Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae

Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae Department of Oral Sciences, School of Candida albicans is a major cause of opportunistic and life-threatening sys- Dentistry, University of Otago, PO Box 647, temic fungal infections, especially in the immunocompromised. The plasma Dunedin 9054, New Zealand. Tel.: +64 3 479 7080; fax: +64 3 479 7078; e-mail: membrane proton-pumping ATPase (Pma1p) is an essential enzyme that gen- [email protected] erates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening Received 11 October 2012; revised 29 for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave January 2013; accepted 30 January 2013. clones expressing CaPma1p that grew slowly at low pH. CaPma1p was Final version published online 15 March expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and DOI: 10.1111/1567-1364.12035 low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric sup- Editor: Jens Nielsen pressor mutants that expressed functional CaPma1p with improved H -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants Keywords identified specific amino acids (between 531 and 595 in CaPma1p) that may yeast; PMA1; plasma membrane H -ATPase; affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified proton pump; antifungal drug target. CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and struc- tural resolution. minimize the risk of host toxicity (Monk & Perlin, 1994). Introduction Fungal Pma1p has been validated as an antifungal drug Candida albicans remains the dominant cause of both target, but has yet to provide drugs used in the clinic opportunistic and life-threatening systemic fungal infec- (Monk et al., 1995, 2005; Perlin et al., 1997; Billack et al., tions, especially in the immunocompromised (Pfaller & 2010). In addition, a structure of Pma1p from a yeast or Diekema, 2007). P-type ATPases are widely used as drug fungal pathogen at a resolution that would aid drug targets for specific interventions in a diverse range of discovery has yet to be determined. human diseases (Yatime et al., 2009). In fungi, the plasma Although the Arabidopsis thaliana AHA2 plasma mem- membrane proton-pumping ATPase (Pma1p) is an essen- brane proton pump (Pedersen et al., 2007) and human 2+ tial enzyme that generates the electrochemical gradient and rabbit Ca -ATPase have been structurally resolved required for nutrient uptake and ionic homeostasis to 3.6 and 2.15 A, respectively (Toyoshima, 2008; Toyo- (Serrano et al., 1986). The synthesis of Pma1p is tightly shima et al., 2011), the only structure available for a fungal transcriptionally regulated, and its activity is controlled P-type ATPase is a low-resolution (8 A) cryo-electron by pH and glucose via phosphorylation of its C-terminal microscopy-based model of Neurospora crassa Pma1p domain (Mason et al., 1998; Martin-Castillo & Portillo, (Auer et al., 1998). We believe that the heterologous 1999; Lecchi et al., 2007). Fungal P-type ATPases are expression of C. albicans Pma1p (CaPma1p) in the model sufficiently conserved within their membrane sector and yeast Saccharomyces cerevisiae should enable new screens ectodomain to provide a broad-spectrum antifungal target for Pma1p-specific inhibitors and structure-directed and differ enough from key human P-type ATPases to antifungal discovery by making cell growth dependent on ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 YEAST RESEARCH Expression of C. albicans Pma1p in S. cerevisiae 303 the target enzyme and by producing homogeneous plements, for the selection of mutants. For liquid assays, enzyme in the quantities needed for structural analysis. buffered CSMYP media (CSM supplemented with 0.1% Our preliminary studies suggested that heterologous yeast extract, 0.2% peptone) allowed cultures to grow to expression would be feasible (Mason et al., 1996). We higher cell densities. The haploid S. cerevisiae strain ADD found that a chimeric S. cerevisiae Pma1p containing (MMLY663, Table 1) used as an expression host (Lam- C. albicans transmembrane loops 1 + 2 and 3 + 4 gave ping et al., 2005, 2007) was derived from strain AD1-8u growth rates, growth yields, glucose-dependent proton- (Decottignies et al., 1998). pumping rates, acid-activated omeprazole sensitivities, salt tolerances, and antifungal sensitivities comparable with Yeast constructs the parental S. cerevisiae enzyme. These experiments demonstrated cross-species complementarity for this Yeasts were transformed using an Alkali–Cation transfor- combination of transmembrane loops. In contrast, single mation kit (Bio 101, Vista, CA). S. cerevisiae MMLY1019 heterologous transmembrane loops caused deleterious (ADD, DPMA2::kanMX4) was derived from ADD by gene phenotypes at either low pH or elevated temperature disruption using a cassette containing the recyclable kan- (Mason et al., 1996). The compatibility of other parts of MX4 marker flanked with loxP repeats and bordered with CaPma1p with the S. cerevisiae enzyme is not known. We ScPMA2-specific arms (Guldener et al., 1996). Transfor- have therefore explored the consequences of expressing mants were selected on CSM media containing geneticin CaPMA1 in place of ScPMA1 and identified structural (200 lgmL G418, Sigma). Transformation cassettes for features needed for Pma1p function. PMA1 replacement were created by fusion PCR using flanking primers, up to 4 DNA fragments containing 25–30 nucleotide overlaps, and KOD Hot Start DNA Materials and methods Polymerase (Novagen ). The CaPMA1 ORF was ampli- fied from C. albicans SC5314. Plasmid pDP100 (Seto- Yeast strains and yeast culture Young et al., 1994) was used as a template for the 584-bp The S. cerevisiae strains used in the study (Table 1) were fragment immediately upstream of the ScPMA1 ORF and grown in YPD medium (1% yeast extract, 2% peptone, a 650-bp fragment immediately downstream of the ORF. and 2% glucose). Synthetic complete supplement mixture The fragment containing the S. cerevisiae PGK1 termina- (CSM, Formedia, UK) containing 10 mM MES and tor plus the S. cerevisiae URA3 gene was amplified using 20 mM HEPES buffered to the indicated pH with Tris the pABC3 vector (Lamping et al., 2007) as template. was used for strain maintenance or, with appropriate sup- Transformants were selected on CSM-Ura dropout med- ium. The sequences of the ORFs, promoters, and down- stream sequences involved in strain construction were confirmed by DNA sequence analysis. Table 1. Saccharomyces cerevisiae strains used in this study. Source or Strain Genotype or description Parent reference Hygromycin B resistance MMLY663 MAT a, PDR1-3, his1, AD1-8u Lamping Yeast cells (200 lL, OD = 0.2, 6 9 10 CFU) 600 nm ADD Dyor1::hisG, et al. (2005) suspended in 5 mL of YPD containing 0.6% (w/v) agarose Dsnq2::hisG, at 49 °C were rapidly poured onto 20 mL of the same med- Dpdr10::hisG, TM ium solidified in a petri dish. Six-millimeter BBL paper Dpdr11::hisG, disks containing 400 nmole of hygromycin B were applied Dycf::hisG, Dpdr3::hisG, Dpdr5::hisG, to the set agarose surface. The plates were incubated at Dpdr15::hisG, 30 °C for 48 h, and the diameters of the growth inhibition Dura3::200 zones (W) were measured. Relative hygromycin B resis- MMLY1019 ADD, DPMA2:: kanMX4 ADD This study tance was calculated as W /W . (ADD) (strain x) MMLY1020 ADD, ADD This study DPMA1::CaPMA1-URA3 MMLY1022 MMLY1019, MMLY1019 This study Acid resistance DPMA1::CaPMA1-URA3 MMLY1021 ADD, MMLY1020 This study CSMYP medium was adjusted with either Tris or HCl to DPMA1::CaPMA1*-URA3 the indicated pH between 3.0 and 7.5. The medium MMLY1087 ADD, MMLY1020 This study (200 lL final volume per well) in 96-well microtiter DPMA1::CaPMA1*-URA3 plates was inoculated with cells in late logarithmic phase *chimeric gene, generated spontaneously (see Table 2, Fig. S1). to OD = 0.08. The optical densities of the cultures 600 nm FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 304 M.V. Keniya et al. were recorded after 24-h incubation at 30 °C with shaking Coomassie R250-stained polyacrylamide gels were (150 r.p.m.) using an EL-340 microplate reader (Bio- analyzed by MALDI-TOF mass spectrometry of tryptic TM Tek ). A final cell density of OD ~ 1.2 indicated fingerprints at the Centre for Protein Research of the 600 nm optimal growth. University of Otago. Glucose-dependent proton pumping Preparation of Pma1p The acidification of the media caused by glucose-depen- Plasma membrane samples (1 mg mL ) were treated + 1 dent H -ATPase activity in live cells was evaluated as with deoxycholate (DOC; 5 mg mL ) in the presence of described previously (Monk et al., 2005). In brief, cells soy phosphatidylcholine (1 mg mL , Sigma) at pH 7.2 grown in CSM at pH 6.5 to mid-log phase (OD for 10 min on ice, and the stripped membranes were pel- 600 nm ~3) were washed and starved overnight in distilled water leted at ~92 000 g for 45 min. The stripped membranes on ice. A microtiter plate assay of glucose-dependent pro- (0.5 mg mL ) were solubilized in 2 mM (5 9 CMC) ton pumping was performed using replicate samples at Zwittergent 3-14 (SB3-14, Sigma) at pH 6.5 in the pres- 30 °C, with the pH of the reaction mixture pre-adjusted ence of bovine brain lipid extract (6 mg mL , Folch to 5.0 with HCl. The incubation premix (150 lL) con- fraction III, Sigma). The solubilized Pma1p-containing tained 50 lL each of yeast cell suspension at vesicles were pelleted at 308 000 g for 16 h, and excess OD = 16, bromophenol blue at 140 lgmL , and lipid was removed by washing with the Zwittergent 600 nm either water or 200 mM KCl. After equilibration to room 3-14-containing buffer. temperature (21 °C), the reaction was initiated by adding 50 lLof8% D-glucose. The OD was measured at 590 nm Size exclusion chromatography 33-s intervals for 40 min using a Synergy 2 microplate TM reader (BioTek ), with the plate shaken prior to each Zwittergent-washed, Pma1p-enriched samples were sepa- measurement. The absorbance of bromophenol dye at rated by size exclusion chromatography using a Superdex TM 590 nm was used to monitor the pH between 5.0 and 200 10/300 (GE Healthcare) column on an ACTA FPLC 3.5. The maximum rate (MaxV) of glucose-dependent system at a flow rate of 0.5 mL min . The buffer con- proton pumping was determined using Gen5 software tained 4 mM (10 9 CMC) Zwittergent 3-14, 0.2 M KCl, TM ). (BioTek 0.3 M NaCl, 0.5 mM PMSF, and 1 tablet of Complete Mini EDTA-free protease inhibitor cocktail (Roche) per 100 mL, in 10 mM HEPES–HCl pH 7.2. Fractions recov- Plasma membrane preparation and analysis ered at an elution volume comparable with the ferritin Yeast cells were grown to early diauxic phase (OD = 5– standard (~440 kDa) were pooled, precipitated using ice- 600 nm 5.5) in CSMYP containing 10 mM MES–HEPES adjusted cold 80% acetone, and analyzed by SDS-PAGE with silver TM with Tris to pH 7.0. Harvested cells were resuspended (60 staining (Merril et al., 1981). The PageRuler Prestained –70 OD mL ) in ice-cold homogenizing medium Protein Ladder Plus (Fermentas) was used as a molecular 600 nm [20% (w/v) glycerol, 0.5 mM EDTA, 1 mM PMSF, weight standard. 50 mM Tris pH 7.5] containing 100 mM glucose. After incubation for 1 h at 4 °C, the pH was adjusted to 7.5, Pma1p ATPase activity TM and the cells were homogenized using a Beadbeater (BioSpec Products Inc, Bartlesville, OK). Crude mem- The ATPase activity and kinetic parameters (K and branes were pelleted by differential centrifugation, and an V ) of Pma1p in enriched plasma membrane max enriched plasma membrane preparation obtained after preparations were measured as previously described acid precipitation (Goffeau & Dufour, 1988). Protein (Monk et al., 1991a) as vanadate (100 lM)-sensitive and concentration was estimated using the Bradford assay oligomycin (20 lM)-insensitive phosphate release from (Bio-Rad, Richmond, CA) with bovine IgG as standard. 15 mM Mg–ATP at pH 6.5 and 7.5 at 30 °C. Plasma membranes dissolved at room temperature in SDS lysis buffer (2% SDS, 50 mM Tris-HC1 pH 6.7, 10% Homology modeling glycerol, 2.5 mM EDTA, 0.01% PMSF, 1 lgmL brom- ophenol blue, 40 mM dithiothreitol) were separated by Homology models of CaPma1p and ScPma1p were gener- SDS-PAGE (Laemmli, 1970). Photographic images of the ated using Modeller 9v9 (Eswar et al., 2006) using the gels were analyzed for Pma1p band density relative to the known structure of the P-type H -ATPase from A. thali- TM whole lane using UN-SCAN-IT gel V.6.1 software (Silk ana (PDB ID: 3B8C) (Pedersen et al., 2007) as a Scientific Corporation). Protein bands excised from template. Sequence alignments between the yeast proteins ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 305 and the template were carried out using T-coffee (Poirot Pma1p migrates as a multimer in size exclusion et al., 2003). Structural figures were prepared using chromatography PyMOL (The PyMOL Molecular Graphics System, version 1.5.0.4 Schrodinger, € LLC). Size exclusion chromatography in Zwittergent 3-14 showed that the three samples obtained from DOC- stripped S. cerevisiae plasma membranes by detergent Results and discussion extraction and washing gave broad peaks with mobilities comparable with ferritin (440 kDa; Fig. 2), which corre- Expression of CaPma1p in S. cerevisiae sponded to at least a tetrameric complex of Pma1p The S. cerevisiae strain ADD was chosen as an expression monomers. A peak corresponding to the predicted Pma1p host for C. albicans Pma1p because it lacks 7 ABC-type monomer (100 kDa, elution volume ~ 12–13 mL) was transporters responsible for the efflux of a wide range of not detected. Bands that matched the expected sizes of xenobiotics. The absence of these transporters was the H -ATPase monomers were detected in silver-stained expected to provide enhanced xenobiotic sensitivity dur- polyacrylamide gels of the Superdex 200 fractions that ing cell-based inhibitor screening, decrease the back- were eluted at 10.5–11.5 mL (Fig. 1b). ground of ATPase activities during in vitro drug screens for Pma1p inhibition, and minimize membrane protein Growth characteristics, hygromycin resistance, contamination during isolation of Pma1p. Heterologous and glucose-dependent proton pumping expression of CaPma1p in S. cerevisiae ADD gave a ~100-kDa band visualized by Coomassie staining of At pH 6.0  0.5, the S. cerevisiae ADD strain expressing SDS-PAGE separated, deoxycholate-extracted plasma CaPma1p (MMLY1022) had growth rates comparable membrane fractions (Fig. 1a). ScPma1p from MMLY1019 with ADD. In unbuffered YPD or unbuffered CSM agar, had a slightly lower mobility (99.6 kDa) than both the CaPma1p-expressing strain showed acid-sensitive CaPma1p (97.5 kDa) extracted from C. albicans SC5314 growth and gave smaller colonies than the unmodified and CaPma1p heterologously expressed in S. cerevisiae S. cerevisiae strain. Expression of the Nicotiana plumba- MMLY1021 (97.7 kDa). The Pma1p from MMLY1021 ginifolia PMA2 gene in S. cerevisiae gave a similar was found to be a chimera of CaPma1p and ScPma2p, as pH-sensitive phenotype (de Kerchove d’Exaerde et al., described below. The identities of the heterologously 1995). The pH sensitivity of the CaPma1p-expressing expressed Pma1ps were confirmed using MALDI-TOF strains correlated with modest expression of CaPma1p in mass spectrometry of the trypsin-digested ~100-kDa plasma membrane fractions detected by SDS-PAGE, low bands excised from the gels after SDS-PAGE (data not ATPase activity in vitro, and increased resistance to shown). hygromycin B compared with the ADD strain (Table 2). (a) (b) Fig. 1. SDS-PAGE analysis/gel of purified Pma1p. (a) Coomassie stained detergent- extracted Pma1p. (b) Silver stained size exclusion chromatography fractions eluting at 10.5–11.5 mL (~490 kDa, see Fig. 2). Lanes of both gels correspond to unmodified ScPma1p from Saccharomyces cerevisiae host strain MMLY 1019; unmodified CaPma1p from Candida albicans donor strain SC5314; and chimeric ScPma2p-CaPma1p from S. cerevisiae MMLY 1021, respectively, along with their theoretical molecular weights. Reference molecular weight markers (Fermentas TM PageRuler ) are shown. The Pma1p monomer is indicated by arrows. FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 306 M.V. Keniya et al. CaPma1p was only half as responsive to the addition of 200 mM KCl, confirming the limited capacity of these cells to produce an electrochemical gradient. Suppressor mutations resulting from recombination with PMA2 Suppressor mutants of MMLY1020 (MMLY1021 and MMLY1087), which showed enhanced growth rates at low pH, reduced resistance to hygromycin B, and KCl- stimulated glucose-dependent proton-pumping rates clo- ser to the native ScPma1p in ADD, were readily obtained as bigger colonies after overnight culture in YPD. DNA Fig. 2. Oligomeric Pma1p is observed during size exclusion sequence analysis of the Pma1p ORF from these mutants chromatography. Yeast strains used were MMLY1019; SC5314 and revealed multiple recombination events between CaPMA1 MMLY1021. O, M, location of Pma1p oligomers or monomers, and ScPMA2 (Table 2). ScPMA2 is a nonessential homo- respectively. The absorbance peak eluting at 15 mL gives a 35 kDa logue of ScPMA1 (Supporting Information, Table S1, band during SDS-PAGE. Fig. S1) that is weakly expressed under normal culture conditions (Supply et al., 1993). ScPma2p expressed under the control of the PMA1 promoter was found to Uptake of the protein synthesis inhibitor hygromycin B partially complement the essential activity of ScPma1p, and the size of the resultant growth inhibition zones in but some acid sensitivity remained. A similar phenome- agarose diffusion assays depend on the electrogenic activ- ity of Pma1p. The increased resistance of S. cerevisiae non of compensatory recombination was encountered strains expressing CaPma1p to hygromycin B indicated during heterologous expression of the N. plumbaginifolia reduced plasma membrane ATPase function (Table 2). PMA gene in S. cerevisiae (Harris et al., 1994; de Kercho- These results were confirmed by an in vivo ATPase ve d’Exaerde et al., 1995). assay measured as glucose-dependent proton efflux. Cells DNA sequence alignment showed that all CaPMA1 expressing CaPma1p acidified the medium at rates that suppressor isolates obtained in the present study had sub- were ~3 times lower than the control strain expressing stitutions from ScPMA2 between nucleotides 1521 and ScPma1p (ADD) (Fig. 3). For ADD, inclusion of 200 mM 1821 of CaPMA1. These resulted in up to 9 amino acid KCl in the assay abolished the membrane potential of the changes in two groups (group 1 and group 2 in Table 2) plasma membrane and doubled the net rate of glucose- within a region of 32 amino acid residues in the CaP- dependent proton pumping. The strain expressing ma1p primary sequence located in the C-terminal part of Table 2. Amino acid substitutions in CaPma1p–ScPma2p chimeric strains. aa in ScPma1p* Protein expression Hygromycin Acid † ‡ Strain (Pma) 554 580 582 587 593 597 617 618 624 level B resistance resistance ADD (ScPma1p) V N E G P L R V N High Low High 554 580 582 587 593 597 617 618 624 1.0 MMLY1022 (CaPma1p) V D D S A I N A S Low High Low 531 557 559 564 570 574 594 595 601 2.9 Group 1 Group 2 N E G P L R V N High Medium High MMLY1021 (chimera) I 531 557 559 564 570 574 594 595 601 1.8 MMLY1087 (chimera) V D D S P L R V N High Low High 531 557 559 564 570 574 594 595 601 1.1 ADD (ScPma2p) I N E G P L R V N 583 609 611 616 622 626 646 647 653 *Amino acid residues that are substituted in the CaPma1p–ScPma1p chimeras are compared by alignment with ScPma1p, ScPma2p, and CaPma1p. The Pma1p expression level was estimated using Coomassie blue-stained gels after SDS-PAGE of plasma membrane fractions. Hygromycin B resistance was expressed as the ratio of the diameters of the zones of inhibition. W /W . Lower resistance results in a (AD D) (strain x) larger zone of inhibition and a smaller ratio. Only I in ScPma2p is unique to this enzyme. ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 307 300 14.7% 9.9% 12.3% 13.2% 14.6% –KCl +KCl 600 571 86 85 200 168 pH 6.5 pH 7.5 Fig. 3. Pma1p-mediated proton pumping activity. The Pma1p Fig. 4. Relative content and ATPase activities of Pma1p in enriched enzyme activity expressed as MaxV (numbers shown above the bars membrane fractions of yeast at pH 6.5 and 7.5. Values are means of and with standard error indicated) was the average of at least three three assays  standard error. Ball: Statistically significant (P < 0.05) assays in the presence of a normal (KCl) or depleted (+KCl) difference between pH 6.5 and 7.5 pairs. Diamond: Statistically membrane potential. Ball: Statistically significant (P < 0.05) difference significant (P < 0.05) difference between heterologously expressed between KCl and +KCl pairs. Diamond: Statistically significant Pma1p and ScPma1p. Bands of Pma1p detected by SDS-PAGE are (P < 0.05) difference between heterologously expressed Pma1p and shown above the bars with relative amount of H -ATPase compared ScPma1p. with total protein (27–300 kDa) indicated. phosphorylation domain, according to AHA2 model and ScPma1p has been detected as functional > 400-kDa alignment (Pedersen et al., 2007). Strain MMLY1021 had oligomers in lipid rafts (Bagnat et al., 2001). Few crystal an additional V186I substitution in actuator domain structures of P-type ATPases are available, and modeling the sequence. Chimeric strain MMLY1087 had the least num- multimeric forms of ScPma1p or CaPma1p is a challenge. ber of amino acid replacements, with 5 ScPma2p-specific Cryo-electron microscopy of purified N. crassa Pma1p substitutions between amino acids 570 and 601 of showed it to be a hexamer (Auer et al., 1998), but the struc- CaPma1p, that is, it was CaPma1p apart from 5 amino ture (PDB ID: 1MHS) is only at 8 A resolution. Higher reso- acids in the C-terminal part of the phosphorylation lution structures are available for P-type ATPases from domain, which came from ScPMA2 (Table 2, Fig. S1). other kingdoms (< 3.6 A for A. thaliana AHA2 and 2.15 A The amino acid substitutions were identical in ScPma1p for Oryctolagus cuniculus SERCA1a), but these have signifi- and ScPma2p. These changes restored wild-type resistance cantly lower homology with the fungal Pma1ps and have to acidic media and gave increased susceptibility to hygro- substantially different C-terminal domains. Homology mycin B. In vitro ATPase activity and the enzyme V in models of ScPma1p and CaPma1p, based on the crystal max enriched plasma membrane fractions increased > 3-fold to structure of AHA2, indicate that the residues E596, R617, ~one-third of the value for CaPma1p fromCalbicans (Fig. 4, and N624 in ScPma1p and the homologous residues E573, Table S2). The relative contribution of the ~100 kDa Pma1p N594, and S601 in CaPma1p are exposed to the surface of a cytoplasmic domain (Fig. 5). Despite ScPma1p E596 and band to all membrane preparations was in the range of 10– 15%. The heterologously expressed unmodified CaPma1p CaPma1p E573 are conserved, the surface orientation of showed the lowest level of correctly trafficked ATPase (10%), this glutamate may be affected by steric clashes with the the chimeras showed intermediate levels (12–13%) and the nearby group 1 residues P593 and L597 in ScPma1p and native enzymes the highest levels (14–15%). The ATPase the corresponding A570 and I574 in CaPma1p. Although activity of chimeric ATPase from MMLY1021 remained low the low resolution of NcPma1p structure not enabling visu- despite five additional amino acid substitutions. The low alization of changing monomer–monomer interactions ATPase activity of MMLY1021 was consistent with the during the reaction cycle or effects due to regulatory modi- higher hygromycin resistance of this construct. fication, the model confirms that the corresponding N. crassa Pma1p residues E596, N617, and Q624 lie at the interface between subunits of a hexamer (Fig. 6, Fig. S2). It Homology modeling of CaPma1p and CaPma1p also shows that the two N. crassa amino acid residues –ScPma2p chimeras (N617 and Q624) with homology to the group 2 chimeric sub- Detergent-extracted ScPma1p and CaPma1p appear to stitutions are in close proximity of the group 2R C-terminal migrate as a multimer during size exclusion chromatography, domain amino acids 893–905 of an adjacent monomer. FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 MaxV + –1 –1 H –ATPase activity (nmol P min mg ) i 308 M.V. Keniya et al. The group 2R region contains charged and polar residues H -ATPase activities of yeast strains and a conserved glutamate (E903 in NcPma1p, E901 in ScPma1p) that is part of a phosphorylation recognition All tested forms of Pma1p were vanadate sensitive with site that regulates the kinetic properties and turnover of IC s2–9 lM (Table S2). Measurement of the vanadate- ATPase (Mason et al., 1998, 2006). Furthermore, an sensitive and oligomycin-insensitive H -ATPase activities 18-amino-acid C-terminal deletion of ScPma1p (E901 of enriched membrane fractions at pH 6.5 (Fig. 4) showed replaced with a stop codon) allowed the ATPase to be ScPma1p from S. cerevisiae ADD had significantly lower properly presented in the plasma membrane and retain ATPase activity than the enzyme from wild-type S. cerevisi- full activity, while a 38-amino-acid truncation (removing ae strain SH122 (de Kerchove d’Exaerde et al., 1995; the entire group 2R residues) was mistrafficked and Mason et al., 1998). This may be due to a D718N mutation 714 720 extensively degraded (Mason et al., 2006). Thus, the C- in the conserved DNSLDID motif between transmem- terminal domain after transmembrane segment 10 regu- brane segments 5 and 6 of ADD Pma1p. Homology model- lates ATPase turnover and activity. Our results therefore ing suggests that this mutation lies in a groove at the suggest that the residues involved in intermolecular con- extracellular vestibule of the proton pore. CaPma1p tacts between the subunits of CaPma1p may have poor expressed in S. cerevisiae MMLY1022 had a significantly compatibility with functional expression in S. cerevisiae at lower plasma membrane H -ATPase activity than CaP- low pH, resulting in modified enzyme activity, inappro- ma1p obtained directly from C. albicans (Fig. 4, Table S2). priate regulation, and protein mislocalization or enhanced All recombinant strains tested, which expressed native and degradation (Chang & Slayman, 1991). chimeric CaPma1p, showed an elevated activity at pH 7.5 closer to the optimum obtained for native CaPma1p (Mason et al., 1996). These results are consistent with the Improved functional expression of CaPma1p in proton-pumping assays described in Fig. 3 except for the S. cerevisiae MMLY1021 preparation, which may not be stable to mem- To heterologously express CaPma1p in S. cerevisiae with- brane purification and/or assay, despite Pma1p being out recombination with ScPMA2, the CaPMA1-URA3 cas- detected in high levels in membrane preparations (Fig. 4). sette obtained by PCR from strain MMLY1020 or the Post-translational C-terminal regulation strongly affects chimeric cassette from strain MMLY1021 was used to ScPma1p activity and turnover (Monk et al., 1991a, b; transform strain ADD DPMA2 (MMLY1019) to Ura . Mason et al., 1998). Glucose metabolism and/or acidifi- The growth rates, acid tolerance, hygromycin B resistance, cation of the cytosol (Eraso & Gancedo, 1987) trigger Pma1p expression, and glucose-dependent proton pump- phosphorylation of residue S899 in S. cerevisiae Pma1p, ing of the transformants were similar to that of their giving a decreased K and an increased V (Eraso & m max donor strain and, as expected, no suppressor mutants Portillo, 1994). Phosphorylation of other C-terminal resi- were obtained (data not shown). The properties of N- dues (E901, S911, T912) may also be involved in Pma1p terminal hexahistidine-tagged and nontagged variants regulation (Mason et al., 1996; Lecchi et al., 2007). Dele- were similar (data not shown). This observation will assist tion of the C-terminus (DE901–918) locks the enzyme in future structure–function studies. an active state (Mason et al., 1998). We hypothesize that Fig. 5. ScPma1p and CaPma1p homology models based on the Arabidopsis thaliana AHA2 structure. Side chain charge and polarity: Red – negative; blue – positive; orange – polar; cyan – neutral. The characteristic extracellular domains and labeled as the actuator (A), nucleotide binding (N) and phosphorylation (P). ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 309 Fig. 6. Neurospora crassa Pma1p (PDB ID: 1mhs) dimer interface. Top view displaying actuator (A) and nucleotide binding (N) domains of the enzymes. The monomers are coloured green or wheat. Marked residues are from the phosphorylation domain obscured by the nucleotide domain and are substitutions in CaPma1p-ScPma2p chimeras that may make intermolecular contacts. Side chain charge and polarity are as for Fig. 5. The C-terminal phosphorylation site residue E903 (equivalent to E901 in Saccharomyces cerevisiae)is coloured in magenta. Primed labels (i.e. A’; Group 1′) represent the same features on the adjacent monomer. group 1 and group 2 residues in CaPma1p provide a C-terminal regulatory domain (Mason et al., 1998). With pH-sensitive platform that promotes blockage of the these insights, we anticipate the design of yeast strains active site at low pH by the C-terminal domain. We expressing CaPma1p that will provide tools for targeted propose that this causes inappropriate regulation of antifungal screens. In addition, the overexpression of a CaPma1p when it is heterologously expressed in S. cerevi- suitable hexahistidine-tagged CaPma1p–ScPma2p chimera siae. The incorporation of two groups of Pma2p residues in S. cerevisiae may enable large-scale production of the allows the chimeric enzyme to work more efficiently than homogenous enzyme for in vitro assays and structural CaPma1p at low pH because the stringency of C-terminal analysis. regulation has been reduced. The conformation of the chimeras might also reduce mistrafficking and enzyme Acknowledgements turnover. This research was supported by NIH grants DE015075 and DE016885 and by the NZ Lottery Grants Board. Conclusion CaPma1p expressed in S. cerevisiae partially complements References the function of the essential, native enzyme ScPma1p at neutral pH. The recombinant enzyme causes acid-sensi- Auer M, Scarborough GA & Kuhlbrandt W (1998) Three- dimensional map of the plasma membrane H -ATPase in tive growth and hygromycin B resistance characteristic of the open conformation. Nature 392: 840–843. limited proton-pumping activity. These effects were Bagnat M, Chang A & Simons K (2001) Plasma membrane moderated by recombination with PMA2, giving rise to proton ATPase Pma1p requires raft association for surface CaPma1p–ScPma2p chimeras, which minimally contain 5 delivery in yeast. Mol Biol Cell 12: 4129–4138. amino acid substitutions that are conserved in ScPma1p Billack B, Pietka-Ottlik M, Santoro M, Nicholson S, and ScPma2p. Differences between native ScPma1p, het- Mlochowski J & Lau-Cam C (2010) Evaluation of the erologously expressed CaPma1p, and CaPma1p–ScPma2p antifungal and plasma membrane H -ATPase inhibitory chimeras did not affect oligomerization of these enzymes. action of ebselen and two ebselen analogs in S. cerevisiae The structural location of the suppressor mutations in cultures. J Enzyme Inhib Med Chem 25: 312–317. the chimeras and the biochemical properties of these Chang A & Slayman CW (1991) Maturation of the yeast enzymes suggest a role for localized interactions with the plasma membrane H -ATPase involves phosphorylation negative regulatory C-terminal domain near a key during intracellular transport. J Cell Biol 115: 289–295. phosphorylation site. The phenotypic effects of these de Kerchove d’Exaerde A, Supply P, Dufour JP, Bogaerts P, interactions may be overcome by simply removing the Thines D, Goffeau A & Boutry M (1995) Functional FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 310 M.V. Keniya et al. complementation of a null mutation of the yeast Mason AB, Allen KE & Slayman CW (2006) Effects of Saccharomyces cerevisiae plasma membrane H -ATPase by a C-terminal truncations on trafficking of the yeast + + plant H -ATPase gene. J Biol Chem 270: 23828–23837. plasma membrane H -ATPase. J Biol Chem 281: Decottignies A, Grant AM, Nichols JW, de Wet H, McIntosh 23887–23898. DB & Goffeau A (1998) ATPase and multidrug transport Merril CR, Dunau ML & Goldman D (1981) A rapid sensitive activities of the overexpressed yeast ABC protein Yor1p. silver stain for polypeptides in polyacrylamide gels. Anal J Biol Chem 273: 12612–12622. Biochem 110: 201–207. Eraso P & Gancedo C (1987) Activation of yeast plasma membrane Monk BC & Perlin DS (1994) Fungal plasma membrane ATPase by acid pH during growth. FEBS Lett 224: 187–192. proton pumps as promising new antifungal targets. Crit Rev Eraso P & Portillo F (1994) Molecular mechanism of Microbiol 20: 209–223. regulation of yeast plasma membrane H -ATPase by Monk BC, Kurtz MB, Marrinan JA & Perlin DS (1991a) glucose. Interaction between domains and identification of Cloning and characterization of the plasma membrane new regulatory sites. J Biol Chem 269: 10393–10399. H -ATPase from Candida albicans. J Bacteriol 173: Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, 6826–6836. Eramian D, Shen MY, Pieper U & Sali A (2006) Monk BC, Montesinos C, Ferguson C, Leonard K & Serrano R Comparative protein structure modeling using Modeller. (1991b) Immunological approaches to the transmembrane Curr Protoc Bioinformatics Chapter 5: Unit 5.6. topology and conformational changes of the carboxyl- Goffeau A & Dufour JP (1988) Plasma membrane ATPase terminal regulatory domain of yeast plasma membrane H - from the yeast Saccharomyces cerevisiae. Methods Enzymol ATPase. J Biol Chem 266: 18097–18103. 157: 528–533. Monk BC, Mason AB, Kardos TB & Perlin DS (1995) Guldener U, Heck S, Fielder T, Beinhauer J & Hegemann JH Targeting the fungal plasma membrane proton pump. Acta (1996) A new efficient gene disruption cassette for repeated Biochim Pol 42: 481–496. use in budding yeast. Nucleic Acids Res 24: 2519–2524. Monk BC, Niimi K, Lin S, Knight A, Kardos TB, Cannon RD, Harris SL, Na S, Zhu X, Seto-Young D, Perlin DS, Teem JH & Parshot R, King A, Lun D & Harding DR (2005) Surface- Haber JE (1994) Dominant lethal mutations in the plasma active fungicidal D-peptide inhibitors of the plasma membrane H -ATPase gene of Saccharomyces cerevisiae. membrane proton pump that block azole resistance. P Natl Acad Sci USA 91: 10531–10535. Antimicrob Agents Chemother 49:57–70. Laemmli UK (1970) Cleavage of structural proteins during Pedersen BP, Buch-Pedersen MJ, Morth JP, Palmgren MG & the assembly of the head of bacteriophage T4. Nature 227: Nissen P (2007) Crystal structure of the plasma membrane 680–685. proton pump. Nature 450: 1111–1114. Lamping E, Tanabe K, Niimi M, Uehara Y, Monk BC & Perlin DS, Seto-Young D & Monk BC (1997) The plasma Cannon RD (2005) Characterization of the Saccharomyces membrane H -ATPase of fungi. A candidate drug target? cerevisiae sec6-4 mutation and tools to create S. cerevisiae Ann NY Acad Sci 834: 609–617. strains containing the sec6-4 allele. Gene 361:57–66. Pfaller MA & Diekema DJ (2007) Epidemiology of invasive Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, Tanabe candidiasis: a persistent public health problem. Clin K, Niimi M, Uehara Y & Cannon RD (2007) Microbiol Rev 20: 133–163. Characterization of three classes of membrane proteins Poirot O, O’Toole E & Notredame C (2003) Tcoffee@igs: a web involved in fungal azole resistance by functional server for computing, evaluating and combining multiple hyperexpression in Saccharomyces cerevisiae. Eukaryot Cell 6: sequence alignments. Nucleic Acids Res 31: 3503–3506. 1150–1165. Serrano R, Kielland-Brandt MC & Fink GR (1986) Yeast Lecchi S, Nelson CJ, Allen KE, Swaney DL, Thompson KL, plasma membrane ATPase is essential for growth and has + + + 2+ Coon JJ, Sussman MR & Slayman CW (2007) Tandem homology with (Na + K ), K - and Ca -ATPases. Nature phosphorylation of Ser-911 and Thr-912 at the C terminus 319: 689–693. of yeast plasma membrane H -ATPase leads to Seto-Young D, Na S, Monk BC, Haber JE & Perlin DS (1994) glucose-dependent activation. J Biol Chem 282: 35471–35481. Mutational analysis of the first extracellular loop region of Martin-Castillo B & Portillo F (1999) Characterization of non- the H -ATPase from Saccharomyces cerevisiae. J Biol Chem dominant lethal mutations in the yeast plasma membrane 269: 23988–23995. H -ATPase gene. Biochim Biophys Acta 1417:32–36. Supply P, Wach A, Thines-Sempoux D & Goffeau A (1993) Mason AB, Kardos TB, Perlin DS & Monk BC (1996) Functional Proliferation of intracellular structures upon overexpression complementation between transmembrane loops of of the PMA2 ATPase in Saccharomyces cerevisiae. J Biol Saccharomyces cerevisiae and Candida albicans plasma Chem 268: 19744–19752. membrane H -ATPases. Biochim Biophys Acta 1284: 181–190. Toyoshima C (2008) Structural aspects of ion pumping by 2+ Mason AB, Kardos TB & Monk BC (1998) Regulation and Ca -ATPase of sarcoplasmic reticulum. Arch Biochem pH-dependent expression of a bilaterally truncated yeast Biophys 476:3–11. plasma membrane H -ATPase. Biochim Biophys Acta 1372: Toyoshima C, Yonekura S, Tsueda J & Iwasawa S (2011) 261–271. Trinitrophenyl derivatives bind differently from parent ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 311 2+ 2+ adenine nucleotides to Ca -ATPase in the absence of Ca . Fig. S1. CLUSTAL W multiple sequence alignment of P Natl Acad Sci USA 108: 1833–1838. CaPma1p, ScPma2p and chimeric proteins from Yatime L, Buch-Pedersen MJ, Musgaard M et al. (2009) P-type MMLY1087 and MMLY1021 strains using GONNET ATPases as drug targets: tools for medicine and science. weight matrix. Biochim Biophys Acta 1787: 207–220. Fig. S2. Neurospora crassa Pma1p (PDB ID: 1mhs) dimer interface. Table S1. PMA genes and predicted gene products from Supporting Information Saccharomyces cerevisiae and Candida albicans. Table S2. Kinetic properties from Saccharomyces cerevisiae Additional Supporting Information may be found in the and Candida albicans plasma membrane ATPases. online version of this article: FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Yeast Research Oxford University Press

Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae

Download PDF
10 pages

Loading next page...
 
/lp/oxford-university-press/heterologous-expression-of-candida-albicans-pma1p-in-saccharomyces-678pC0f94p

References (42)

Publisher
Oxford University Press
Copyright
© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
ISSN
1567-1356
eISSN
1567-1364
DOI
10.1111/1567-1364.12035
pmid
23374681
Publisher site
See Article on Publisher Site

Abstract

Department of Oral Sciences, School of Candida albicans is a major cause of opportunistic and life-threatening sys- Dentistry, University of Otago, PO Box 647, temic fungal infections, especially in the immunocompromised. The plasma Dunedin 9054, New Zealand. Tel.: +64 3 479 7080; fax: +64 3 479 7078; e-mail: membrane proton-pumping ATPase (Pma1p) is an essential enzyme that gen- [email protected] erates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening Received 11 October 2012; revised 29 for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave January 2013; accepted 30 January 2013. clones expressing CaPma1p that grew slowly at low pH. CaPma1p was Final version published online 15 March expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and DOI: 10.1111/1567-1364.12035 low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric sup- Editor: Jens Nielsen pressor mutants that expressed functional CaPma1p with improved H -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants Keywords identified specific amino acids (between 531 and 595 in CaPma1p) that may yeast; PMA1; plasma membrane H -ATPase; affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified proton pump; antifungal drug target. CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and struc- tural resolution. minimize the risk of host toxicity (Monk & Perlin, 1994). Introduction Fungal Pma1p has been validated as an antifungal drug Candida albicans remains the dominant cause of both target, but has yet to provide drugs used in the clinic opportunistic and life-threatening systemic fungal infec- (Monk et al., 1995, 2005; Perlin et al., 1997; Billack et al., tions, especially in the immunocompromised (Pfaller & 2010). In addition, a structure of Pma1p from a yeast or Diekema, 2007). P-type ATPases are widely used as drug fungal pathogen at a resolution that would aid drug targets for specific interventions in a diverse range of discovery has yet to be determined. human diseases (Yatime et al., 2009). In fungi, the plasma Although the Arabidopsis thaliana AHA2 plasma mem- membrane proton-pumping ATPase (Pma1p) is an essen- brane proton pump (Pedersen et al., 2007) and human 2+ tial enzyme that generates the electrochemical gradient and rabbit Ca -ATPase have been structurally resolved required for nutrient uptake and ionic homeostasis to 3.6 and 2.15 A, respectively (Toyoshima, 2008; Toyo- (Serrano et al., 1986). The synthesis of Pma1p is tightly shima et al., 2011), the only structure available for a fungal transcriptionally regulated, and its activity is controlled P-type ATPase is a low-resolution (8 A) cryo-electron by pH and glucose via phosphorylation of its C-terminal microscopy-based model of Neurospora crassa Pma1p domain (Mason et al., 1998; Martin-Castillo & Portillo, (Auer et al., 1998). We believe that the heterologous 1999; Lecchi et al., 2007). Fungal P-type ATPases are expression of C. albicans Pma1p (CaPma1p) in the model sufficiently conserved within their membrane sector and yeast Saccharomyces cerevisiae should enable new screens ectodomain to provide a broad-spectrum antifungal target for Pma1p-specific inhibitors and structure-directed and differ enough from key human P-type ATPases to antifungal discovery by making cell growth dependent on ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 YEAST RESEARCH Expression of C. albicans Pma1p in S. cerevisiae 303 the target enzyme and by producing homogeneous plements, for the selection of mutants. For liquid assays, enzyme in the quantities needed for structural analysis. buffered CSMYP media (CSM supplemented with 0.1% Our preliminary studies suggested that heterologous yeast extract, 0.2% peptone) allowed cultures to grow to expression would be feasible (Mason et al., 1996). We higher cell densities. The haploid S. cerevisiae strain ADD found that a chimeric S. cerevisiae Pma1p containing (MMLY663, Table 1) used as an expression host (Lam- C. albicans transmembrane loops 1 + 2 and 3 + 4 gave ping et al., 2005, 2007) was derived from strain AD1-8u growth rates, growth yields, glucose-dependent proton- (Decottignies et al., 1998). pumping rates, acid-activated omeprazole sensitivities, salt tolerances, and antifungal sensitivities comparable with Yeast constructs the parental S. cerevisiae enzyme. These experiments demonstrated cross-species complementarity for this Yeasts were transformed using an Alkali–Cation transfor- combination of transmembrane loops. In contrast, single mation kit (Bio 101, Vista, CA). S. cerevisiae MMLY1019 heterologous transmembrane loops caused deleterious (ADD, DPMA2::kanMX4) was derived from ADD by gene phenotypes at either low pH or elevated temperature disruption using a cassette containing the recyclable kan- (Mason et al., 1996). The compatibility of other parts of MX4 marker flanked with loxP repeats and bordered with CaPma1p with the S. cerevisiae enzyme is not known. We ScPMA2-specific arms (Guldener et al., 1996). Transfor- have therefore explored the consequences of expressing mants were selected on CSM media containing geneticin CaPMA1 in place of ScPMA1 and identified structural (200 lgmL G418, Sigma). Transformation cassettes for features needed for Pma1p function. PMA1 replacement were created by fusion PCR using flanking primers, up to 4 DNA fragments containing 25–30 nucleotide overlaps, and KOD Hot Start DNA Materials and methods Polymerase (Novagen ). The CaPMA1 ORF was ampli- fied from C. albicans SC5314. Plasmid pDP100 (Seto- Yeast strains and yeast culture Young et al., 1994) was used as a template for the 584-bp The S. cerevisiae strains used in the study (Table 1) were fragment immediately upstream of the ScPMA1 ORF and grown in YPD medium (1% yeast extract, 2% peptone, a 650-bp fragment immediately downstream of the ORF. and 2% glucose). Synthetic complete supplement mixture The fragment containing the S. cerevisiae PGK1 termina- (CSM, Formedia, UK) containing 10 mM MES and tor plus the S. cerevisiae URA3 gene was amplified using 20 mM HEPES buffered to the indicated pH with Tris the pABC3 vector (Lamping et al., 2007) as template. was used for strain maintenance or, with appropriate sup- Transformants were selected on CSM-Ura dropout med- ium. The sequences of the ORFs, promoters, and down- stream sequences involved in strain construction were confirmed by DNA sequence analysis. Table 1. Saccharomyces cerevisiae strains used in this study. Source or Strain Genotype or description Parent reference Hygromycin B resistance MMLY663 MAT a, PDR1-3, his1, AD1-8u Lamping Yeast cells (200 lL, OD = 0.2, 6 9 10 CFU) 600 nm ADD Dyor1::hisG, et al. (2005) suspended in 5 mL of YPD containing 0.6% (w/v) agarose Dsnq2::hisG, at 49 °C were rapidly poured onto 20 mL of the same med- Dpdr10::hisG, TM ium solidified in a petri dish. Six-millimeter BBL paper Dpdr11::hisG, disks containing 400 nmole of hygromycin B were applied Dycf::hisG, Dpdr3::hisG, Dpdr5::hisG, to the set agarose surface. The plates were incubated at Dpdr15::hisG, 30 °C for 48 h, and the diameters of the growth inhibition Dura3::200 zones (W) were measured. Relative hygromycin B resis- MMLY1019 ADD, DPMA2:: kanMX4 ADD This study tance was calculated as W /W . (ADD) (strain x) MMLY1020 ADD, ADD This study DPMA1::CaPMA1-URA3 MMLY1022 MMLY1019, MMLY1019 This study Acid resistance DPMA1::CaPMA1-URA3 MMLY1021 ADD, MMLY1020 This study CSMYP medium was adjusted with either Tris or HCl to DPMA1::CaPMA1*-URA3 the indicated pH between 3.0 and 7.5. The medium MMLY1087 ADD, MMLY1020 This study (200 lL final volume per well) in 96-well microtiter DPMA1::CaPMA1*-URA3 plates was inoculated with cells in late logarithmic phase *chimeric gene, generated spontaneously (see Table 2, Fig. S1). to OD = 0.08. The optical densities of the cultures 600 nm FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 304 M.V. Keniya et al. were recorded after 24-h incubation at 30 °C with shaking Coomassie R250-stained polyacrylamide gels were (150 r.p.m.) using an EL-340 microplate reader (Bio- analyzed by MALDI-TOF mass spectrometry of tryptic TM Tek ). A final cell density of OD ~ 1.2 indicated fingerprints at the Centre for Protein Research of the 600 nm optimal growth. University of Otago. Glucose-dependent proton pumping Preparation of Pma1p The acidification of the media caused by glucose-depen- Plasma membrane samples (1 mg mL ) were treated + 1 dent H -ATPase activity in live cells was evaluated as with deoxycholate (DOC; 5 mg mL ) in the presence of described previously (Monk et al., 2005). In brief, cells soy phosphatidylcholine (1 mg mL , Sigma) at pH 7.2 grown in CSM at pH 6.5 to mid-log phase (OD for 10 min on ice, and the stripped membranes were pel- 600 nm ~3) were washed and starved overnight in distilled water leted at ~92 000 g for 45 min. The stripped membranes on ice. A microtiter plate assay of glucose-dependent pro- (0.5 mg mL ) were solubilized in 2 mM (5 9 CMC) ton pumping was performed using replicate samples at Zwittergent 3-14 (SB3-14, Sigma) at pH 6.5 in the pres- 30 °C, with the pH of the reaction mixture pre-adjusted ence of bovine brain lipid extract (6 mg mL , Folch to 5.0 with HCl. The incubation premix (150 lL) con- fraction III, Sigma). The solubilized Pma1p-containing tained 50 lL each of yeast cell suspension at vesicles were pelleted at 308 000 g for 16 h, and excess OD = 16, bromophenol blue at 140 lgmL , and lipid was removed by washing with the Zwittergent 600 nm either water or 200 mM KCl. After equilibration to room 3-14-containing buffer. temperature (21 °C), the reaction was initiated by adding 50 lLof8% D-glucose. The OD was measured at 590 nm Size exclusion chromatography 33-s intervals for 40 min using a Synergy 2 microplate TM reader (BioTek ), with the plate shaken prior to each Zwittergent-washed, Pma1p-enriched samples were sepa- measurement. The absorbance of bromophenol dye at rated by size exclusion chromatography using a Superdex TM 590 nm was used to monitor the pH between 5.0 and 200 10/300 (GE Healthcare) column on an ACTA FPLC 3.5. The maximum rate (MaxV) of glucose-dependent system at a flow rate of 0.5 mL min . The buffer con- proton pumping was determined using Gen5 software tained 4 mM (10 9 CMC) Zwittergent 3-14, 0.2 M KCl, TM ). (BioTek 0.3 M NaCl, 0.5 mM PMSF, and 1 tablet of Complete Mini EDTA-free protease inhibitor cocktail (Roche) per 100 mL, in 10 mM HEPES–HCl pH 7.2. Fractions recov- Plasma membrane preparation and analysis ered at an elution volume comparable with the ferritin Yeast cells were grown to early diauxic phase (OD = 5– standard (~440 kDa) were pooled, precipitated using ice- 600 nm 5.5) in CSMYP containing 10 mM MES–HEPES adjusted cold 80% acetone, and analyzed by SDS-PAGE with silver TM with Tris to pH 7.0. Harvested cells were resuspended (60 staining (Merril et al., 1981). The PageRuler Prestained –70 OD mL ) in ice-cold homogenizing medium Protein Ladder Plus (Fermentas) was used as a molecular 600 nm [20% (w/v) glycerol, 0.5 mM EDTA, 1 mM PMSF, weight standard. 50 mM Tris pH 7.5] containing 100 mM glucose. After incubation for 1 h at 4 °C, the pH was adjusted to 7.5, Pma1p ATPase activity TM and the cells were homogenized using a Beadbeater (BioSpec Products Inc, Bartlesville, OK). Crude mem- The ATPase activity and kinetic parameters (K and branes were pelleted by differential centrifugation, and an V ) of Pma1p in enriched plasma membrane max enriched plasma membrane preparation obtained after preparations were measured as previously described acid precipitation (Goffeau & Dufour, 1988). Protein (Monk et al., 1991a) as vanadate (100 lM)-sensitive and concentration was estimated using the Bradford assay oligomycin (20 lM)-insensitive phosphate release from (Bio-Rad, Richmond, CA) with bovine IgG as standard. 15 mM Mg–ATP at pH 6.5 and 7.5 at 30 °C. Plasma membranes dissolved at room temperature in SDS lysis buffer (2% SDS, 50 mM Tris-HC1 pH 6.7, 10% Homology modeling glycerol, 2.5 mM EDTA, 0.01% PMSF, 1 lgmL brom- ophenol blue, 40 mM dithiothreitol) were separated by Homology models of CaPma1p and ScPma1p were gener- SDS-PAGE (Laemmli, 1970). Photographic images of the ated using Modeller 9v9 (Eswar et al., 2006) using the gels were analyzed for Pma1p band density relative to the known structure of the P-type H -ATPase from A. thali- TM whole lane using UN-SCAN-IT gel V.6.1 software (Silk ana (PDB ID: 3B8C) (Pedersen et al., 2007) as a Scientific Corporation). Protein bands excised from template. Sequence alignments between the yeast proteins ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 305 and the template were carried out using T-coffee (Poirot Pma1p migrates as a multimer in size exclusion et al., 2003). Structural figures were prepared using chromatography PyMOL (The PyMOL Molecular Graphics System, version 1.5.0.4 Schrodinger, € LLC). Size exclusion chromatography in Zwittergent 3-14 showed that the three samples obtained from DOC- stripped S. cerevisiae plasma membranes by detergent Results and discussion extraction and washing gave broad peaks with mobilities comparable with ferritin (440 kDa; Fig. 2), which corre- Expression of CaPma1p in S. cerevisiae sponded to at least a tetrameric complex of Pma1p The S. cerevisiae strain ADD was chosen as an expression monomers. A peak corresponding to the predicted Pma1p host for C. albicans Pma1p because it lacks 7 ABC-type monomer (100 kDa, elution volume ~ 12–13 mL) was transporters responsible for the efflux of a wide range of not detected. Bands that matched the expected sizes of xenobiotics. The absence of these transporters was the H -ATPase monomers were detected in silver-stained expected to provide enhanced xenobiotic sensitivity dur- polyacrylamide gels of the Superdex 200 fractions that ing cell-based inhibitor screening, decrease the back- were eluted at 10.5–11.5 mL (Fig. 1b). ground of ATPase activities during in vitro drug screens for Pma1p inhibition, and minimize membrane protein Growth characteristics, hygromycin resistance, contamination during isolation of Pma1p. Heterologous and glucose-dependent proton pumping expression of CaPma1p in S. cerevisiae ADD gave a ~100-kDa band visualized by Coomassie staining of At pH 6.0  0.5, the S. cerevisiae ADD strain expressing SDS-PAGE separated, deoxycholate-extracted plasma CaPma1p (MMLY1022) had growth rates comparable membrane fractions (Fig. 1a). ScPma1p from MMLY1019 with ADD. In unbuffered YPD or unbuffered CSM agar, had a slightly lower mobility (99.6 kDa) than both the CaPma1p-expressing strain showed acid-sensitive CaPma1p (97.5 kDa) extracted from C. albicans SC5314 growth and gave smaller colonies than the unmodified and CaPma1p heterologously expressed in S. cerevisiae S. cerevisiae strain. Expression of the Nicotiana plumba- MMLY1021 (97.7 kDa). The Pma1p from MMLY1021 ginifolia PMA2 gene in S. cerevisiae gave a similar was found to be a chimera of CaPma1p and ScPma2p, as pH-sensitive phenotype (de Kerchove d’Exaerde et al., described below. The identities of the heterologously 1995). The pH sensitivity of the CaPma1p-expressing expressed Pma1ps were confirmed using MALDI-TOF strains correlated with modest expression of CaPma1p in mass spectrometry of the trypsin-digested ~100-kDa plasma membrane fractions detected by SDS-PAGE, low bands excised from the gels after SDS-PAGE (data not ATPase activity in vitro, and increased resistance to shown). hygromycin B compared with the ADD strain (Table 2). (a) (b) Fig. 1. SDS-PAGE analysis/gel of purified Pma1p. (a) Coomassie stained detergent- extracted Pma1p. (b) Silver stained size exclusion chromatography fractions eluting at 10.5–11.5 mL (~490 kDa, see Fig. 2). Lanes of both gels correspond to unmodified ScPma1p from Saccharomyces cerevisiae host strain MMLY 1019; unmodified CaPma1p from Candida albicans donor strain SC5314; and chimeric ScPma2p-CaPma1p from S. cerevisiae MMLY 1021, respectively, along with their theoretical molecular weights. Reference molecular weight markers (Fermentas TM PageRuler ) are shown. The Pma1p monomer is indicated by arrows. FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 306 M.V. Keniya et al. CaPma1p was only half as responsive to the addition of 200 mM KCl, confirming the limited capacity of these cells to produce an electrochemical gradient. Suppressor mutations resulting from recombination with PMA2 Suppressor mutants of MMLY1020 (MMLY1021 and MMLY1087), which showed enhanced growth rates at low pH, reduced resistance to hygromycin B, and KCl- stimulated glucose-dependent proton-pumping rates clo- ser to the native ScPma1p in ADD, were readily obtained as bigger colonies after overnight culture in YPD. DNA Fig. 2. Oligomeric Pma1p is observed during size exclusion sequence analysis of the Pma1p ORF from these mutants chromatography. Yeast strains used were MMLY1019; SC5314 and revealed multiple recombination events between CaPMA1 MMLY1021. O, M, location of Pma1p oligomers or monomers, and ScPMA2 (Table 2). ScPMA2 is a nonessential homo- respectively. The absorbance peak eluting at 15 mL gives a 35 kDa logue of ScPMA1 (Supporting Information, Table S1, band during SDS-PAGE. Fig. S1) that is weakly expressed under normal culture conditions (Supply et al., 1993). ScPma2p expressed under the control of the PMA1 promoter was found to Uptake of the protein synthesis inhibitor hygromycin B partially complement the essential activity of ScPma1p, and the size of the resultant growth inhibition zones in but some acid sensitivity remained. A similar phenome- agarose diffusion assays depend on the electrogenic activ- ity of Pma1p. The increased resistance of S. cerevisiae non of compensatory recombination was encountered strains expressing CaPma1p to hygromycin B indicated during heterologous expression of the N. plumbaginifolia reduced plasma membrane ATPase function (Table 2). PMA gene in S. cerevisiae (Harris et al., 1994; de Kercho- These results were confirmed by an in vivo ATPase ve d’Exaerde et al., 1995). assay measured as glucose-dependent proton efflux. Cells DNA sequence alignment showed that all CaPMA1 expressing CaPma1p acidified the medium at rates that suppressor isolates obtained in the present study had sub- were ~3 times lower than the control strain expressing stitutions from ScPMA2 between nucleotides 1521 and ScPma1p (ADD) (Fig. 3). For ADD, inclusion of 200 mM 1821 of CaPMA1. These resulted in up to 9 amino acid KCl in the assay abolished the membrane potential of the changes in two groups (group 1 and group 2 in Table 2) plasma membrane and doubled the net rate of glucose- within a region of 32 amino acid residues in the CaP- dependent proton pumping. The strain expressing ma1p primary sequence located in the C-terminal part of Table 2. Amino acid substitutions in CaPma1p–ScPma2p chimeric strains. aa in ScPma1p* Protein expression Hygromycin Acid † ‡ Strain (Pma) 554 580 582 587 593 597 617 618 624 level B resistance resistance ADD (ScPma1p) V N E G P L R V N High Low High 554 580 582 587 593 597 617 618 624 1.0 MMLY1022 (CaPma1p) V D D S A I N A S Low High Low 531 557 559 564 570 574 594 595 601 2.9 Group 1 Group 2 N E G P L R V N High Medium High MMLY1021 (chimera) I 531 557 559 564 570 574 594 595 601 1.8 MMLY1087 (chimera) V D D S P L R V N High Low High 531 557 559 564 570 574 594 595 601 1.1 ADD (ScPma2p) I N E G P L R V N 583 609 611 616 622 626 646 647 653 *Amino acid residues that are substituted in the CaPma1p–ScPma1p chimeras are compared by alignment with ScPma1p, ScPma2p, and CaPma1p. The Pma1p expression level was estimated using Coomassie blue-stained gels after SDS-PAGE of plasma membrane fractions. Hygromycin B resistance was expressed as the ratio of the diameters of the zones of inhibition. W /W . Lower resistance results in a (AD D) (strain x) larger zone of inhibition and a smaller ratio. Only I in ScPma2p is unique to this enzyme. ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 307 300 14.7% 9.9% 12.3% 13.2% 14.6% –KCl +KCl 600 571 86 85 200 168 pH 6.5 pH 7.5 Fig. 3. Pma1p-mediated proton pumping activity. The Pma1p Fig. 4. Relative content and ATPase activities of Pma1p in enriched enzyme activity expressed as MaxV (numbers shown above the bars membrane fractions of yeast at pH 6.5 and 7.5. Values are means of and with standard error indicated) was the average of at least three three assays  standard error. Ball: Statistically significant (P < 0.05) assays in the presence of a normal (KCl) or depleted (+KCl) difference between pH 6.5 and 7.5 pairs. Diamond: Statistically membrane potential. Ball: Statistically significant (P < 0.05) difference significant (P < 0.05) difference between heterologously expressed between KCl and +KCl pairs. Diamond: Statistically significant Pma1p and ScPma1p. Bands of Pma1p detected by SDS-PAGE are (P < 0.05) difference between heterologously expressed Pma1p and shown above the bars with relative amount of H -ATPase compared ScPma1p. with total protein (27–300 kDa) indicated. phosphorylation domain, according to AHA2 model and ScPma1p has been detected as functional > 400-kDa alignment (Pedersen et al., 2007). Strain MMLY1021 had oligomers in lipid rafts (Bagnat et al., 2001). Few crystal an additional V186I substitution in actuator domain structures of P-type ATPases are available, and modeling the sequence. Chimeric strain MMLY1087 had the least num- multimeric forms of ScPma1p or CaPma1p is a challenge. ber of amino acid replacements, with 5 ScPma2p-specific Cryo-electron microscopy of purified N. crassa Pma1p substitutions between amino acids 570 and 601 of showed it to be a hexamer (Auer et al., 1998), but the struc- CaPma1p, that is, it was CaPma1p apart from 5 amino ture (PDB ID: 1MHS) is only at 8 A resolution. Higher reso- acids in the C-terminal part of the phosphorylation lution structures are available for P-type ATPases from domain, which came from ScPMA2 (Table 2, Fig. S1). other kingdoms (< 3.6 A for A. thaliana AHA2 and 2.15 A The amino acid substitutions were identical in ScPma1p for Oryctolagus cuniculus SERCA1a), but these have signifi- and ScPma2p. These changes restored wild-type resistance cantly lower homology with the fungal Pma1ps and have to acidic media and gave increased susceptibility to hygro- substantially different C-terminal domains. Homology mycin B. In vitro ATPase activity and the enzyme V in models of ScPma1p and CaPma1p, based on the crystal max enriched plasma membrane fractions increased > 3-fold to structure of AHA2, indicate that the residues E596, R617, ~one-third of the value for CaPma1p fromCalbicans (Fig. 4, and N624 in ScPma1p and the homologous residues E573, Table S2). The relative contribution of the ~100 kDa Pma1p N594, and S601 in CaPma1p are exposed to the surface of a cytoplasmic domain (Fig. 5). Despite ScPma1p E596 and band to all membrane preparations was in the range of 10– 15%. The heterologously expressed unmodified CaPma1p CaPma1p E573 are conserved, the surface orientation of showed the lowest level of correctly trafficked ATPase (10%), this glutamate may be affected by steric clashes with the the chimeras showed intermediate levels (12–13%) and the nearby group 1 residues P593 and L597 in ScPma1p and native enzymes the highest levels (14–15%). The ATPase the corresponding A570 and I574 in CaPma1p. Although activity of chimeric ATPase from MMLY1021 remained low the low resolution of NcPma1p structure not enabling visu- despite five additional amino acid substitutions. The low alization of changing monomer–monomer interactions ATPase activity of MMLY1021 was consistent with the during the reaction cycle or effects due to regulatory modi- higher hygromycin resistance of this construct. fication, the model confirms that the corresponding N. crassa Pma1p residues E596, N617, and Q624 lie at the interface between subunits of a hexamer (Fig. 6, Fig. S2). It Homology modeling of CaPma1p and CaPma1p also shows that the two N. crassa amino acid residues –ScPma2p chimeras (N617 and Q624) with homology to the group 2 chimeric sub- Detergent-extracted ScPma1p and CaPma1p appear to stitutions are in close proximity of the group 2R C-terminal migrate as a multimer during size exclusion chromatography, domain amino acids 893–905 of an adjacent monomer. FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 MaxV + –1 –1 H –ATPase activity (nmol P min mg ) i 308 M.V. Keniya et al. The group 2R region contains charged and polar residues H -ATPase activities of yeast strains and a conserved glutamate (E903 in NcPma1p, E901 in ScPma1p) that is part of a phosphorylation recognition All tested forms of Pma1p were vanadate sensitive with site that regulates the kinetic properties and turnover of IC s2–9 lM (Table S2). Measurement of the vanadate- ATPase (Mason et al., 1998, 2006). Furthermore, an sensitive and oligomycin-insensitive H -ATPase activities 18-amino-acid C-terminal deletion of ScPma1p (E901 of enriched membrane fractions at pH 6.5 (Fig. 4) showed replaced with a stop codon) allowed the ATPase to be ScPma1p from S. cerevisiae ADD had significantly lower properly presented in the plasma membrane and retain ATPase activity than the enzyme from wild-type S. cerevisi- full activity, while a 38-amino-acid truncation (removing ae strain SH122 (de Kerchove d’Exaerde et al., 1995; the entire group 2R residues) was mistrafficked and Mason et al., 1998). This may be due to a D718N mutation 714 720 extensively degraded (Mason et al., 2006). Thus, the C- in the conserved DNSLDID motif between transmem- terminal domain after transmembrane segment 10 regu- brane segments 5 and 6 of ADD Pma1p. Homology model- lates ATPase turnover and activity. Our results therefore ing suggests that this mutation lies in a groove at the suggest that the residues involved in intermolecular con- extracellular vestibule of the proton pore. CaPma1p tacts between the subunits of CaPma1p may have poor expressed in S. cerevisiae MMLY1022 had a significantly compatibility with functional expression in S. cerevisiae at lower plasma membrane H -ATPase activity than CaP- low pH, resulting in modified enzyme activity, inappro- ma1p obtained directly from C. albicans (Fig. 4, Table S2). priate regulation, and protein mislocalization or enhanced All recombinant strains tested, which expressed native and degradation (Chang & Slayman, 1991). chimeric CaPma1p, showed an elevated activity at pH 7.5 closer to the optimum obtained for native CaPma1p (Mason et al., 1996). These results are consistent with the Improved functional expression of CaPma1p in proton-pumping assays described in Fig. 3 except for the S. cerevisiae MMLY1021 preparation, which may not be stable to mem- To heterologously express CaPma1p in S. cerevisiae with- brane purification and/or assay, despite Pma1p being out recombination with ScPMA2, the CaPMA1-URA3 cas- detected in high levels in membrane preparations (Fig. 4). sette obtained by PCR from strain MMLY1020 or the Post-translational C-terminal regulation strongly affects chimeric cassette from strain MMLY1021 was used to ScPma1p activity and turnover (Monk et al., 1991a, b; transform strain ADD DPMA2 (MMLY1019) to Ura . Mason et al., 1998). Glucose metabolism and/or acidifi- The growth rates, acid tolerance, hygromycin B resistance, cation of the cytosol (Eraso & Gancedo, 1987) trigger Pma1p expression, and glucose-dependent proton pump- phosphorylation of residue S899 in S. cerevisiae Pma1p, ing of the transformants were similar to that of their giving a decreased K and an increased V (Eraso & m max donor strain and, as expected, no suppressor mutants Portillo, 1994). Phosphorylation of other C-terminal resi- were obtained (data not shown). The properties of N- dues (E901, S911, T912) may also be involved in Pma1p terminal hexahistidine-tagged and nontagged variants regulation (Mason et al., 1996; Lecchi et al., 2007). Dele- were similar (data not shown). This observation will assist tion of the C-terminus (DE901–918) locks the enzyme in future structure–function studies. an active state (Mason et al., 1998). We hypothesize that Fig. 5. ScPma1p and CaPma1p homology models based on the Arabidopsis thaliana AHA2 structure. Side chain charge and polarity: Red – negative; blue – positive; orange – polar; cyan – neutral. The characteristic extracellular domains and labeled as the actuator (A), nucleotide binding (N) and phosphorylation (P). ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 309 Fig. 6. Neurospora crassa Pma1p (PDB ID: 1mhs) dimer interface. Top view displaying actuator (A) and nucleotide binding (N) domains of the enzymes. The monomers are coloured green or wheat. Marked residues are from the phosphorylation domain obscured by the nucleotide domain and are substitutions in CaPma1p-ScPma2p chimeras that may make intermolecular contacts. Side chain charge and polarity are as for Fig. 5. The C-terminal phosphorylation site residue E903 (equivalent to E901 in Saccharomyces cerevisiae)is coloured in magenta. Primed labels (i.e. A’; Group 1′) represent the same features on the adjacent monomer. group 1 and group 2 residues in CaPma1p provide a C-terminal regulatory domain (Mason et al., 1998). With pH-sensitive platform that promotes blockage of the these insights, we anticipate the design of yeast strains active site at low pH by the C-terminal domain. We expressing CaPma1p that will provide tools for targeted propose that this causes inappropriate regulation of antifungal screens. In addition, the overexpression of a CaPma1p when it is heterologously expressed in S. cerevi- suitable hexahistidine-tagged CaPma1p–ScPma2p chimera siae. The incorporation of two groups of Pma2p residues in S. cerevisiae may enable large-scale production of the allows the chimeric enzyme to work more efficiently than homogenous enzyme for in vitro assays and structural CaPma1p at low pH because the stringency of C-terminal analysis. regulation has been reduced. The conformation of the chimeras might also reduce mistrafficking and enzyme Acknowledgements turnover. This research was supported by NIH grants DE015075 and DE016885 and by the NZ Lottery Grants Board. Conclusion CaPma1p expressed in S. cerevisiae partially complements References the function of the essential, native enzyme ScPma1p at neutral pH. The recombinant enzyme causes acid-sensi- Auer M, Scarborough GA & Kuhlbrandt W (1998) Three- dimensional map of the plasma membrane H -ATPase in tive growth and hygromycin B resistance characteristic of the open conformation. Nature 392: 840–843. limited proton-pumping activity. These effects were Bagnat M, Chang A & Simons K (2001) Plasma membrane moderated by recombination with PMA2, giving rise to proton ATPase Pma1p requires raft association for surface CaPma1p–ScPma2p chimeras, which minimally contain 5 delivery in yeast. Mol Biol Cell 12: 4129–4138. amino acid substitutions that are conserved in ScPma1p Billack B, Pietka-Ottlik M, Santoro M, Nicholson S, and ScPma2p. Differences between native ScPma1p, het- Mlochowski J & Lau-Cam C (2010) Evaluation of the erologously expressed CaPma1p, and CaPma1p–ScPma2p antifungal and plasma membrane H -ATPase inhibitory chimeras did not affect oligomerization of these enzymes. action of ebselen and two ebselen analogs in S. cerevisiae The structural location of the suppressor mutations in cultures. J Enzyme Inhib Med Chem 25: 312–317. the chimeras and the biochemical properties of these Chang A & Slayman CW (1991) Maturation of the yeast enzymes suggest a role for localized interactions with the plasma membrane H -ATPase involves phosphorylation negative regulatory C-terminal domain near a key during intracellular transport. J Cell Biol 115: 289–295. phosphorylation site. The phenotypic effects of these de Kerchove d’Exaerde A, Supply P, Dufour JP, Bogaerts P, interactions may be overcome by simply removing the Thines D, Goffeau A & Boutry M (1995) Functional FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 310 M.V. Keniya et al. complementation of a null mutation of the yeast Mason AB, Allen KE & Slayman CW (2006) Effects of Saccharomyces cerevisiae plasma membrane H -ATPase by a C-terminal truncations on trafficking of the yeast + + plant H -ATPase gene. J Biol Chem 270: 23828–23837. plasma membrane H -ATPase. J Biol Chem 281: Decottignies A, Grant AM, Nichols JW, de Wet H, McIntosh 23887–23898. DB & Goffeau A (1998) ATPase and multidrug transport Merril CR, Dunau ML & Goldman D (1981) A rapid sensitive activities of the overexpressed yeast ABC protein Yor1p. silver stain for polypeptides in polyacrylamide gels. Anal J Biol Chem 273: 12612–12622. Biochem 110: 201–207. Eraso P & Gancedo C (1987) Activation of yeast plasma membrane Monk BC & Perlin DS (1994) Fungal plasma membrane ATPase by acid pH during growth. FEBS Lett 224: 187–192. proton pumps as promising new antifungal targets. Crit Rev Eraso P & Portillo F (1994) Molecular mechanism of Microbiol 20: 209–223. regulation of yeast plasma membrane H -ATPase by Monk BC, Kurtz MB, Marrinan JA & Perlin DS (1991a) glucose. Interaction between domains and identification of Cloning and characterization of the plasma membrane new regulatory sites. J Biol Chem 269: 10393–10399. H -ATPase from Candida albicans. J Bacteriol 173: Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, 6826–6836. Eramian D, Shen MY, Pieper U & Sali A (2006) Monk BC, Montesinos C, Ferguson C, Leonard K & Serrano R Comparative protein structure modeling using Modeller. (1991b) Immunological approaches to the transmembrane Curr Protoc Bioinformatics Chapter 5: Unit 5.6. topology and conformational changes of the carboxyl- Goffeau A & Dufour JP (1988) Plasma membrane ATPase terminal regulatory domain of yeast plasma membrane H - from the yeast Saccharomyces cerevisiae. Methods Enzymol ATPase. J Biol Chem 266: 18097–18103. 157: 528–533. Monk BC, Mason AB, Kardos TB & Perlin DS (1995) Guldener U, Heck S, Fielder T, Beinhauer J & Hegemann JH Targeting the fungal plasma membrane proton pump. Acta (1996) A new efficient gene disruption cassette for repeated Biochim Pol 42: 481–496. use in budding yeast. Nucleic Acids Res 24: 2519–2524. Monk BC, Niimi K, Lin S, Knight A, Kardos TB, Cannon RD, Harris SL, Na S, Zhu X, Seto-Young D, Perlin DS, Teem JH & Parshot R, King A, Lun D & Harding DR (2005) Surface- Haber JE (1994) Dominant lethal mutations in the plasma active fungicidal D-peptide inhibitors of the plasma membrane H -ATPase gene of Saccharomyces cerevisiae. membrane proton pump that block azole resistance. P Natl Acad Sci USA 91: 10531–10535. Antimicrob Agents Chemother 49:57–70. Laemmli UK (1970) Cleavage of structural proteins during Pedersen BP, Buch-Pedersen MJ, Morth JP, Palmgren MG & the assembly of the head of bacteriophage T4. Nature 227: Nissen P (2007) Crystal structure of the plasma membrane 680–685. proton pump. Nature 450: 1111–1114. Lamping E, Tanabe K, Niimi M, Uehara Y, Monk BC & Perlin DS, Seto-Young D & Monk BC (1997) The plasma Cannon RD (2005) Characterization of the Saccharomyces membrane H -ATPase of fungi. A candidate drug target? cerevisiae sec6-4 mutation and tools to create S. cerevisiae Ann NY Acad Sci 834: 609–617. strains containing the sec6-4 allele. Gene 361:57–66. Pfaller MA & Diekema DJ (2007) Epidemiology of invasive Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, Tanabe candidiasis: a persistent public health problem. Clin K, Niimi M, Uehara Y & Cannon RD (2007) Microbiol Rev 20: 133–163. Characterization of three classes of membrane proteins Poirot O, O’Toole E & Notredame C (2003) Tcoffee@igs: a web involved in fungal azole resistance by functional server for computing, evaluating and combining multiple hyperexpression in Saccharomyces cerevisiae. Eukaryot Cell 6: sequence alignments. Nucleic Acids Res 31: 3503–3506. 1150–1165. Serrano R, Kielland-Brandt MC & Fink GR (1986) Yeast Lecchi S, Nelson CJ, Allen KE, Swaney DL, Thompson KL, plasma membrane ATPase is essential for growth and has + + + 2+ Coon JJ, Sussman MR & Slayman CW (2007) Tandem homology with (Na + K ), K - and Ca -ATPases. Nature phosphorylation of Ser-911 and Thr-912 at the C terminus 319: 689–693. of yeast plasma membrane H -ATPase leads to Seto-Young D, Na S, Monk BC, Haber JE & Perlin DS (1994) glucose-dependent activation. J Biol Chem 282: 35471–35481. Mutational analysis of the first extracellular loop region of Martin-Castillo B & Portillo F (1999) Characterization of non- the H -ATPase from Saccharomyces cerevisiae. J Biol Chem dominant lethal mutations in the yeast plasma membrane 269: 23988–23995. H -ATPase gene. Biochim Biophys Acta 1417:32–36. Supply P, Wach A, Thines-Sempoux D & Goffeau A (1993) Mason AB, Kardos TB, Perlin DS & Monk BC (1996) Functional Proliferation of intracellular structures upon overexpression complementation between transmembrane loops of of the PMA2 ATPase in Saccharomyces cerevisiae. J Biol Saccharomyces cerevisiae and Candida albicans plasma Chem 268: 19744–19752. membrane H -ATPases. Biochim Biophys Acta 1284: 181–190. Toyoshima C (2008) Structural aspects of ion pumping by 2+ Mason AB, Kardos TB & Monk BC (1998) Regulation and Ca -ATPase of sarcoplasmic reticulum. Arch Biochem pH-dependent expression of a bilaterally truncated yeast Biophys 476:3–11. plasma membrane H -ATPase. Biochim Biophys Acta 1372: Toyoshima C, Yonekura S, Tsueda J & Iwasawa S (2011) 261–271. Trinitrophenyl derivatives bind differently from parent ª 2013 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 302–311 Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018 Expression of C. albicans Pma1p in S. cerevisiae 311 2+ 2+ adenine nucleotides to Ca -ATPase in the absence of Ca . Fig. S1. CLUSTAL W multiple sequence alignment of P Natl Acad Sci USA 108: 1833–1838. CaPma1p, ScPma2p and chimeric proteins from Yatime L, Buch-Pedersen MJ, Musgaard M et al. (2009) P-type MMLY1087 and MMLY1021 strains using GONNET ATPases as drug targets: tools for medicine and science. weight matrix. Biochim Biophys Acta 1787: 207–220. Fig. S2. Neurospora crassa Pma1p (PDB ID: 1mhs) dimer interface. Table S1. PMA genes and predicted gene products from Supporting Information Saccharomyces cerevisiae and Candida albicans. Table S2. Kinetic properties from Saccharomyces cerevisiae Additional Supporting Information may be found in the and Candida albicans plasma membrane ATPases. online version of this article: FEMS Yeast Res 13 (2013) 302–311 ª 2013 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Downloaded from https://academic.oup.com/femsyr/article-abstract/13/3/302/541176 by Ed 'DeepDyve' Gillespie user on 13 April 2018

Journal

FEMS Yeast Research – Oxford University Press

Published: May 1, 2013

Keywords: yeast; PMA1; plasma membrane H + -ATPase; proton pump; antifungal drug target

There are no references for this article.