Analysis of the Antimalarial Drug Resistance Protein Pfcrt Expressed in Yeast
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m204005200
ISSN1083-351X
AutoresHanbang Zhang, Ellen M. Howard, Paul D. Roepe,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoMutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000) Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiaeand Pichia pastoris yeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversus CQR-conferring mutant Pfcrt function, which may include effects on H+ transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000) Mol. Biochem. Parasitol. 110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparum transport proteins and membrane associated vaccine candidates. Mutations in the novel membrane protein Pfcrt were recently found to be essential for chloroquine resistance (CQR) in Plasmodium falciparum, the parasite responsible for most lethal human malaria (Fidock, D. A., Nomura, T., Talley, A. K., Cooper, R. A., Dzekunov, S. M., Ferdig, M. T., Ursos, L. M., Sidhu, A. B., Naude, B., Deitsch, K. W., Su, X. Z., Wootton, J. C., Roepe, P. D., and Wellems, T. E. (2000) Mol. Cell6, 861–871). Pfcrt is localized to the digestive vacuolar membrane of the intraerythrocytic parasite and may function as a transporter. Study of this putative transport function would be greatly assisted by overexpression in yeast followed by characterization of membrane vesicles. Unfortunately, the very high AT content of malarial genes precludes efficient heterologous expression. Thus, we back-translated Pfcrt to design idealized genes with preferred yeast codons, no long poly(A) sequences, and minimal stem-loop structure. We synthesized a designed gene with a two-step PCR method, fused this to N- and C-terminal sequences to aid membrane insertion and purification, and now report efficient expression of wild type and mutant Pfcrt proteins in the plasma membrane of Saccharomyces cerevisiaeand Pichia pastoris yeast. To our knowledge, this is the first successful expression of a full-length malarial parasite integral membrane protein in yeast. Purified membranes and inside-out plasma membrane vesicle preparations were used to analyze wild typeversus CQR-conferring mutant Pfcrt function, which may include effects on H+ transport (Dzekunov, S., Ursos, L. M. B., and Roepe, P. D. (2000) Mol. Biochem. Parasitol. 110, 107–124), and to perfect a rapid purification of biotinylated Pfcrt. These data expand on the role of Pfcrt in conferring CQR and define a productive route for analysis of importantP. falciparum transport proteins and membrane associated vaccine candidates. Malaria causes ∼2.5 million deaths annually, mostly children. Four malarial species infect humans, the most deadly beingPlasmodium falciparum. For decades, malaria has been treated effectively with the 4-aminoquinoline chloroquine (CQ) 1The abbreviations used are: CQ, chloroquine; CQR, chloroquine resistant (resistance); DV, digestive vacuole; DHFR-TS, dihydrofolate thymidylate synthase; bad, biotin acceptor domain; DM, dodecyl maltoside; tcbd, transcarboxylase biotin acceptor domain; ISOV, inside-out plasma membrane vesicles; PBS, phosphate-buffered saline; NaPi, sodium phosphate buffer; HRP, horseradish peroxidase; AO, acridine orange; VPL, verapamil; PMA1, plasma membrane ATPase 1; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; Ab, antibody; however, CQ-resistant (CQR) strains of P. falciparum have spread with considerable success and continue to evolve. Mutations in two genes (pfmdr and cg2) were previously suggested to mediate CQR, but more recently CQR was found to be caused by mutations in the Pfcrt gene (1Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. Sidhu A.B. Naude B. Deitsch K.W. Su X.Z. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar). Pfmdr1 protein likely plays a modulatory role that influences the drug resistance profile (2Reed M.B. Saliba K.J. Caruana S.R. Kirk K. Cowman A.F. Nature. 2000; 403: 906-909Crossref PubMed Scopus (716) Google Scholar). Pfcrt protein is localized to the membrane of the parasite digestive vacuole (DV). The DV is the site of hemoglobin digestion, which is a principle source of food for the parasite during rapid intraerythrocytic development. Mutant Pfcrt may confer drug resistance by directly or indirectly lowering DV pH (3Dzekunov S. Ursos L.M.B. Roepe P.D. Mol. Biochem. Parasitol. 2000; 110: 107-124Crossref PubMed Scopus (113) Google Scholar), which quite effectively titrates soluble drug target (heme released from hemoglobin) out of solution without compromising detoxification of heme to hemozoin (4Ursos L.M.B. Dzekunov S. Roepe P.D. Mol. Biochem. Parasitol. 2000; 110: 125-134Crossref PubMed Scopus (55) Google Scholar, 5Ursos L.M.B. Roepe P.D. Med. Res. Rev. 2002; 22: 465-491Crossref PubMed Scopus (99) Google Scholar). There are actually multiple mutant Pfcrt alleles that appear to cause drug resistance and that arose in a geographically distinct pattern (1Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. Sidhu A.B. Naude B. Deitsch K.W. Su X.Z. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar). Further elucidating the molecular mechanism of CQR is essential. Pfcrt plays a critical role in CQR, probably via some yet to be determined transport function. The protein resides in a subcellular membrane within an intracellular parasite, so to experimentally study Pfcrt transport function requires that transported substrates cross three membranes in a coordinated fashion. This is extremely difficult to manipulate experimentally. Since the technology required for fabricating membrane vesicles of various types (e.g.secretory, inside out plasma, right side out plasma, vacuolar) and for purifying and reconstituting polytopic integral membrane proteins is well developed for yeast, heterologous expression of Pfcrt in yeast would obviously greatly assist further analysis of Pfcrt transport function. Unfortunately, a literature survey for successful heterologous expression of P. falciparum genes is not encouraging. There is but one successful report of low level heterologous expression of a native P. falciparum cDNA (dihydrofolate reductase-thymidylate synthase (DHFR-TS) protein (6Sirawaraporn W. Sirawaraporn R. Cowman A.F. Yuthavong Y. Santi D.V. Biochemistry. 1990; 29: 10779-10785Crossref PubMed Scopus (68) Google Scholar)). A notorious feature of the P. falciparum genome is its very high A,T content (7Gardner M.J. Curr. Opin. Genet. Dev. 1999; 9: 704-708Crossref PubMed Scopus (28) Google Scholar, 8Bowman S. Lawson D. Basham D. Brown D. Chillingworth T. Churcher C.M. Craig A. Davies R.M. Devlin K. Feltwell T. Gentles S. Gwilliam R. Hamlin N. Harris D. Holroyd S. Hornsby T. Horrocks P. Jagels K. Jassal B. Kyes S. McLean J. Moule S. Mungall K. Murphy L. Barrell B.G. et al.Nature. 1999; 400: 532-538Crossref PubMed Scopus (279) Google Scholar), thus, P. falciparum genes reveal a markedly biased codon usage. Coding regions are ∼70% A+T, and are flanked by A+T-rich regions as high as 86% (9Saul A. 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Thus, expression of a synthetic DHFR-TS gene with optimized codon usage was found to increase the level of expressed protein (21Prapunwattana P. Sirawaraporn W. Yuthavong Y. Santi D.V. Mol. Biochem. Parasitol. 1996; 83: 93-106Crossref PubMed Scopus (30) Google Scholar). This result recently enticed others to construct genes with optimized yeast codon usage for pfsub1 (a subtilisin-like protease),pfmsp-1 (a merozoite stage-specific surface protein complex), and the antigen Pfs48/45 (22Withers-Martinez C. Carpenter E.P. Hackett F. Ely B. Sajid M. Grainger M. Blackman M.J. Protein Eng. 1999; 12: 1113-1120Crossref PubMed Scopus (84) Google Scholar, 23Pan W. Ravot E. Tolle R. Frank R. Mosbach R. Turbachova I. Bujard H. Nucleic Acids Res. 1999; 27: 1094-1103Crossref PubMed Scopus (59) Google Scholar, 24Milek R.L. Stunnenberg H.G. Konings R.N. Vaccine. 2000; 18: 1402-1411Crossref PubMed Scopus (40) Google Scholar). These three studies have yielded some additional success; however, heterologous expression ofP. falciparum genes remains extremely difficult, and no successful overexpression of a polytopic integral membrane protein has yet been reported. Polytopic integral membrane proteins are typically encoded by large genes that are more difficult to assemble synthetically. Also, aside from codon usage and poly(A) termination issues, the encoded proteins contain folding, membrane translocation, and membrane insertion sequences that can be species- and membrane-specific. These are not well defined for malarial membrane proteins. S. cerevisiae yeast have been used as an important tool in the heterologous expression of proteins (25Romanos M.A. Scorer C.A. Clare J.J. Yeast. 1992; 8: 423-488Crossref PubMed Scopus (857) Google Scholar). Several membrane proteins have been heterologously expressed, among them, human mdr1 (26Kuchler K. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2302-2306Crossref PubMed Scopus (89) Google Scholar, 27Fritz F. Howard E.M. Hoffman M.M. Roepe P.D. Biochemistry. 1999; 38: 4214-4226Crossref PubMed Scopus (25) Google Scholar), mouse mdr3 (28Evans G.L. Ni B. Hrycyna C.A. Chen D. Ambudkar S.V. Pastan I. Germann U.A. Gottesman M.M. J. Bioenerg. Biomembr. 1995; 27: 43-52Crossref PubMed Scopus (46) Google Scholar), CFTR (cystic fibrosis transmembrane conductance regulator) (29Kiser G.L. Gentzsch M. Kloser A.K. Balzi E. Wolf D.H. Goffeau A. Riordan J.R. Arch. Biochem. Biophys. 2001; 390: 195-205Crossref PubMed Scopus (36) Google Scholar), monoamine transporter (30Yelin R. Schuldiner S. Biochim. Biophys. Acta. 2001; 1510: 426-441Crossref PubMed Scopus (17) Google Scholar), Na+ channel (31Gupta S.S. Canessa C.M. FEBS Lett. 2000; 481: 77-80Crossref PubMed Scopus (10) Google Scholar), and dopamine receptor (32Andersen B. Stevens R.C. Protein Expression Purif. 1998; 13: 111-119Crossref PubMed Scopus (17) Google Scholar). Very few polytopic integral membrane proteins have been successfully expressed in P. pastorisyeast (33Doring F. Michel T. Rosel A. Nickolaus M. Daniel H. Mol. Membr. Biol. 1998; 15: 79-88Crossref PubMed Scopus (32) Google Scholar, 34Lerner-Marmarosh N. Gimi K. Urbatsch I.L. Gros P. Senior A.E. J. Biol. Chem. 1999; 274: 34711-34718Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 35Weiss H.M. Haase W. Michel H. Reilander H. Biochem. J. 1998; 330: 1137-1147Crossref PubMed Scopus (85) Google Scholar). We have examined these issues and have constructed a syntheticPfcrt gene that harbors appropriate base pair content and other necessary features. We have modified the N and C termini regions based on previous work wherein we were able to functionally express the human multidrug resistance protein (humdr1, P-glycoprotein) in S. cerevisiae (27Fritz F. Howard E.M. Hoffman M.M. Roepe P.D. Biochemistry. 1999; 38: 4214-4226Crossref PubMed Scopus (25) Google Scholar), have subcloned the resulting constructs into appropriate yeast expression vectors, and have achieved high level inducible expression of Pfcrt and Pfcrt-biotin acceptor domain fusion (Pfcrt-bad) proteins in the plasma membrane of P. pastoris. More modest constitutive expression in S. cerevisiae was also achieved. Using the successfully expressed wild type gene as template, we have also created a CQR-associated mutant Pfcrtallele and have overexpressed the mutant Pfcrt-bad protein. Analysis of membrane vesicles from these yeast supports the earlier suggestion (1Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. Sidhu A.B. Naude B. Deitsch K.W. Su X.Z. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar,3Dzekunov S. Ursos L.M.B. Roepe P.D. Mol. Biochem. Parasitol. 2000; 110: 107-124Crossref PubMed Scopus (113) Google Scholar, 5Ursos L.M.B. Roepe P.D. Med. Res. Rev. 2002; 22: 465-491Crossref PubMed Scopus (99) Google Scholar) that Pfcrt is involved in modulating H+transport. Cloned Pfu polymerase was from Stratagene (La Jolla, CA). Rabbit anti-Pfcrt IgG was a kind gift from Dr. T. Wellems (NIAID/National Institutes of Health, Bethesda, MD). HRP-conjugated monkey anti-rabbit IgG, HRP-conjugated streptavidin and ECL detection reagents were from Amersham Biosciences. Prestained SDS-PAGE molecular markers were from Bio-Rad. Immobilized monomeric avidin resin was from Pierce. Yeast and bacteria growth media reagents were from Difco. Oligonucleotides were custom made by MWG-Biotech (High Point, NC). Dodecyl maltoside (DM) was fromCalbiochem (San Diego, CA). Plasmids and sequencing primers relevant for subcloning and gene expression in P. pastoris yeast were purchased from Invitrogen (version L Picchia expression kit). All other reagents were reagent grade or better and were purchased from Sigma. The Escherichia coli strain NM522 (hsd Δ 5 Δ (lac-pro)F− lacI lacZ ΔM15 pro supE) was used for all bacterial work. S. cerevisiae strain 9.3 (Mat a leu2 ura3 trp1 ade2 trk1Δ trk2Δ ena1::His3::ena4) was kindly provided by Dr. Alonso Rodriguez-Navarro, Universidad Politechnica, Madrid, Spain.P. pastoris strains KM71 and GS115 (arg4 his4 aox1::ARG4 and his4) were from Invitrogen. 9.3 yeast harboring pYHZHB3crt, pYHZHB3crt-bad, pYHZDd2crt-bad, or pYKM77 were selected for growth in synthetic complete medium lacking uracil supplemented with 100 mm KCl. P. pastoris strains KM71 and GS115 harboring pPIC35HB3crt, pPIC35HB3crt-bad, pPIC35Dd2crt-bad, or pPIC35 were selected for growth in minimal glycerol medium lacking histidine. The Pfcrt-hb3 gene sequence (wild typePfcrt) was obtained from GenBankTM(www.ncbi.nlm.nih.gov) and we used the CODOP program (generously provided by Dr. Elisabeth P Carpenter, Division of Protein Structure, National Institute for Medical Research, London, UK (22Withers-Martinez C. Carpenter E.P. Hackett F. Ely B. Sajid M. Grainger M. Blackman M.J. Protein Eng. 1999; 12: 1113-1120Crossref PubMed Scopus (84) Google Scholar)) to back-translate the encoded protein sequence. We allowed CODOP to back-translate Pfcrt into a theoretically optimized Pfcrtgene using a S. cerevisiae yeast codon usage table (www.kazusa.or.jp/codon). The process was repeated many times with different random seeding values. All the poly(A) and premature codon patterns in a selected sequence were then screened and destroyed by a second layer of codon engineering. We suspect disrupting poly(A) is more important than 100% optimization of codon usage (see "Results," Table I caption). A Kozak consensus (GCCGCCACCAUGG) was included and adjusted at −3 (to A) and +4 (to G). Finally, this theoretically optimized Pfcrt gene was divided up into a collection of 40 base fragments encoding both DNA strands and the melting temperature (T m) for the 20-bp overlap regions in each primer set (see "Results") were calculated. All primer set T m were then adjusted to reside in the range 56–64 °C by yet a third round of codon adjustment. Vector NTI software was also used to check all primers for repeats, palindromes, hairpins, and dimers. In the final theoretical optimizedPfcrt gene AT% is reduced from 72 to 55% (see "Results").Table IComparison of malarial versus yeast codon preferences and codon usage for native versus yeast optimized PfcrtAmino acidCodonP. falciparumusageS. cerevisiae usageNumber used in nativePfcrtNumber used post-optimizationAfter CODOPFinal geneArgCGA2.43.0200CGC0.52.6000CGG0.21.7000CGU3.46.5355AGA16.721.31189AGG4.09.3143LeuCUA5.313.4166CUC1.75.4100CUG1.410.401013CUU8.612.21031UUA49.326.329125UUG10.227.151521SerUCA18.018.8754UCC5.414.2669UCG2.88.6000UCU15.223.6675AGC3.89.7358AGU21.714.2763ThrACA22.717.79106ACC5.612.65410ACG3.88.0000ACU12.820.2775ProCCA13.318.2445CCC2.66.8021CCG1.05.3000CCU9.213.6311AlaGCA12.816.2776GCC3.412.6435GCG1.26.1000GCU12.521.1787GlyGGA16.610.91033GGC1.79.72106GGG2.96.0000GGU16.923.9111014ValGUA17.911.81363GUC2.611.64310GUG4.910.7144GUU17.7227128LysAAA90.542.123150AAG19.430.841227AsnAAC18.824.971632AAU106.13625160GlnCAA24.827.51086CAG3.312.2024HisCAC3.97.8334CAU19.613.7221GluGAA63.745.9172013GAG10.019.1307AspGAC8.620.44611GAU5537.81083TyrUAC5.714.751018UAU46.218.81380CysUGC2.54.7269UGU15.58.01285PheUUC7.318.2131938UUU34.52625190IleAUA44.417.81390AUC6.017.141441AUU33.730.229235MetAUG21.120.9121212TrpUGG5.310.3222TerUAA1.11.0111UAG0.20.5000UGA0.20.6000Total425425425Use per 1000 codons is listed for P. falciparum and S. cerevisiae (www.kazusa.or.jp/codon) (third and fourth columns). Codon usage in native pfcrt (fifth column) was compared with preferred usage for S. cerevisiae (fourth column). Subsequently, the optimized gene was analyzed for poly(A) and premature termination sequences, and the sequence was further adjusted. Thus, for example, even though the yeast preferred codon for lysine is AAA, all lysine residues were coded with AAG to avoid poly(A) sequences (compare last two columns) in the final optimized gene. Open table in a new tab Use per 1000 codons is listed for P. falciparum and S. cerevisiae (www.kazusa.or.jp/codon) (third and fourth columns). Codon usage in native pfcrt (fifth column) was compared with preferred usage for S. cerevisiae (fourth column). Subsequently, the optimized gene was analyzed for poly(A) and premature termination sequences, and the sequence was further adjusted. Thus, for example, even though the yeast preferred codon for lysine is AAA, all lysine residues were coded with AAG to avoid poly(A) sequences (compare last two columns) in the final optimized gene. A total of 66 40-mers were made that encoded both strands of the theoretically optimized Pfcrt gene. We then followed procedures suggested in Refs. 22Withers-Martinez C. Carpenter E.P. Hackett F. Ely B. Sajid M. Grainger M. Blackman M.J. Protein Eng. 1999; 12: 1113-1120Crossref PubMed Scopus (84) Google Scholar and 36Stemmer W.P. Crameri A. Ha K.D. Brennan T.M. Heyneker H.L. Gene (Amst.). 1995; 164: 49-53Crossref PubMed Scopus (568) Google Scholar with some modifications. Equal volumes of all 66 40-mers (1.5 μm each) were combined, and the resultant mixture was diluted 10-fold in 100 μl of a PCR mixture (20 mm Tris-HCl (pH 8.8), 2 mm MgSO4, 10 mm KCl, 10 mm(NH4)2SO4, 0.1% Triton X-100, 0.1 mg/μl nuclease-free bovine serum albumin, 0.2 mm each dNTP, 2.5 units of Pfu polymerase). The initial PCR program was one denaturation step at 94 °C for 1 min, followed by 25 cycles of 94 °C (30 s), 52 °C (30 s), and 72 °C (2 min) and then a final incubation at 72 °C for 10 min. 10 μl of this "assembly solution" was then diluted 10-fold in 100 μl of similar PCR mixture, but with a 1 μm concentration each of the 5′- and 3′-flanking primers (1 and 34 in the sequence of 66, respectively). The amplifying PCR program used was one denaturation step at 94 °C for 1 min, followed by 25 cycles at 94 °C (45 s), 68 °C (45 s), and 72 °C (5 min) and then a final incubation cycle at 72 °C for 10 min. A fragment from the Propionibacterium shermaniitranscarboxylase biotin acceptor domain (tcbd) is a convenient biotin tag for monitoring protein expression and facilitating purification (37Glerum D.M. Muroff I. Jin C. Tzagoloff A. J. Biol. Chem. 1997; 272: 19088-19094Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Isolated plasmid YEp352/BIO6 containing this fragment (kindly provided by Dr. A. Tzagoloff, Columbia University) was PCR-amplified with two designed primers that created convenient overlap and restriction sites (tcbd 5′ primer (5′): GACAGTAT CATCACTC AAGCGGCCGCAG GCTTCGAG CTCGGTAC CCGGGGAT CCGGT (3′);tcbd 3′ primer (5′): GGCCAGTG CCAAGCTTGC ATGCTTGC AGGT (3′)). A NotI restriction site was engineered, a PstI site was destroyed, and aHindIII site was retained (underlined). Purified syntheticPfcrt and purified tcbd fragment were combined and PCR-amplified to form one larger PCR product containing an in-frame fusion of Pfcrt and tcbd. Several different mutant Pfcrt alleles have been associated with chloroquine resistance (CQR) (1Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. Sidhu A.B. Naude B. Deitsch K.W. Su X.Z. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar), including the "Dd2" allele. This allele differs from the CQS-associated allele (what we will call the "HB3" allele) at 8 codons (74–76, 220, 271, 326, 356, 371). Thus, we designed a collection of 12 oligonucleotides (sequences available from the authors as supplemental information) that overlapped in six locations to create 8 codon mutations (encoding M74I, N75E, K76T, A220S, Q271E, N326S, I356T, and R371I amino acid substitutions, see Ref. 1Fidock D.A. Nomura T. Talley A.K. Cooper R.A. Dzekunov S.M. Ferdig M.T. Ursos L.M. Sidhu A.B. Naude B. Deitsch K.W. Su X.Z. Wootton J.C. Roepe P.D. Wellems T.E. Mol. Cell. 2000; 6: 861-871Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar). These oligonucleotides also directed silent codon point mutations that created restriction sites flanking each codon mutation (or group of mutations). The created restriction sites (which facilitated subcloning and which will facilitate future work studying other resistance-associated Pfcrt alleles) wereNruI (flanking codon 74–76), EcoRV (flanking codon 220), AfIII (flanking codon 271),ClaI (flanking codon 326), ApaI (flanking codon 356), and SacII (flanking codon 371). A sequence of PCR reactions was required to synthesize the full-length optimized Dd2crt-bad gene using the optimizedHB3crt-bad gene (wild type Pfcrt-tcbdfusion gene described above) as initial template. First, six PCR fragments (A–F) were amplified from HB3crt-bad using the six complimentary sets of 12 oligonucleotides that created the codon and restriction site mutations described above. These were purified, combined in pairs of two, and PCR-amplified to yield three larger fragments I, II, III (e.g. fragment I encoding codons 1–225 was created using fragments A and B as template), etc. By using this sequential strategy, the entire Dd2crt-bad gene was constructed via tiered PCR, subcloned into pYKM77 as described below, and colony-amplified and purified. Plasmid pYKM77 (kindly provided by Drs. K. Kuchler and J. Thorner) was isolated and restricted with PstI and HindIII. The syntheticHB3crt gene, the synthetic HB3crt-bad fusion gene, and the synthetic Dd2crt-bad gene were each trimmed with PstI and HindIII and ligated to the 5.4-kbp vector. Recombinant plasmids were isolated from bacterial transformants and analyzed by restriction. Candidate pHZHB3crtbad (encoding the wild type fusion protein under control of a modified Ste6 promoter), pHZDd2crtbad (encoding mutant crt fusion protein), and pHZHB3crt (encoding wild type Pfcrt) plasmids were sequenced in both directions using oligonucleotides from the gene assembly steps, the BigDye Terminator cycle sequencing program, and ABI prism 373 software. None of the dozen or so sequences that were fully sequenced in each case properly encoded the proteins of interest. Typically, two to three unwanted point mutations were found in the fully assembled recombinant clones. However, by combining restriction fragments from these constructs (using sites fortuitously placed during the initial gene design) our final synthetic genes encoding full-length proteins of the correct sequence were ligated together. These genes were then also subcloned into the vector pPIC3.5 to create pPIC35hb3crt, pPIC35hb3crtbad, and pPIC35dd2crtbad for expression inP. pastoris. The pPIC vector harbors an inducible promoter activated by MeOH, as described under "Results." Yeast were transformed by the lithium acetate method with 2 μg of target plasmid and 10 μg of carrier plasmid to enhance transformation efficiency. Transformants were plated on selective synthetic complete medium lacking uracil (S. cerevisiae) or MGM (P. pastoris) agar plates. Yeast cells were grown to midlog phase, and crude cellular membranes were isolated via a glass bead lysis protocol (27Fritz F. Howard E.M. Hoffman M.M. Roepe P.D. Biochemistry. 1999; 38: 4214-4226Crossref PubMed Scopus (25) Google Scholar) and stored at −80 °C. Plasma membranes were purified via the acid precipitation method of Goffeau and Dufour (38Goffeau A. Dufour J.P. Methods Enzymol. 1988; 157: 528-533Crossref PubMed Scopus (83) Google Scholar), and clear plasma membrane pellets were resuspended in glycerol-containing solution and stored at −80 C. Crude membranes were resuspended to a protein concentration of 1 mg/ml in solubilization buffer (0.75% DM, 500 mm NaCl, 50 mm Tris-Cl (pH 7.50), 250 mm sucrose, 20% (v/v) glycerol, 1 mm MgATP, 1 mm MgCl2, 6.5 mm dithiothreitol) and mixed gently for 1 h at 4 °C. The unsolubilized material was removed by centrifugation (100,000 × g/1 h/4 °C). DM extracts were loaded on immobilized monomeric avidin resin that had been pre-equilibrated in column wash buffer (0.1% DM, 250 mm NaCl, 50 mm Tris-Cl (pH 7.5), 250 mm sucrose, 20% (v/v) glycerol, 1 mmMgCl2, 6.5 mm dithiothreitol). The column was washed with 6 bed volumes of column wash buffer. Biotinylated wild type Pfcrt-bad protein was eluted with 2 bed volumes of elution buffer (2 mm d-biotin in column wash buffer). Inside-out plasma membrane vesicles (ISOV) were isolated following the procedure described by Menendez et al. (39Menendez A. Larsson C. Ugalde U. Anal. Biochem. 1995; 230: 308-314Crossref PubMed Scopus (21) Google Scholar) with some modifications (27Fritz F. Howard E.M. Hoffman M.M. Roepe P.D. Biochemistry. 1999; 38: 4214-4226Crossref PubMed Scopus (25) Google Scholar). In particular, ISOV were fabricated with 100 mm KCl inside the vesicles so that H+pumping could be analyzed with symmetrical high Cl− on either side of the plasma membrane. When elevating [salt], [sucrose] was adjusted to conserve osmolality. Our method is adapted from Ohsumi and Anraku (40Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar) with some minor modifications. The P. falciparum DV was isolated following procedures described in Ref. 41Bray P.G. Janneh O. Raynes K.J. Mungthin M. Ginsburg H. Ward S.A. J. Cell Biol. 1999; 145: 363-376Crossref PubMed Scopus (147) Google Scholar, with some modifications. 5 ml of culture suspensions of twice synchronized midtrophozoites of the Sudan 106/1 strain (at ∼12% parasitemia) were washed three times in PBS (pH 7.4). Each 5-ml sample was resuspended in PBS containing 0.15% saponin, incubated for 5 min, and centrifuged to collect trophozoites. The isolated trophozoites were washed repeatedly in ice-cold PBS until the supernatant w
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