functional complementation of the yeast p-type h+-atpase, pma1, by the pneumocystis carinii p-type...

Functional Complementation of the Yeast P-type H1-ATPase, PMA1, by thePneumocystis carinii P-type H1-ATPase, PCA1

DANIELA GRIGORE and JOHN C. MEADE

Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505

ABSTRACT. The opportunistic fungus Pneumocystis is the etiologic agent of an interstitial plasma cell pneumonia that primarily afflictsimmunocompromised individuals. Like other fungi Pneumocystis maintains a H1 plasma membrane gradient to drive nutrient uptake andregulates intracellular pH by ATP-dependent proton efflux. Previously, we identified a Pneumocystis gene, PCA1, whose predicted proteinproduct was homologous to fungal proton pumps. In this study, we show by functional complementation in a Saccharomyces strain whoseendogenous PMA1 proton pump activity is repressed that the Pneumocystis PCA1 encodes a H1-ATPase. The properties of PCA1 char-acterized in this system closely resemble those of yeast PMA1. Yeast expressing PCA1 grow at low pH and are able to acidify the externalmedia. Maximal enzyme activity (Vmax) and efficiency of substrate utilization (Km) in plasma membranes were nearly identical for PCA1and PMA1. PCA1 contains an inhibitory COOH-terminal domain; removal of the final 40 amino acids significantly increased Vmax andgrowth at pH 6.5. PCA1 activity was inhibited by proton pump inhibitors omeprazole and lansoprazole, but was unaffected by H1/K1-ATPase inhibitor SCH28080. Thus, H1 homeostasis in Pneumocystis is likely regulated as in other fungi. This work also establishes asystem for screening PCA1 inhibitors to identify new anti-Pneumocystis agents.

Key Words. COOH-terminal inhibitory domain, omeprazole, proton pump, P-type ATPase, Saccharomyces complementation.

PNEUMOCYSTIS jirovecii (formerly P. carinii f. sp. hominis)is a significant opportunistic pathogen that causes Pneumo-

cystis pneumonia (PCP) in immunosuppressed hosts. Throughoutthe AIDS epidemic PCP has been the most common AIDS-defin-ing opportunistic infection to occur first, the most common inci-dent AIDS-defining opportunistic infection, and the most commonof AIDS-defining opportunistic infections that have occurred dur-ing the course of AIDS (Centers for Disease Control and Preven-tion 1997; Jones et al. 1999). Despite recent advances in humanimmunodeficiency virus (HIV)-related therapy and opportunisticinfection regimens, PCP is still a significant cause of morbidityand mortality in HIV-infected patients (Fisk, Meshnick, andKazanjian 2003; Kaplan et al. 1998; Morris et al. 2004). Thecombination of trimethoprim-sulfamethoxazole remains the pre-ferred agent for both therapy and prophylaxis, but failures oftreatment are common, resulting both from poor patient compli-ance because of drug toxicity and an increasing incidence of drugresistance of P. jirovecii, a trait associated with increased mortal-ity (Crothers et al. 2005; Helwig-Larsen et al. 1999; Kazanjian etal. 2004). Also of concern is the link between prophylaxis failures,severity of pneumonia, and Pneumocystis genotype, suggestingthe existence of mutation-prone strains (Hauser et al. 2001; Millerand Wakefield 1999; Nahimana et al. 2004). Clearly, there is anurgent need to identify novel anti-Pneumocystis therapeutic tar-gets with improved efficacy and reduced toxicity. However, manyaspects of Pneumocystis biology, ecology, and pathogenesis arepoorly understood owing to the lack of an adequate in vitro cul-tivation system and difficulties in isolating organisms. In turn,these have hampered identification, characterization, and devel-opment of new anti-Pneumocystis cellular targets.

P-type ATPases form a ubiquitous family of proteins involvedin active transport of charged ions, Ca21, Na1, K1, H1, Mg1, andheavy metals, across biological membranes to maintain intra-cellular ion homeostasis, and in the preservation of membraneasymmetry via phospholipid translocation (Kuhlbrandt 2004).Fungi and plants utilize P-type H1-ATPases to transport protonsout of the cell in order to control intracellular pH (pHi) and es-tablish an electrochemical proton gradient to drive nutrient uptake(Lefebvre, Boutry, and Morsomme 2003). These pumps havebeen proposed as potential anti-fungal targets as analogous elec-

trogenic proton pumps are absent in vertebrates (Monk et al. 1995;Seto-Young et al. 1997). Indeed, inhibition of the cardiac Na1/K1-ATPase by glycosides to regulate impulse conduction, inhi-bition of the gastric H1/K1-ATPase by omeprazole derivatives tocontrol stomach acidity, and inhibition of the Plasmodium SE-RCA pump by artemisinin in anti-malarial treatment illustrate thetherapeutic potential of targeting members of the P-type ATPasefamily (Eckstein-Ludwig et al. 2004; Kjeldsen and Bundgaard2003; Vanderhoff and Tahboub 2002).

The maintenance of pHi and an electrogenic plasma membranepotential in P. carinii have been shown to be dependent ona transporter whose properties are consistent with P-type H1-ATPases (Docampo et al. 1996; VanderHeyden, McLaughlin, andDocampo 2000). A closely related gene homolog, PCA1, of theyeast proton pump, which presumably mediates pHi and the elec-trogenic plasma membrane potential, has been isolated from aPneumocystis carinii genomic library (Meade and Stringer 1995).PCA1 encodes a 101.4-kDa protein composed of 927 amino acids.It closely resembles—in size, structure, and sequence—H1-ATPases reported for 14 proton pumps from 12 species of fungi.The predicted Pneumocystis PCA1 peptide also contains 115/121amino acids found to be conserved in H1-ATPases (Wach,Schlesser, and Goffeau 1992).

In this work, we confirm sequence predictions and demonstrateby functional complementation of a recipient Saccharomycesstrain lacking endogenous proton pump activity that PCA1encodes a H1-ATPase. The properties of PCA1 expressed inSaccharomyces resemble those of other fungal H1-ATPases. Thisyeast expression system now provides a mechanism for the iden-tification and development of anti-PCA1 inhibitors as well as pro-viding a source of peptide for future structure-function studies ofPCA1 and its inhibitors.

MATERIALS AND METHODS

Yeast strains. Saccharomyces cerevisiae strain YAK2 (MATa,ade2-101, leu2D1, his3D200, ura3-52, trp1D63, lys2-801,pma1D::HIS3, pma2D::TRP1) is a haploid yeast strain with de-letions/mutations in genes for adenine, leucine, histidine, uracil,tryptophan, and lysine biosynthesis (obtained from M. Boutry). Itsendogenous PMA1 and PMA2 proton pump genes are replacedwith histidine (HIS3) and tryptophan (TRP1) biosynthesis genes.PMA1 is an essential gene so YAK2 survival requires a cent-romeric plasmid, cp(GAL1)PMA1(URA3), which contains thePMA1 gene under control of a galactose-inducible promoter and

Corresponding Author: J. MEADE, Department of Microbiology,University of Mississippi Medical Center, 2500 North State Street,Jackson, Mississippi 39216-4505—Telephone number: 601-984-1914;FAX number: 601-984-1708; e-mail: [email protected]

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J. Eukaryot. Microbiol., 53(3), 2006 pp. 157–164r 2006 The Author(s)Journal compilation r 2006 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2006.00089.x

a URA3 gene for cp plasmid retention during culture in uracil-deficient media (de Kerchove d’Exaerde et al. 1995). YAK2growth and survival requires galactose in the medium for PMA1expression, the absence of both histidine and tryptophan to main-tain genomic PMA1 and PMA2 deletions, and the absence ofuracil to maintain the centromeric plasmid.

Media. Yeast was grown at 30 1C on synthetic medium (QBio-gene, Carlsbad, CA) containing (per liter): 7 g of yeast nitrogenbase, 5 g ammonium sulfate, 0.60–0.70 g of a supplement mix(CSM) with all the amino acids except, in some cases, those usedfor selection (histidine, leucine, uracil, or tryptophan), and 20 g ofeither glucose (DOBGlu) or galactose (DOBGal). Solid mediacontained, in addition, 17 g/l agar. These media were supplement-ed with 20 mM KH2PO4 and their pH adjusted with 1 N HCl or1 N NaOH. YAK2 was grown on DOBGal plus CSM minus his-tidine, tryptophan, and uracil (DOBGal-His, -Trp, -Ura). YAK2transformed with the 2-m plasmid containing Pneumocystis PCA1were cultured in DOBGal plus CSM minus histidine, tryptophan,uracil, and leucine (DOBGal-His, -Trp, -Ura, -Leu). Complement-ation of yeast PMA1 activity by PCA1 was assessed by growth onDOBGlu plus CSM minus histidine, tryptophan, uracil, andleucine (DOBGlu-His, -Trp, -Ura, -Leu). The PMA1 containingcp plasmid was removed from yeast transformants containing 2-mplasmids by growth in DOBGlu plus CSM minus histidine,tryptophan, and leucine supplemented with 0.1% (w/v) 5-fluoro-orotic acid (DOBGlu-His, -Trp, -Leu, 15-FOA). Transformantswith 2-m plasmids, which had lost the cp plasmid, were culturedfor experiments in DOBGlu plus CSM minus histidine, try-ptophan, and leucine (DOBGlu-His, -Trp, -Leu).

Plasmid constructions. The Pneumocystis PCA1-coding re-gion, including 50- and 30-flanking sequences (Meade and Stringer1995), was available in vector pBluescript (Stratagene, La Jolla,CA). The 50- and 30-ends of PCA1 were modified by PCR toinclude 50 Xho I-Bam HI restriction sites and a consensus yeasttranslation initiation sequence (CACC), appended immediatelyupstream of the start codon (ATG), and a 30 BamHI restriction siteimmediately downstream of the stop codon. The new PCA1 50-end, as a 1,186-nucleotide (nt) XhoI–BlpI fragment, and 30-end, asa 349-nt Eco72I–BamHI fragment, were used to replace the cor-responding fragments of PCA1 in pBluescript. The new 50 and 30

ends were sequenced to verify the fidelity of PCR modifications.The modified PCA1 was excised from pBluescript as a 2.94-kilobase pair (kb) BamHI fragment and used to replace the Nicotianaplumbaginifolia proton pump gene PMA4, excised by BamHI–HindIII digestion, in the 9.6-kb yeast 2-m plasmid, 2 m(PMA1)PMA4 (obtained from M. Boutry) (de Kerchove d’Exaerde et al.1995), to produce 2 m(PMA1)PCA1. Incompatible restriction ter-mini in this procedure were blunt-ended with Klenow fragment ofEscherichia coli DNA polymerase I before ligation. 2 m(PMA1)PCA1 has PCA1 under the control of the strong, constitutiveSaccharomyces PMA1 transcriptional promoter, a 2-m yeast plasmidorigin of replication for high copy maintenance (20 copies/cell), aLEU2 gene as selection marker for growth in media lacking le-ucine, and a bacterial origin of replication and ampicillin resist-ance gene for propagation and selection in E. coli. A COOH-truncated version of PCA1 with the sequence encoding the last 40amino acids deleted was produced by digestion with EcoRI andBamHI followed by a Klenow fill in reaction to blunt the ends.The resulting 2.7-kb PCA1 fragment was then cloned into2 m(PMA1)PMA4 as described. The 2-m plasmids with PCA1 wereverified by comparing their predicted restriction digest patternsversus those actually produced using a battery of restriction endo-nucleases.

PCA1 expression in yeast. The 2 m(PMA1)PCA1 plasmidswere transformed into YAK2 using lithium/cesium acetate treat-ment (Zymo Research, Orange, CA). YAK2 cells that acquired

2 m(PMA1)PCA1 were selected by growth on media withoutleucine (DOBGal-His, -Tryp, -Ura, -Leu). To assess the abilityof PCA1 to complement PMA1 activity, YAK2 transformantswere plated on glucose-containing media to repress expression ofPMA1 (DOBGlu-His, -Trp, -Ura, -Leu). Centromeric plasmidcp(GAL1)PMA1(URA3) was then removed from YAK2 trans-formants by culture in DOBGlu-His, -Trp, -Leu, 15-FOA. Cent-romeric plasmids are spontaneously lost at a rate of approximately1% per generation and cells that lose cp(GAL1)PMA1(URA3)were selected by inclusion of 5-FOA in the media, which is me-tabolized to a toxic product by URA3 (Boeke, LaCroute, and Fink1984). Loss of cp(GAL1)PMA1(URA3) plasmid and PMA1 tran-scripts in YAK2 transformants was verified by Southern andNorthern blot analysis.

Yeast nucleic acid isolation. Yeast cell walls were digestedwith zymolyase to produce spheroplasts using a Yeast Cell LysisPreparation kit (Qbiogene, Carlsbad, CA). Yeast DNA (genomicand plasmid) was isolated from yeast spheroplasts using the Gen-ome DNA kit (Qbiogene). Yeast RNA was isolated from yeastspheroplasts with the RNA Easy kit (Qiagen Inc., Valencia, CA).

Plasma membrane preparation. Yeast plasma membraneswere prepared as described by Serrano (1983) with minor mod-ifications. Yeast were harvested in mid-log phase at 1,000 g,washed 3 times in ice-cold water, and resuspended in 1-ml lysisbuffer (250 mM sucrose, 1 mM EGTA, 2 mM MgCl2, 25 mMHEPES, 10 mM benzamidine, 15 mM dithiothreithol, 1.5% (v/v)fungal protease inhibitor mixture (Sigma, St. Louis, MO), pH 7.5).An equal vol. of 0.5-mm glass beads was added and the mixturevortexed until �90% of cells were lysed, as quantified by mi-croscopy. The glass beads were gravity-washed in glycerol buffer(20% (v/v) glycerol, 1 mM EGTA, 2 mM MgCl2, 25 mM HEPES,15 mM dithiothreithol, pH 6.5). Unbroken cells and debris wereremoved at 3,000 g for 3 min, the supernatant decanted, and mem-branes pelleted by centrifugation for 30 min at 20,000 g. The pel-let (total membrane fraction) was resuspended in 4 ml of glycerolbuffer, layered on a sucrose step gradient (3 ml of 53.5% sucrose(w/w) and 6 ml of 43.5% sucrose (w/w)) and centrifuged for 6 h at80,000 g. The plasma membranes were colleted at the interphase,diluted fivefold with water, and again centrifuged for 30 min at80,000 g. Pellets were resuspended in glycerol buffer and stored at� 80 1C until use. Protein concentration was determined using theBio-Rad DC Protein colorimetric assay with bovine serum albu-min as a standard (Bio-Rad, Hercules, CA). All operations wereperformed at 0–4 1C. Mitochondrial contamination in membranepreparations was determined in assay reactions at pH 9.0 with thesodium azide removed and 200 mM vanadate added to inhibitP-type ATPase activity. The Pi released because of mitochondrialATPase was less than 8% of the H1-ATPase activity detected(data not shown).

H1-ATPase assays. ATPase activity in plasma membranepreparations was assayed as orthophosphate released from subst-rate ATP using a colorimetric procedure as described previously(Chifflet et al. 1988; Luo, Scott, and Docampo 2002). The assaymixture contained 50 mM MES, adjusted to pH 6.5 with TRIS,5 mM MgSO4, 50 mM KNO3 (a vacuolar ATPase inhibitor),5 mM sodium azide (a mitochondrial ATPase inhibitor), 2 mMsodium molybdate (an acid phosphatase inhibitor), and 2 mMATP. Reactions were performed at room temperature in 96-wellmicro-plates using 4 mg of plasma membrane protein per 50 mlreaction vol. Reactions were initiated by the addition of ATP andat designated intervals 50 ml of 12% (w/v) sodium dodecyl sulfate(SDS) were added to stop the reaction. Free phosphate (Pi) wasdetected by addition of 100-ml detection reagent, equal parts offresh 6% (w/v) ascorbic acid in 1 N HCl and 1% (w/v) ammoniummolybdate, followed after 3–10 min by a solution containing2% (w/v) sodium citrate, 2% (w/v) sodium meta-arsenite, and 2%

158 J. EUKARYOT. MICROBIOL., VOL. 53, NO. 3, MAY– JUNE 2006

(v/v) acetic acid. Reactions were read, after 10-min incubation, at750 nm on a BIO-TEK Elx800 microplate reader (Bio-Tek In-struments Inc., Winooski, VT), calibrated using phosphate stand-ards (10–1,000 nM Pi/well). Enzyme Vmax and Km weredetermined using an ATP regeneration system, 100 mg/ml pyruv-ate kinase, and 5 mM phosphoenol pyruvate with ATP concent-rations varying from 0.05 to 10 mM. The MgSO4 concentration inthese assays was increased to 10 mM. Km and Vmax values werecalculated using the Solver function in MS Excel. Specific activ-ities were expressed in mmoles Pi released/minute/mg protein andall experiments were performed in triplicate. Control reactions tomeasure contaminating phosphate in the wells were performed byadding 50 ml of 12% (w/v) SDS to stop the reaction at the sametime as the 2-mM ATP used to initiate the reaction.

Measurement of glucose-induced acid efflux from yeast.YAK2 and the 2 m PCA1 transformants were grown in 100 ml ofthe appropriate media at pH 6.5, harvested in late exponentialphase at 1,000 g, washed 4 times with ice-cold water, and storedon ice for 4 h to reduce cellular metabolism to a minimum. Then,109 yeast cells were resuspended in 10 ml of 250 mM sorbitol atroom temperature in a vial with a magnetic stir bar and a pHelectrode. A stable pH baseline was established (�5 min),250 mM glucose were added as an energy source, and the de-crease in pH was recorded over time. The drop in pH was fol-lowed until the pH of the media reached a constant value, afterabout 10 min. The magnitude of the glucose-induced pH change isa measure of the ability of the H1-ATPases to pump protons.

Statistical analysis. Significant differences at Po0.05 andstandard error (SE) were determined using Sigma Plot 9.0 (SystatSoftware, Richmond, CA).

RESULTS

Expression of Pneumocystis PCA1 in yeast. PCA1 was in-serted into a high copy number 2-m yeast plasmid under control ofthe strong constitutive PMA1 promoter to permit its expression inyeast. 2 m(PMA1)PCA1 was transformed into YAK2 and trans-formants containing 2-m plasmids were selected by growth onDOBGal-His, -Trp, -Ura, -Leu agar plates. Yeast transformantscontaining 2 m(PMA1)PCA1, designated YAK2-PCA1, were thentested for their ability to complement PMA1 activity by replicatransfer to DOBGlu-His, -Trp, -Leu plates. The replacement ofgalactose with glucose in this medium represses expression ofPMA1 from cp(GAL1)PMA1(URA3) and YAK2 transformantswill survive only if the 2-m plasmid-borne PCA1 is expressedand can functionally substitute for PMA1 proton pump activity.Complementation efficiency was 100% of the transferred YAK2-PCA1 colonies indicating that true functional complementationhad occurred (data not shown). If the occurrence of PMA1–PCA1recombination events or cp(GAL1)PMA1(URA3) mutation eventswere responsible for the growth of YAK2 transformants on glu-cose media then complementation efficiency would be greatly re-duced, reflecting the rare nature of these events. The functionalcomplementation of PMA1 by PCA1 is illustrated in Fig 1A.YAK2 grows in media containing galactose (Fig. 1A1), but is un-able to grow in glucose containing media (Fig. 1A2, A3) whereasYAK2-PCA1 transformants support growth in glucose media atboth pH 6.5 (Fig. 1A2) and pH 4.5 (Fig. 1A3).

YAK2-PCA1 transformants were next transferred to mediacontaining 0.1% (w/v) 5-FOA in order to remove centromericplasmid cp(GAL1)PMA1(URA3). This eliminates the potential for

Fig. 1. Functional complementation of yeast PMA1 H1-ATPase activity by Pneumocystis carinii P-type H1-ATPase PCA1 expressed in Sac-charomyces strain YAK2. (A) PCA1 complements PMA1 activity and sustains yeast growth. Strains: YAK2 with cp(GAL1)PMA1(URA3), YAK2-PCA1with 2m(PMA1)PCA1. Yeasts were grown to saturation in permissive media, DOBGal-His, -Trp, -Ura for YAK2 or DOBGal-His, -Trp, -Ura, -Leu forYAK2-PCA1; 10 ml aliquots containing 103 washed cells were spotted unto solid media containing DOBGal-His, -Trp, -Ura, (panel A1), DOBGlu-His, -Trp, -Ura, pH 6.5 (panel A2), or DOBGlu-His, -Trp, -Ura, pH 4.5 (panel A3). Growth was recorded after 72 h at 30 1C. Growth in glucose-containingmedia indicates successful complementation of yeast PMA1 function by PCA1. (B) Removal of cp(GAL1)PMA1(URA3) plasmid by treatment with 5-fluoroorotic acid (5-FOA). Strains: YAK2 with cp(GAL1)PMA1(URA3), YAK2-PCA1 with 2m(PMA1)PCA1,1FOA indicates treatment of YAK2-PCA1with 5-fluoroorotic acid, �FOA indicates untreated cells. Yeast were grown to saturation in permissive media, galactose media (DOBGal-His, -Trp,Ura) at pH 6.5 for YAK2 and in glucose media (DOBGlu-His, -Trp, -Leu) at pH 6.5 for YAK2-PCA1; 10-ml inoculums containing 103 cells were spottedonto solid medium containing DOBGal-His, -Trp, -Leu, -Ura, pH 6.5 (panel B1) or DOBGal-His, -Trp, -Leu, pH 6.5 (panel B2). Growth was recordedafter 3 d at 30 1C. The inability of YAK2-PCA1 treated with 5-FOA to grow in media lacking uracil (panel B1) indicates the loss of cp(GAL1)PMA1(URA3) plasmid as these cells no longer contain the URA3 gene necessary for de novo uracil synthesis.

159GRIGORE & MEADE—PNEUMOCYSTIS PCA1 PROTON PUMP

homologous recombination between 2-m plasmid-borne PCA1 andcp plasmid-borne PMA1, which might occur during long-termculture owing to the high degree of homology in their gene se-quences. The presence of 5-FOA in the media selects for yeastcells that have lost cp(GAL1)PMA1(URA3) because of mis-seg-regation during cell division (cp plasmids only replicate duringyeast mitosis). Metabolism of 5-FOA by URA3 produces a toxicproduct and cells containing cp(GAL1)PMA1(URA3) will not sur-vive in this medium. Loss of cp(GAL1)PMA1(URA3) from 5-FOA-treated YAK2-PCA1 is shown in Fig 1B. The 5-FOA-treatedYAK2-PCA1 grows in medium containing uracil (Fig. 1B2), butwas unable to grow in medium lacking uracil (Fig. 1B1) becauseof the absence of the URA3 gene necessary for uracil biosynthesis,proving that cp(GAL1)PMA1(URA3) had been eliminated fromthese cells. YAK2-PCA1 that had not been treated with 5-FOAgrows in the absence of uracil because of the presence of a URA3gene on its cp(GAL1)PMA1(URA3) plasmid (Fig. 1B1). YAK2did not grow in either media because of the absence of leucine inthe media as YAK2 lacks a functional LEU2 gene for leucinebiosynthesis (Fig. 1B1, B2). A LEU2 gene is present on PCA1containing 2-m plasmids in YAK2-PCA1 and these cells can growin media lacking leucine.

The loss of PMA1 containing cp(GAL1)PMA1(URA3) plasmidand the presence of 2 m(PMA1)PCA1 plasmids in 5-FOA-treatedYAK2-PCA1 transformants was confirmed by Southern blotting(Fig. 2A). After digested with restriction endonuclease EcoRV,hybridization of YAK2 DNA to a radiolabelled 540-nt fragmentof PMA1 identified a 5-kb fragment whose size was consistentwith that predicted (4.98 kb) from the cp(GAL1)PMA1(URA3)plasmid sequence (Fig. 2A, center panel, lane 1). No hybridizationsignal was observed in 5-FOA-treated YAK2-PCA1 DNA indi-cating the loss of cp(GAL1)PMA1(URA3) from these cells (Fig.2A, center panel, lane 2). Probing of the same blot after strippingwith a radiolabeled 490-nt fragment of PCA1 identified a 5.1-kbfragment of 2 m(PMA1)PCA, in agreement with the size predicted(5.13 kb), showing that PCA1 DNA was present in 5-FOA-treatedYAK2-PCA1 (Fig. 2A, right panel, lane 2). The hybridizationsignal in lane 1 (Fig. 2A, right panel) is due to cross-hybridizationof the PCA1 probe with PMA1 in YAK2. The absence of PMA1

expression in YAK2-PCA1 was demonstrated by Northern blot-ting (Fig. 2B). A 3.3-kb PMA1 message was detected in RNAisolated from YAK2 probed with a 1.8-kb SalI DNA fragment ofPMA1 but PMA1 message was not detected in RNA from 5-FOA-treated YAK2-PCA1 (Fig. 2B, upper panel, lanes 1 and 2). Theblot was stripped and re-probed with a 600-nt LEU2-specific geneprobe (Cla I-Eco RV fragment). A 1.4-kb LEU2 message waspresent only in RNA from YAK2-PCA1 because of the presenceof a LEU2 gene on the 2-m plasmids (Fig. 2B, bottom panel, lane2). This result showed that 2m(PMA1)PCA1 transcription was oc-curring and also verified the presence of RNA in this lane. YAK2lacks a functional LEU2 gene and no LEU2 transcription was de-tected in YAK2 RNA (Fig. 2B, lower panel, lane 1).

PCA1 in YAK2-PCA1 encodes an H1-ATPase. The activityof the Pneumocystis proton pump in yeast was characterized bygrowth at different pH, its ability to acidify external media, andpH optimum and kinetic properties in isolated membranes. Theability of the YAK2-PCA1 transformant to grow at low pH wasassessed by calculating duplication time during log phase growth(Fig. 3A). Saccharomyces characteristically grows well at low pH,and YAK2 (Fig. 3A, black bars) grew as well at pH 3.0 as at pH6.5, with generation times of 2.16 h at pH 3.0 and 1.93 h at pH 6.5.YAK2-PCA1 (Fig. 3A, white bars) grew significantly more slow-ly than YAK2 at each pH, with duplication times 2-fold greater atpH 3.0, 4.0, and 6.5. The Pneumocystis proton pump transformantgrew best at pH 5.0 where its duplication time was only 1.5-foldgreater than YAK2. Growth of the YAK2-PCA1 transformant ingalactose media (DOBGal-His, -Trp, -Leu) generated results ateach pH similar to those illustrated in Fig. 3A (data not shown).

The Saccharomyces PMA1 H1-ATPase has the ability to acidifythe external medium after addition of an energy source such asglucose (Foury, Boutry, and Goffeau 1976; Serrano, Kielland-Brandt, and Fink 1986). The PCA1 transformant (Fig. 3B, whitebars) can also mediate acidification of the external medium al-though not to the same level as the pH decrease mediated by YAK2cells expressing PMA1 (Fig. 3B, black bars). Despite a similarity intheir initial rate (data not shown), the steady-state level of acidifi-cation by YAK2 of 1.31 pH units was approximately 50% greaterthan the PCA1 driven pH decrease of 0.84 units. These results were

Fig. 2. Southern blot analysis (A) demonstrating the presence of PCA1 DNA and absence of PMA1 DNA in YAK2-PCA1 and Northern blot analysis(B) demonstrating the absence of PMA1 message in RNA from YAK2-PCA1. (A) Autoradiogram of a Southern blot containing 5mg DNA per lanehybridized to a 32P-labeled 540-nt SalI fragment of PMA1 (center panel) or a 32P-labeled 490-nt Eco 72I–BamHI fragment of PCA1 (right panel). Lane 1,YAK2 DNA digested with EcoRV; lane 2, YAK2-PCA1 (5-FOA treated) DNA digested with EcoRV. The kilo base pair size of the molecular weightmarkers in lane MW (left panel) are shown in the left margin. (B) Autoradiogram of a Northern blot containing 4mg total RNA per lane hybridized to a1.7-kb SalI fragment of Saccharomyces cerevisiae PMA1 (top panel) or a 600-nt ClaI–EcoRV fragment of yeast LEU2 (bottom panel) as a control forRNA loading. Lane 1, YAK2 RNA; lane 2, YAK2-PCA1 RNA. The size of the transcripts detected, based on RNA molecular mass markers, is shown inthe right margin.


consistent with the ability of YAK2 to grow better at low pH thanthe YAK2-PCA1 transformant (cf. Fig. 3A).

The pH dependence of ATP hydrolysis catalyzed by PMA1 andPCA1 was determined in enzymatic ATPase assays at differentpH, using isolated membranes from YAK2 and YAK2-PCA1. Theactivity of yeast PMA1 was optimal at pH 6.0 and was broadlyactive throughout the pH range tested (Fig. 4, filled circles). Theseresults correlate with the ability of YAK2 to grow over a broadlyacidic pH range and to grow optimally at pH 6.5 (Fig. 3A). PCA1

ATPase activity exhibited a lower pH optimum of 5.5 and wasalso active from pH 3.5 to 7.5 (Fig. 4, open circles). This resultagrees with the ability of PCA1 to support growth of YAK2-PCA1cells at low pH (cf. Fig. 3A). The ATPase activity of both PMA1and PCA1 proton pumps was lowest when the assay pH was aboveneutral pH.

The kinetic properties (Vmax and Km) of the Pneumocystis pro-ton pump in YAK2-PCA1 were determined in isolated yeast mem-brane fractions by measuring phosphate release from ATP in anATP-regenerating system (100 mg/ml pyruvate kinase and 5 mMPEP) over a range of 0.05–10 mM MgATP. Pneumocystis PCA1in YAK2-PCA1 has a maximal ATP hydrolysis value (Vmax) of0.622 mmol Pi/min/mg protein and an apparent Km of 0.434 mMMg21-ATP (Table 1). The maximal activity and efficiency ofMg21-ATP utilization of the Pneumocystis H1-ATPase were seento be nearly identical to the values for the yeast PMA1 pump inYAK2, Vmax of 0.651 mmole Pi/min/mg protein and Km of0.434 mM. Expression of heterologous H1-ATPases in prior yeastcomplementation studies has most often been less robust than the

Fig. 3. Comparison of the ability to grow and acidify media by YAK2, YAK2-PCA1, and COOH-truncated YAK2-PCA1. Yeast strains: YAK2(yeast PMA1 under control of the Gal1 promoter), black bars; YAK2-PCA1 (PCA1 under PMA1 promoter), white bars; YAK2-PCA1-COOHD(PCA1with a 40-amino acids C-terminal deletion), white cross-hatched bars. All data represent the means � SE of at least 3 individual experiments. Thepresence of an asterisk (�) indicates that the difference in YAK2 and YAK2-PCA1 was significant at Po0.05, the cross (w) indicates significance atPo0.05 for the difference between YAK2-PCA1 and YAK2-PCA1-COOHat pH 6.5. (A) Duplication times calculated at mid-log phase growth fromOD600 readings of yeast grown at 30 1C from an initial inoculum of 103 cells in 30 ml of media at pH 6.5, pH 5.0, pH 4.0, or pH 3.0. YAK2 was grown ingalactose media (DOBGal-His, -Trp, -Ura) and YAK2-PCA1 and YAK2-PCA1-COOHwere grown in glucose media (DOBGlu-His, -Trp, -Leu). (B)Media acidification, measured by pH drop, produced by starved yeast cells after addition of 250 mM glucose.

Fig. 4. pH dependence of ATPase activity measured in yeastmembranes isolated from YAK2 (�), YAK2-PCA1 (�), and YAK2-PCA-COOH (}). Assays were conducted in an ATPase reaction medium,buffered with 50 mM MES, 50 mM MOPS, and 50 mM Tris, adjusted tothe indicated pH with 1 N HCl or 1 N KOH. Data points indicate meanATPase activity from 3 to 5 separate preparations � SE.

Table 1. Comparison of kinetic values, Vmax and Km of ATP hydrolysis,for YAK 2, PCA1, and COOH-truncated PCA1, expressed in YAK2.

Yeast strain H1-ATPase Vmax Km

Expressed mmol/min/mg protein

mM

YAK2 PMA1 0.651 � 0.065 0.433 � 0.029YAK2-PCA1 PCA1 0.622 � 0.040 0.434 � 0.110YAK2-PCA1-COOH

PCA1-COOH 0.977 � 0.067� 0.636 � 0.098�

Data are expressed as mean Vmax and Km values � SE calculated fromATP hydrolysis assays on three independent plasma membrane prepara-tions of each yeast strain.�The difference between Vmax values of PCA1-COOH and PCA1 is

significant at Po0.05.


endogenous PMA1 activity, reflecting the inefficiencies in ex-pression, peptide processing, and membrane insertion because ofamino-acid sequence differences in heterologous pumps as com-pared with the endogenous pumps. However, the high degree ofhomology of the Pneumocystis PCA1 pump with yeast PMA1(70% identity, 81% amino-acid similarity) enables PneumocystisPCA1 to function as well in yeast membranes as its endogenousPMA1 pump.

PCA1 contains a COOH-terminal inhibitory domain. Thecarboxyl terminal amino-acid domains of yeast and plant H1-ATPases regulate enzyme activity by an inhibitory interaction withthe remainder of the protein. This inhibition is removed by bindingof activator molecules to the C-terminal domain. Removal of thecytoplasmic, COOH-terminal amino acids from these pumps alsoresults in a constitutively activated enzyme (Morsomme, Slayman,and Goffeau 2000; Portillo 2000). In order to determine if thePCA1-encoded peptide contained a similar regulatory domain, aCOOH-truncated version was constructed and tested for increasedH1-ATPase activity by measuring growth rate, media acidifica-tion, and enzyme kinetics. The YAK2 transformant of PCA1 witha deletion of the sequence encoding the 40 amino acids at thePCA1 COOH terminus was produced in a similar fashion as wildtype YAK2-PCA1. The COOH-truncated version, YAK2-PCA1-COOHDgrew significantly better at pH 6.5 than the wild-typeYAK2-PCA1 with a greater than 25% reduction in duplicationtime (Fig. 3A). This improvement in growth rate however, was notobserved at lower pH. The PCA1-truncated protein also showed anincreased ability to acidify media, although the difference was notsignificant at Po0.05 (Fig. 3B). Activation of yeast or plant H1-ATPases is also characterized by a rise in enzyme maximal ve-locity (Vmax), which can be accompanied by a reduction in thesubstrate concentration necessary to achieve half maximal activity(Km). The Vmax for H1-ATPase activity in membrane preps fromYAK2-PCA1-COOHDwas significantly increased (57%) over thatobtained with YAK2-PCA1 membranes (Table 1). The Km forMg21 ATP in assays with YAK2-PCA1-COOHD membranes wasalso increased as compared with wild-type clones but this differ-ence was not significant.

Effect of proton pump inhibitors on PCA1. The sensitivityof PMA1 and PCA1 ATPase activity in yeast plasma membranefractions to classical inhibitors of H1-ATPases and H1/K1-ATPases was also determined. Vanadate, a specific inhibitor ofP-type ATPases, was a potent inhibitor of both pumps with 50%inhibitory concentrations (IC50) of 12.95 and 10.24 mM, respec-tively (Table 2). SCH28080, a potent, reversible inhibitor of thegastric H1/K1-ATPase, binds near the luminal K1 high affinitysite of H1/K1-ATPases (Asano et al. 1997). No inhibition of AT-Pase activity was observed in yeast plasma membranes containingPMA1 and PCA1 after 20 min pre-incubation even using concen-trations of SCH28080 as high as 500 mM (Table 2). The ben-zimidazole drugs omeprazole and lansoprazole, which are bothpotent inhibitors of mammalian H1/K1-ATPases widely used todecrease gastric acidity, had an inhibitory effect on all the pumpssimilar to the effect of vanadate (IC50 6.38–28.07 mM range,

Table 2). The seleno-organic drug ebselen [2-phenyl-1-2-benzoisoselenazole-3(2H)-one], which also inhibits H1/K1-AT-Pases as well as a number of other enzymes such as protein kin-ases, was also seen to be a potent inhibitor of PCA1 with an IC50

value of less than 1 mM (data not shown).

DISCUSSION

The H1-ATPases in fungi are high-capacity plasma membraneproton pumps that play a critical role in their cellular physiologythrough regulation of intracellular pH and maintenance of a trans-membrane electrochemical proton gradient for nutrient uptake.We have demonstrated that the P. carinii PCA1 gene encodes aplasma membrane P-type H1-ATPase, confirming predictionsbased on the PCA1 sequence. Pneumocystis PCA1 complementsthe YAK2 yeast strain whose endogenous proton pump activityhas been repressed, supports growth of transformed yeast at lowpH, mediates acidification of the external media, and is sensitiveto the P-type ATPase inhibitor vanadate. The properties of thePCA1 H1-ATPase characterized here closely resemble the Sac-charomyces PMA1 proton pump. Both pumps support yeastgrowth at low pH (3.0), their enzyme kinetics (Vmax and Km) arenearly identical, and they have similar pH optima for enzyme ac-tivity. Both possess an inhibitory COOH-terminal domain whoseremoval activates the enzyme, and surprisingly, they both are in-hibited by the human proton pump inhibitors omeprazole and la-nsoprazole. However, recent evidence questions the specificity ofthese benzimidazole drugs for H1/K1-ATPases (Magalhaes et al.2003; Riel et al. 2002; Sutak et al. 2004) and omeprazole has alsobeen shown to inhibit H1-ATPases in other yeast and protozoans(Biswas et al. 2000; Jiang et al. 2002; Monk et al. 1995). Neitherpump is inhibited by SCH28080, another gastric H1/K1-ATPaseinhibitor, which is consistent with the lack of a K1 ion-bindingsite in PMA1 and PCA1 and their identity as H1-ATPases and notH1/K1-ATPases.

These data suggest that PCA1 function, activity, and regulationin Pneumocystis will be analogous to that observed for protonpumps in other fungal species. For instance, although little isknown of Pneumocystis growth and survival outside mammalianhosts, the ability of PCA1 to support yeast growth at low pH andthe acidic pH optima for PCA1 activity imply that Pneumocystis,like yeast, is able to subsist in acidic pH environments. Likewise,the presence of an inhibitory COOH-terminal domain in PCA1also suggests similarity to PMA1 activation in yeast. This in-volves binding of activator molecules induced by glucose metab-olism and phosphorylation of serine/threonine residues in theCOOH-terminal domain to release inhibitory interactions withother protein domains in the molecule. The strong conservationbetween PCA1 and other fungal proton pumps in their carboxylsequences including critical serine and threonine residues thatare phosphorylated supports this interpretation (Meade andStringer 1995). The stable expression system for PCA1 in yeastestablished here (in cultivation for greater than 1 yr) will provide asystem amenable to the detailed genetic and biochemical analysis

Table 2. Effect of inhibitors on the H1-ATPase activity of the YAK2 PMA1 H1-ATPase and the PCA1 H1-ATPase in YAK2, expressed as themicromolar drug concentration required for 50% inhibition of ATP hydrolysis activity (IC50).

Yeast strain H1-ATPase IC50 (mM) IC50 (mM) IC50 (mM) IC50 (mM)Expressed Vanadate SCH28080 Omeprazole Lansoprazole

YAK2 PMA1 12.95 >500 6.38 11.93YAK2-PCA1 PCA1 10.24 >500 7.56 28.07

Values are means calculated from assays using six drug concentrations with isolated membranes from three independent preparations of each yeaststrain. Standard error varied from 3% to 9%.


necessary for resolution of mechanistic aspects of PCA1 functionand regulation.

In addition to its utility in elucidating PCA1 enzymatic prop-erties, this complementation system also has potential value inadvancing anti-Pneumocystis drug research. There is an urgentneed for the identification of new cellular targets and novel pilotcompounds for the development of supplementary anti-Pneumo-cystis therapeutic options. Current drug treatments for this path-ogen are toxic and only partially effective owing to increasingPneumocystis drug resistance and poor patient compliance andtolerance. The essential nature of H1-ATPase function in Pneu-mocystis and the accessibility of PCA1 as a surface protein at thecritical interface between host and pathogen, coupled with theabsence of similar electrogenic H1-ATPases in mammalian cells,makes PCA1 an attractive target for anti-PCP chemotherapy. Thedemonstration of omeprazole and lansoprazole inhibition ofPCA1 activity in this study shows the feasibility of utilizing thisclass of inhibitors as lead compounds to advance research in anti-Pneumocystis therapy. However, the modification of existingH1-ATPase agonists, such as omeprazole, as well as the ration-al design of new anti-proton pump derivatives requires an accuratedepiction of PCA1 structure and its interactions with inhibitors; anundertaking dependent on isolation of purified peptide. Unfortu-nately, biohazard concerns and the lack of an adequate in vitrocultivation system hinder characterization of PCA1 properties andscreening of PCA1 inhibitors in Pneumocystis.

Complementation of S. cerevisiae proton pump function byPCA1 in the YAK2 knockout yeast strain offers a unique oppor-tunity to overcome these obstacles. Expression of PCA1 in Sac-charomyces will permit the exploitation of a class of drugs withwell-documented pharmacokinetic properties and well-estab-lished safety profiles. Purification of isolated PCA1 protein fromthe complementation clones generated here could also providepeptide for structure-function studies via X-ray diffraction cry-stallography.

ACKNOWLEDGMENTS

This work was supported by the Southeastern Center forEmerging Biologic Threats (SECEBT), Grant No. U38/CCU423095-01 from the Centers for Disease Control and Pre-vention (CDC), Atlanta, GA. Its contents are solely the responsi-bility of the authors and do not necessarily represent the officialviews of SECEBT or CDC.

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Received: 09/26/05, 12/15/05; accepted: 12/20/05


functional complementation of the yeast p-type h+-atpase, pma1, by the pneumocystis carinii p-type...

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