchased from Genosys Biotechnologies Inc. (Woodlands, TX). The pET-28a(+) expression vector, the Escherichia coli DE3 strain, the Quick 900 cartridge and the His-bind buffer kit were from Novagen (Madison, WI). The Protease Inhibitor Mixture Set III was from Calbiochem (La Jolla, CA). The pYES2 vector and the pBluescript II KS(-) vector were from Stratagene (La Jolla, CA). Monoclonal antibody BB2 against the Saccharomyces cerevisiae Ty1 virus-like particle (23Bastin P. Bagherzadeh A. Matthews K.R. Gull K. Mol. Biochem. Parasitol. 1996; 77: 235-239Google Scholar) was a gift from Keith Gull (University of Oxford). Alexa 488 and Alexa 546-labeled antibodies were from Molecular Probes Inc. (Eugene, OR). All other reagents were analytical grade. Nucleic Acid Analysis—DNA was isolated by a standard procedure (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Total RNA was isolated with Trizol reagent according to the manufacturer's recommendations. The polyadenylated RNA was obtained using the poly(A) tract mRNA isolation system. mRNA was electrophoresed in 1% agarose gels with 2.2 m formaldehyde, 40 mm sodium acetate, 5 mm EDTA, 100 mm MOPS, pH 8.0. Northern hybridization was done by a standard procedure (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) using probes a (315 bp, N-terminal flanking and coding regions of TbPMC1), b (557 bp, N-terminal flanking and coding regions of TbPMC2), and c (TbPMC1.0, 1.0 kb, corresponding to the central regions of TbPMC1 and TbPMC2) (see Fig. 2B). The α-tubulin (TUB1) fragment used as a control in Northern blotting was obtained from the pZJM vector (25Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Google Scholar). Comparison of levels of TbPMC1 and TbPMC2 transcripts in procyclic and bloodstream forms was done taking as a reference the densitometric values obtained with the TUB1 transcripts and assuming a similar level of expression of this gene in both stages. Similar results were obtained when the densitometric values were compared by taking into account the amount of RNA added to each lane in three different experiments. DNA was run in 1.0% agarose gels with TAE (40 mm Tris, 20 mm acetic acid, 1 mm EDTA pH 8.0) buffer and transferred to Zeta Probe GT nylon membranes. DNA probes were prepared using random hexanucleotide primers, Klenow fragment of DNA polymerase I (Prime-a-Gene Labeling System) and [α-32P]dCTP. The hybridized filters were washed under high stringency conditions (0.1% standard saline citrate-0.1% SDS at 65 °C), unless otherwise indicated. Oligonucleotide primers were designed to recognize the ATP phosphorylation site and the ATP binding site of cationic ATPase genes (26Allen G. Green N.M. FEBS Lett. 1976; 63: 188-192Google Scholar, 27Bassilian R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Google Scholar), i.e. 5′-CGGGATCCGTNATNTGYWSNGAYAA-3′ and 5′-CGGAATTCGSRTCRTTNRYNCCR-3′ as the 5′- and 3′-primer, respectively. PCR was performed in a PTC-100 Programmable Thermal Controller (MJ Research, Inc., Watertown, MA) at 94 °C for 1 min, 55-62 °C for 2 min, and 72 °C for 3 min/cycle (30 cycles) using Taq polymerase. PCR products were cloned into the pGEM-T or the PCR 2.1-TOPO vector according to the manufacturer's instructions. The cloned PCR products were sequenced and the deduced amino acid sequences were compared with the data base in DDBJ/EMBL/GenBank™. A 1.0-kb PCR clone with identity to organelle-type Ca2+-ATPases was used as a probe to screen T. brucei 927 P1 genomic libraries (28Pierce J.C. Sauer B. Sternberg N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2056-2060Google Scholar). DNA sequencing was performed by the dideoxynucleotide chain termination method of Sanger et al. (29Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Google Scholar) with a 373A DNA Automatic Sequencer (PerkinElmer Applied Biosystems, Foster City, CA). Internal oligonucleotide primers were designed to complete the DNA sequence in both directions. DNA and deduced amino acid sequence analyses were performed using the University of Wisconsin Genetics Computer Group software package (GCG program, version 8.0). Hydropathy analysis was done using the method of Kyte and Doolittle (30Kyte J. Doolittle D.F. J. Mol. Biol. 1982; 157: 105-132Google Scholar). The genomic sequences of TbPMC1 and TbPMC2 were deposited in DDBJ/EMBL/GenBank™ under the accession numbers: AF359561 and AF507073. 5′-RACE, 3′-RACE, and Reverse Transcriptase-PCR—To obtain full-length cDNA, rapid amplification of cDNA ends (RACE) technique (31Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Google Scholar) was performed using a Marathon cDNA amplification kit (Clontech). First strand cDNA synthesis was primed with an oligonucleotide that annealed to 836-bp downstream of the putative start codon of the TbPMC1 ORF (RTP3′-1,5′-AAGAAGACTCACCGACGGCG-3′, 1011-1030 nt) in a reaction containing 1 mm dNTPs, 2.5 mm MgCl2, 10 mm dithiothreitol, 1× SuperScript PCR buffer, and 200 units of SuperScript II reverse transcriptase. Target sequences were amplified in a standard PCR using the first strand cDNA as template and primers Tb5SL (5′-AACGCTATTATTAGAACAGTTTCTGT-3′), and a specific primer that annealed to a sequence just 421-bp upstream of the primer used for first strand cDNA synthesis (RTP3′-2,5′-GGATAAGCGACACACATGCG-3′, 570- 589 nt). Oligo dT primer (5′-ATGGATGCCTACAGCGCTCTTTTTTTTTTTTTTTTTTTTTTT-3′) and specific primers, RTP3 (5′-AAGTGTTGGCCATCTGCACC-3′, 3071-3090 nt) and RTP4 (5′-TGTCGACGGTAGTGCCTCTC-3′, 3240-3249 nt), for 3′-RACE. PCR conditions were the same as described above except that the annealing temperature was 60 °C. The product of the amplification reaction was ligated into vector pCR2.1TOPO for sequence analysis. The complete cDNA sequence of TbPMC1 and TbPMC2 were deposited in DDBJ/EMBL/GenBank™ under the accession numbers AY065988 and AY065989. Preparation of Antibodies—Two primers, EXP1 (5′-GGATCCCGGACTGTGAATGACAA-3′) and EXP2 (5′-CTCGAGTCATACCATCCTTAATGTG-3′) derived from the TbPMC1 sequence (identical to TbPMC2) 796RTVNDN753 and 1100DTRMV1106, encoding 310 amino acids of the C-terminal region, were used to amplify a 933-bp fragment of the TbPMC1 or TbPMC2 gene. The excised fragment was ligated to the pET-28a(+) expression vector at the BamHI and XhoI sites, resulting in a construct that encoded the protein fused to a His6 tag that allowed its purification on nickel-agarose columns. DNA sequencing of the plasmid was done to ensure that the correct construct had been obtained. The recombinant plasmid was transfected into the DE3 strain of E. coli, the fusion protein was induced, and the expressed protein of 33 kDa, present in inclusion bodies, was solubilized and purified according to the manufacturer's instructions. Rabbits were injected subcutaneously with 100 μg of fusion protein emulsified in Freund's complete adjuvant, followed 2 weeks later by subcutaneous injection of 100 μg of fusion protein in Freund's incomplete adjuvant. At 6, 8, and 10 weeks following the initial injection, rabbits were boosted with 100 μg of fusion protein in PBS containing a 10 mg/ml suspension of Al(OH)3. Serum was collected before the initial injection (pre-immune serum) and 10 days after each boost. The antiserum was aliquoted and stored at -80 °C. Affinity purification of anti-TbPMC antibodies was carried out by elution from a column to which the His6-TbPMC fusion protein had been coupled. The affinity column was prepared by conjugating purified His6-tagged TbPMC (derived from a 1-liter culture of the transformed E. coli) to CNBr-activated 4B (0.5 ml of beads) as described by the manufacturer. The affinity matrix was incubated with 10 ml of anti-TbPMC1 antiserum 1:2 dilution in PBS for 16 h at 4 °C, washed four times with 20 vol of PBS, and the antibodies eluted in 0.2 m glycine, pH 2.8, 1 mm EDTA. The antibodies were immediately neutralized with 0.1 volume of 1 m Tris, pH 9.5, supplemented with sodium azide to a final concentration of 0.05%, and stored at 4 °C. Polyclonal antiserum from Balb/c mice was raised against a keyhole limpet hemocyanin (KLH)-conjugated synthetic peptide corresponding to the T. cruzi V-H+-PPase (NH2-734CNTGGAWDNAKKYIEKGGLRDKNKGKGS761-COOH) at the University of Illinois Biotechnology Center. SDS Electrophoresis and Preparation of Western Blots—The electrophoretic system used was essentially the same as that described by Laemmli (32Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). Freshly prepared T. brucei procyclic and bloodstream forms (1 × 108) were centrifuged at 2,500 × g for 10 min, and resuspended in 300 μl of Dulbecco's PBS containing proteinase inhibitors (1 μg of aprotinin per ml, 1 μg of leupeptin per ml, 1 μg of pepstatin per ml, and 1 mm phenylmethylsulfonyl fluoride). Aliquots of parasites (20 μl; 30 μg of protein) were mixed with 20 μl of 125 mm Tris-HCl, pH 7, 10% (w/v) β-mercaptoethanol, 20% (v/v) glycerol, 6.0% (w/v) SDS, and 0.4% (w/v) bromphenol blue as tracking dye, and boiled for 5 min prior to application to 10% SDS-polyacrylamide gels. Electrophoresed proteins were transferred to nitrocellulose by the method of Towbin et al. (33Towbin H. Stahelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar), with a Bio-Rad (Richmond, CA) transblot apparatus. Following transfer, the nitrocellulose was blocked in 5% nonfat dry milk in TPBS (0.1% Tween 20, 80 mm Na2HPO4, 20 mm NaH2PO4, 100 mm NaCl, pH 7.5) overnight at 4 °C. A 1:5,000 dilution of polyclonal antiserum in TPBS was then applied at room temperature for 60 min. The nitrocellulose was washed three times for 15 min each with TPBS. After incubating with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:20,000) and washing three times for 15 min each with TPBS, immunoblots were visualized on blue-sensitive x-ray film (Midwest Scientific, St Louis, MO) using the ECL chemiluminescence detection kit and following the instructions of the manufacturer. Functional Complementation of the PMC1 Gene of S. cerevisiae with TbPMC1 and TbPMC2—We transformed the S. cerevisiae vcx1 pmc1 strain K665 with the yeast expression vectors pYES2 and pYES2-TbPMCA1 and TbPMCA2 by a standard lithium acetate transformation procedure (34Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Google Scholar). The Ura+ transformants were selected by plating on synthetic-complete Ura medium (34Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Google Scholar). The TbPMC1 and TbPMC2 coding regions were amplified by the PCR technique using cDNA as template and HindIII and XbaI sites created on the PCR primers: YESP1 and YESP2 for TbPMC1, YESP3 and YESP2 for TbPMC2 (YESP1, 5′-AAGCTTACGATGGTTTCCCCAAAAGACAC-3′; YESP2, 5′-TCTAGATCATACCATCCTTAATGTG-3′ YESP3, 5′-AAGCTTAGCATGGATCCACTTGAGTCACC-3′). The TbPMC1 and TbPMC2 coding regions were placed at the HindIII and XbaI sites of pYES2 with the same orientation as the GAL1 promoter. The cultures were grown in YPD medium pH 5.5 containing 0, 50, 100, 200, and 400 mm CaCl2 to identify Ca2+-tolerant transformants. Yeast strains K665 MATa (vcx1::hisGpmc1::TRP1) and K661 MATa (vcx1::hisG), kindly provided by Kyle W. Cunningham (Johns Hopkins University) (35Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Google Scholar), were grown at 30 °C in standard YPD medium (1% Difco yeast extract, Bacto Peptone, 2% galactose) or in YPD medium pH 5.5 (adjusted with succinic acid), supplemented with 0, 50, 100, 200, and 400 mm CaCl2. Cell growth was assessed by measuring the optical density of the liquid cultures at 600 nm or by counting the number of colonies in plates. Both strains are isogenic and harbor the following additional mutations: ade2-1, can1-100, his3-11,15 leu2-3,112 trp1-1, and ura3-1. Immunofluorescence Microscopy—Parasites fixed with 4% formaldehyde were allowed to adhere to poly-l-lysine-coated coverslips, permeabilized with 0.3% Triton X-100 for 5 min, blocked with 3% bovine serum albumin, 1% fish gelatin, 50 mm NH4Cl, and 5% goat serum in PBS for 1 h. The cells were stained with anti-TbPMC (1:100) against the 33 kDa expressed protein or polyclonal antibody against T. cruzi V-H+-PPase (1:100) and monoclonal antibody BB2 (1:100) against S. cerevisiae Ty1 (23Bastin P. Bagherzadeh A. Matthews K.R. Gull K. Mol. Biochem. Parasitol. 1996; 77: 235-239Google Scholar) followed by Alexa 546 goat anti-rabbit or Alexa 488-conjugated goat anti-mouse antibodies (1:1000, Molecular Probes). Control preparations were incubated with preimmune serum (1:100) or without the primary antibody. Confocal images were collected with a Leica laser scanning confocal microscope (TCS SP2) using a 63′ Plan-Apo objective with NA 1.32. Single optical sections were recorded with an optimal pinhole of 0.000293 according to Leica instructions. Adobe Photoshop was used for image processing. Double-stranded RNA Expression and Trypanosome Transfection—DNA fragments corresponding to the N-terminal flanking and coding regions of TbPMC1 from nucleotides 41 to 355 and to TbPMC2 from nucleotides 150 to 606 were amplified by RT-PCR from T. brucei procyclic cDNA, using primers: P15, 5′-CTCGAGCGGTAGGTGTCATTTGC-3′ and P13, 5′-AAGCTTCGACGCAACTGAACAAGGTG-3′; P25, 5′- CTCGAGGAGAGGAACGGATCGCTTGC-3′ and P23, 5′-AAGCTTTGTGTCTTATGTTCCTGCGG-3′ containing XhoI and HindIII linkers (underlined). To inhibit the expression of both TbPMC1 and TbPMC2 simultaneously, DNA fragments corresponding to the central coding regions of TbPMC1 from nucleotides 1468 to 2136 nt and TbPMC2 from nucleotides 1784 to 2452 nt were amplified by RT-PCR from T. brucei procyclic cDNA, using primers: P5, 5′-GCTCGAGATGTTCCGTGTCTCGAATCC-3′, and P3, 5′-CAAGCTTCTGGCACATTCGCACAG-3′, containing XhoI and HindIII linkers. The PCR fragments were cloned into the TA-cloning pCRII vector (Invitrogen) and sequenced using BigdyeÔ Terminator v3.0 Ready Reaction Cycle Sequencing kit (ABI PRISM), and ligated into the XhoI and HindIII sites of the RNA interference (RNAi) vector pZJM (25Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Google Scholar), kindly provided by Paul Englund (Johns Hopkins University). For stable transfection of the procyclic host strain 29-13, the protocol was performed as described by Wang et al. (25Wang Z. Morris J.C. Drew M.E. Englund P.T. J. Biol. Chem. 2000; 275: 40174-40179Google Scholar). Briefly, procyclic forms (1 × 108) were washed once in 5 ml of cytomix (36Van den Hoff M.J. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Google Scholar) and resuspended in 0.5 ml of cytomix containing 10 μg of plasmid that had been linearized by NotI digestion, so that it could target the rDNA spacer region (37Wirtz E. Clayton C. Science. 1995; 268: 1179-1183Google Scholar). Transfections were carried out in 2-mm cuvettes using a BTX electroporator with peak discharge at 1.6 kV, 50 microfarads. Immediately following transfection, parasites were transferred into 10 ml of SDM-79 supplemented with G418 and hygromycin. After 20 h, selection was applied by culturing in the presence of 2.5 μg/ml phleomycin, and the parasites were grown for 2 or 3 weeks to form stable lines. For induction of dsRNA, parasites were cultured in the same medium containing 1.0 μg/ml tetracycline. Parasites were diluted 10-fold when densities reached a minimum of 1 × 106 cells/ml. They were not allowed to grow beyond 5 × 106 cells/ml before diluting again. For the bloodstream host strain BF cell line, the protocol was performed as described by Morris et al. (38Morris J.C. Wang Z. Drew M.E. Paul K.S. Englund P.T. Mol. Biochem. Parasitol. 2001; 117: 111-113Google Scholar). Briefly, for each transfection, BF parasites were grown to mid-log phase (5 × 105 cells/ml) and 1 × 108 cells were washed once in 5 ml of cytomix and resuspended in 0.5 ml of cytomix containing 100 μg of plasmid that had been linearized with NotI. Electroporations were carried out at room temperature in 4-mm cuvettes using a BTX electroporator (peak discharge at 1.7 kV, 50 μF). Immediately following transfection, parasites were transferred into 24
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