ilarity was analyzed by TFast A in combination with manual ... carried out by using the DNA Strider program (29). Isolation .... Titertek Microtiter Plus plate reader.
JOURNAL OF BACTERIOLOGY, Nov. 1991, p. 6826-6836
Vol. 173, No. 21
0021-9193/91/216826-11$02.00/0 Copyright © 1991, American Society for Microbiology
Cloning and Characterization of the Plasma Membrane H+-ATPase from Candida albicans BRIAN C. MONK,' MYRA B. KURTZ,2 JEAN A. MARRINAN,2 AND DAVID S. PERLIN1* Public Health Research Institute, 455 First Avenue, New York, New York 10016,1 and Antifungal Drug Discovery Group, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065_09002 Received 18 June 1991/Accepted 23 August 1991
The Candida albicans PMAI gene was isolated from a genomic library by using a hybridization probe obtained from the PMAI gene of Saccharomyces cerevisiae. The gene was localized to chromosome m of the Candida genome. An open reading frame of 2,685 nucleotides predicts an amino acid sequence of 895 amino acids that is 83% homologous at both the DNA and protein levels to its S. cerevisiae equivalent. A polyadenylated mRNA transcript of about 4,000 nucleotides contains a highly folded AU-rich leader of 242 nucleotides. The structure of the gene, codon bias, and levels of -100-kDa H+-ATPase protein recovered in plasma membranes indicate a highly expressed gene. The plasma membrane ATPase was purified to about 90% homogeneity and appeared to be blocked at the amino terminus. Three hydrophobic membrane sector tryptic fragments from the partially digested ATPase provided internal sequence information for over 50 amino acids, which agrees with the sequence predicted by the cloned gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the C. albicans enzyme is about 3 kDa smaller than its Saccharomyces counterpart and was consistent with a predicted M, of 97,398. Antibodies to the S. cerevisiae whole ATPase or its carboxyl terminus bound to the C. albicans enzyme but with lower avidity. Kinetic analysis showed that the Candida and Saccharomyces ATPases respond to glucose activation-starvation in nonidentical fashions. The amino-terminal domain of the C. albicans ATPase is marked by a net deletion of 23 amino acids in comparison with the S. cerevisiae ATPase. These diferences maintain net charge, occur in nonconserved regions of fungal ATPases, and are sufficient to account for the observed difference in electrophoretic mobility between the two yeast ATPases.
The fungal plasma membrane H+-ATPase is an electrogenic proton pump with a Mr of -100 kDa that regulates intracellular pH, maintains ion balance, and generates the electrochemical proton gradient necessary for nutrient uptake (39, 44). The H+-ATPase is encoded by the PMAJ gene (47) and is a member of the extended family of the P-type ATPases that mediate cation transport. The PMA genes from several fungal species are highly conserved at both the DNA and amino acid levels, despite the long evolutionary history of the group (13, 15, 46, 47). The PMAI gene of Saccharomyces cerevisiae is essential (47), and molecular genetic manipulation of PMAI expression (37, 53) demonstrated that the enzyme regulates the rate of cell growth. Genetic perturbation of ATPase catalytic activity was found to confer cellular resistance to Dio-9 (52) and hygromycin B (35) and highlighted the role of the enzyme in intracellular pH regulation (38). Intracellular pH transients have been observed during morphogenesis of the human pathogen Candida albicans, and ATPase inhibitors have been used to block germ tube formation (51). These experiments, and related experiments with Dictyostelium discoideum (14), suggest that the plasma membrane H+-ATPase plays a role in morphogenic choice. The factors which determine morphogenic choice are important aspects in the pathogenic mechanism for C. albicans, since the blastoconidial morphology characterizes topical colonization, the mycelial form of the organism is required for tissue penetration, and both morphologies are generally observed in disseminated infections (34). The diploid opportunistic fungal pathogen C. albicans is of *
Corresponding author. 6826
increasing importance to modern medicine (34, 48). Those at greatest risk from life-threatening infection include immunocompromised individuals, such as victims of AIDS, cancer patients, transplant recipients, and neonates, while others with a predisposition to infection include patients undergoing antibiotic therapy, pregnant women, infants, intensive care patients with permanently implanted catheters, and burn victims. Candidal infections are frequently acquired in hospital environments and significantly extend the length of hospitalization (8). The limited efficacy, considerable side-
effects, and toxicity of existing therapeutic regimens, particularly for disseminated candidal infections, have prompted the search for new drugs targeted at the organism. The mammalian P-type ATPases are well established as therapeutic targets. Stomach acidity can be limited by drugs such as omeprazole, which inhibits gastric H+,K+-ATPase from the luminal acidic face of the membrane (41) and appears to bind to a cysteine residue located at the luminal end of transmembrane segment 3 of the enzyme (32). Heart attack is frequently treated with cardiac glycosides which specifically inhibit Na+,K+-ATPase by intercalating into the plasma membrane from outside the cell. The affinity of ouabain for Na+,K+-ATPase is governed by an extracytoplasmic sequence located between transmembrane segments 1 and 2 (40). These precedents, the genetic diversity of the extracytoplasmic faces of P-type ATPases, and the physiological role of fungal plasma membrane ATPase suggest C. albicans plasma membrane ATPase as a target for the biologically based rational design of soluble and highly specific antifungal agents. Drugs targeted at this enzyme can be expected to limit both fungal growth and morphogenic choice and therefore have potential as prophylactic agents.
VOL. 173, 1991
CLONING AND CHARACTERIZATION OF CANDIDA H+-ATPase
However, the plasma membrane ATPase of C. albicans has received limited attention at the biochemical and genetic levels (18, 19). As first steps towards obtaining genetic, structural, and functional information on the plasma membrane ATPase of C. albicans, we report on the molecular and immunological properties of the enzyme, describe a facile purification of the protein, and detail the cloning and characterization of the PMAI gene of the pathogenic fungus. MATERIALS AND METHODS Strains. Escherichia coli JM109 [recA supE44 endAl hsdRJ7 gyrA96 relAl thi A(lac-proAB) F(traD36 proAB+ lacPl lacZAM15)] and DH5 (recAl endAl gyrA96 thi hsdRJ7 supE44 relAl) were used for library construction, amplification, and subcloning. Yeast strains Y55 (S. cerevisiae HO gal3 MAL1 SUCI), B311 (C. albicans), and ATCC 10261 (C. albicans) were used in this study. Growth conditions. C. albicans wild-type strains B311 and ATCC 10261 and S. cerevisiae wild-type strain Y55 were grown at 25°C in YEPD (1% yeast extract, 2% peptone, 2% glucose) in vigorously aerated 11 flasks for use in biochemical and molecular biological studies. Uridine (50 p.g/ml) was used to supplement Candida ura3 mutants. Synthetic medium (SD) with the necessary supplements (49) was used as a defined medium. Southern analysis. Genomic DNA was isolated from C. albicans ATCC 10261 by the method of Sherman et al. (49). DNA (3 ,ug) was cut with the indicated restriction enzymes, separated by electrophoresis on a 1% agarose gel, and transferred to a nylon membrane (GeneScreen; DuPont-New England Nuclear). Prehybridization was performed in accordance with the manufacturer's recommendation. Hybridization was performed for 20 h at 60°C in a solution containing Denhardt's salts, 1% sodium dodecyl sulfate (SDS), 0.33 M NaCl, 10% dextran sulfate, 0.1% Na4P207, 0.5 M Tris-HCl (pH 7.5), and 0.1 mg of salmon sperm DNA per ml (28). The probes were labelled with 32P by the random-priming procedure of Feinberg and Vogelstein (11). After hybridization, membranes were washed at room temperature four times in 2x SSC (0.15 M NaCl, 0.015 M sodium citrate)-0.1% SDS for 5 min and twice at 600C with 0.1 x SSC-0.1% SDS for 15 min. Screening of genomic libraries. A library of C. albicans genomic DNA constructed from BamHI-HindIII-digested DNA isolated from strain WO-1 and inserted into the same sites in plasmid pEMBLY-23 was kindly provided by P. T. Magee (University of Minnesota). A total of 4,700 clones in E. coli were screened for homology to the S. cerevisiae PMAJ gene by colony hybridization on NEN Colony/Plaque Screen filters (New England Nuclear). Duplicate filters were probed with the 2.1-kb KpnI fragment of plasmid B1138, which contains the central part of the S. cerevisiae PMAI gene (kindly provided by G. Fink, Whitehead Institute; 47). The hybridization conditions were the same as for Southern analysis. One clone (1-10) was positive through all rounds of screening. A library enriched for the Candida plasma membrane H+-ATPase gene was constructed on the basis of the observation that the Candida and Saccharomyces probes hybridize to a single ClaI fragment of about 12 kb. A portion of C. albicans ATCC 10261 chromosomal DNA was completely digested with ClaI. A DNA fraction enriched for 10to 15-kb fragments was obtained by electroelution of the appropriate segment of electrophoretically separated digested DNA and ligated into ClaI-digested YEp24 (4). A total of 35,000 E. coli colonies transformed with the library
6827
was screened as described above, by using a 0.7-kb EcoRVEcoRI fragment from clone 1-10 as the probe. Six colonies reproducibly hybridized to the probe, and plasmids recovered from them all had the same restriction enzyme pattern. Restriction mapping and sequencing. Portions of the 12-kb ClaI insert of the positive YEp24 library were subcloned into Promega vectors pGEM3z(+) and pGEM5zf(+) for restriction mapping and sequencing. Sequencing was initiated with the internal EcoRI restriction fragment subcloned into pGEM3z(+) (pJAM25) and the internal EcoRV fragment subcloned into pGEM5zf(+) (pJAM27). Double-stranded sequencing with the T7 and SP6 primers (Promega), by using the method of Lim and Pene (26) and the Sequenase kit protocol (U.S. Biochemical Corp.), generated 250-base sequences from both ends of the 2.0-kb EcoRV Candida fragment. Regions not accessible with the T7 and SP6 primers were sequenced by using synthetic oligonucleotide primers (17-mers) based on the sequence acquired. Approximately 300 bases were read from each primer, and each newly synthesized primer overlapped the previous sequence by approximately 50 bases. Both strands of DNA were sequenced in their entirety, and all overlaps between partial sequences were established. Sequence analysis. University of Wisconsin Genetics Computer Group programs were used for sequence comparisons and protein secondary structure predictions. Sequence similarity was analyzed by TFast A in combination with manual alignment procedures. Other analyses of the sequence were carried out by using the DNA Strider program (29). Isolation of candida mRNA and Northern (RNA) analysis. Washed late-log-phase C. albicans cells were broken in a French press (20,000 lb/in2) and immediately extruded into 5 M guanidinium thiocyanate containing 10 mM EDTA, 8% (wt/vol) 2-mercaptoethanol, and 50 mM Tris-HCl (pH 7.8) (7). Total RNA was recovered by LiCi precipitation, extracted with phenol-chloroform, and precipitated with ethanol. The RNA was stored in 70% ethanol at -20°C until required for further purification or analysis. Poly(A) RNA was purified by using a poly(dT)-based purification kit (Clontech). RNA samples for Northern analysis were denatured, mixed with ethidium bromide, separated in 1.2% agarose gels containing formaldehyde for 5 h, vacuum blotted to Nytran nylon filters for 1 h, and fixed with UV light for 5 min as described by Kroczek and Siebert (22). A DNA probe for Northern analysis was isolated as a 1.2-kB AvaIEcoRV fragment of pJAM25 by agarose gel electrophoresis and electroelution. The 1.2-kB fragment was labeled with [a-32P]dCTP by using a random primer extension kit obtained from Pharmacia. Oligonucleotides were prepared in the Public Health Research Institute Microchemical Facility and end labeled by using T4 polynucleotide kinase and [y-32P]ATP. The 1.2-kb probe was hybridized with the filter at 65°C in the presence of 6x SSC-5x Denhardt's salts0.1% SDS-10 mM EDTA-100 ,ug of sheared and denatured herring sperm DNA per ml. After overnight incubation, the filter was washed twice for 1 h each time at room temperature with 2x SSC-0.1% SDS and twice for 1 h each time at 650C with 0.1 x SSC-0.1% SDS. The same conditions were used for the oligonucleotide probes, except that the hybridization was carried out at 37°C and washes were for 5 min each, with two washes at room temperature and two at 37°C. Primer extension analysis of RNA. C. albicans total RNA (50 ,ug) was treated with RQ1 DNase (40 U), extracted with phenol-chloroform, and precipitated with ethanol. The RNA was suspended in a 16-pl volume containing 1.25 mM dithiothreitol, 10 mM MgCl2, 37.5 mM KCl, 62.5 mM Tris
6828
J. BACTERIOL.
MONK ET AL.
(pH 8), 40 U of RNasin (Promega), and 1 ,ul of a 5' 32P-labelled oligonucleotide probe (5 ng). The mixture was incubated at 65°C for 10 min and cooled slowly to 42°C, and 4 p.l of a 10 mM deoxynucleoside triphosphate mixture was added. Reverse transcriptase (37 U; Life Sciences) was added to the reaction mixture, which was then incubated at 42°C for 35 min. The reaction was stopped with 2 ,ul of 0.5 M EDTA. The mixture was made up to 50 ,ul with distilled water, and the nucleic acid was recovered after phenolchloroform extraction and ethanol precipitation. The sample was suspended in 20 ,u1 of TE buffer (28). One 10-,ul aliquot was treated with 2 U of RNase before dissolution in DNA sequencing gel loading buffer (Pharmacia). The primer extension samples were analyzed on a DNA sequencing gel in parallel with DNA sequencing samples initiated from pJAM25 by using the same primer. Chromosomal blots. Chromosomes from C. albicans ATCC 10261 were separated by the contour-clamped homogeneous electric field technique (27); the gel was stained with ethidium bromide, photographed, and transferred to Nytran (Schleicher & Schuell, Inc.); and the filter was subjected to Southern analysis by using probes specific for chromosome 7 (ARG57), chromosome 3 (ADE2), and the S. cerevisiae and C. albicans PMAI genes. The ARG57 gene was kindly provided by B. B. Magee (University of Minnesota). Filters were probed as described for Northern analysis. Plasma membrane isolation. Gradient-purified yeast plasma membranes were prepared by a modification of the method described by Serrano (45), except that cells were broken in a French pressure cell at a pressure of 19,500 lb/in2. Cells (late-log phase) were washed twice with distilled water and stored on ice prior to homogenization (glucosestarved cells) or vigorously aerated for 10 min at room temperature in homogenization medium supplemented with 4% glucose (glucose-metabolizing cells). The glucosestarved cells showed no basal medium acidification properties but rapidly acidified the medium following addition of glucose. It is assumed that these cells were efficiently starved of glucose. The homogenization medium contained 2.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 50 mM Tris, pH 7.5. Immediately after cell disruption, the homogenate was adjusted to pH 7.25 with 2 M Tris. After two centrifugations at 5,000 x g for 10 min, a crude membrane fraction was pelleted from the supernatant by centrifugation at 30,000 x g for 1 h. The crude membranes were suspended in 20% glycerol-1 mM EDTA-1 mM PMSF-10 mM Tris (pH 7.0) and washed once by centrifugation. Purified plasma membranes were recovered at the 53.5/43.5% (wt/wt) sucrose interface of a step gradient containing 1 mM EDTA and 10 mM Tris (pH 7.0) after centrifugation for 5 h at 35,000 rpm in an SW41 rotor. ATPase assays. Assays of plasma membrane ATPase activity were scaled down and conducted in microtiter plates. The basic ATPase assay medium contained 15 mM MgSO4 and 15 mM ATP in 50 mM MES-Tris buffer at pH 6.0 but also included 50 mM KNO3, 0.2 mM ammonium molybdate, and 5 mM NaN3 to eliminate possible contributions from residual vacuolar ATPase, nonspecific phosphatase, and mitochondrial ATPase activities, respectively. The 120-,ul reaction mixture was incubated for 15 min at 28°C, and the reaction was stopped by addition of 130 ,ul of a stopdeveloping reagent containing 1% SDS, 100 mM sodium molybdate, 0.6 M H2S04, and 0.8% ascorbate. After a 10 min of incubation at 25°C, the A620 was determined in a Titertek Microtiter Plus plate reader. The amount of phosphate liberated was estimated by using a linear standard
curve containing up to 100 nmol of Pi in the reaction mixture, and suitable blanks were used to correct for nonspecific hydrolysis. Protein was determined by the Bradford method with bovine gamma globulin as the standard (5). Antibodies and Western blot (immunoblot) analysis. Antibodies against the whole S. cerevisiae ATPase and its carboxyl-terminal domain were affinity purified and used to analyze Western blotted plasma membranes on nitrocellulose as described by Monk et al. (31). Plasma membrane proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 8% acrylamide gels in a Bio-Rad minigel system and transferred to nitrocellulose by semidry blotting using the buffers described by Dunn (10). Tryptic digestion of purified plasma membrane ATPase and recovery of membrane sector polypeptides. Gradient-purified plasma membranes (300 mg) were washed by pelleting in 20 mM HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.0) and extracted with 25 ml of 0.5% deoxycholate in the same buffer. A deoxycholate-insoluble membrane fraction of about 150 mg of protein was recovered by centrifugation at 100,000 x g for 45 min, suspended in 9 ml of 20% glycerol-1 mM EDTA-10 mM Tris (pH 7.5), and extracted with 9 ml of 2% octylglucoside in the same medium. Aliquots (3 ml) of the supernatant, recovered after centrifugation at 100,000 x g for 30 min, were applied to gradients comprising 2-ml steps of 70, 50, 45, and 35% (wt/vol) glycerol in 1 mM EDTA-10 mM Tris (pH 7.5) and centrifuged in an SW41 rotor (Beckman) at 39,000 rpm for 16 h. The -100-kDa ATPase band was localized by SDSPAGE. Fractions containing the most purified material were pooled, dialyzed against 2 liters of 50 mM ammonium bicarbonate overnight, and concentrated to a suitable small volume by vacuum centrifugation (SpeedVac; Savant). The -100-kDa ATPase recovered by this technique, although inactive, was about 90% homogeneous and reacted with antibodies prepared against the whole S. cerevisiae ATPase or its carboxyl-terminal domain. Purified plasma membrane ATPase (2 mg) in 2.2 ml of 50 mM ammonium bicarbonate at pH 7.8 was digested for 30 min at room temperature with 40 ,ug of sequencing grade chemically modified trypsin (Promega). The reaction was stopped by addition of 40 gl of 0.1 M PMSF. The samples were dispensed into 10 tubes and dried under vacuum. The pellets were combined and suspended in 1 ml of 0.75 M ammonium bicarbonate containing 1 mM PMSF and incubated on ice for 1 h. The insoluble material was recovered by centrifugation at 13,000 x g for 20 min. The pellet was suspended in 1 ml of the ammonium bicarbonate medium, distributed into four tubes, incubated for 15 min on ice, and centrifuged as described above. The pellets were stored at
-800C. Gas phase protein microsequencing. Ammonium bicarbonate-insoluble tryptic ATPase fragments (-100 p.g) were dissolved in 150 p.l of SDS lysis buffer (25) at 40C in the presence of 1 mM PMSF and 1 mM EDTA and separated by SDS-PAGE on 15% acrylamide gels by using the protocol of Kratzin et al. (21). Purified plasma membrane ATPase was separated by SDS-PAGE (25). BRL prestained molecular weight markers (Mr range, 14,000 to 200,000) and CNBrdigested myoglobin (21) were used as molecular weight markers as appropriate. Protein bands were electrotransferred to polyvinylidene difluoride membranes (Problot; Applied Biosystems Inc.) in 10 mM CAPS (pH 11)-10% methanol and stained with Coomassie blue R250 as described by Matsudaira (30). Strips containing suitably sized tryptic fragments or the intact 100-kDa ATPase molecule
CLONING AND CHARACTERIZATION OF CANDIDA H+-ATPase
VOL. 173, 1991 Kpn I Pvu II EcoR I EcoRV ii DraI Afiho 1Hind UI Hind Il BamH I
6829
EooRV
cerevisiae probe, which was consistent with a single hybridizing region. A BamHI-HindIII C. albicans library was screened with the 2.1-kb KpnI probe, and one clone, 1-10, hybridized reproducibly. Restriction mapping of the plasmid 5, 3 from this clone indicated that a strongly hybridizing 1-~~~~~~~~~~~~ isolated 1 Kb 0 2 1.9-kb EcoRV fragment detected in the Southern analysis 3 was not contained within the clone. Since a second screening FIG. 1. Restriction map of the C. albicans PMAI gene. The of the library with a probe prepared from 1-10 did not positions of selected restriction enzyme sites are shown on a 3.25-kb produce clones with the missing portions, another library segment of DNA. The open box indicates the coding region of the PMAI gene. enriched for the ATPase was prepared. On the basis of the observation that a single 12-kb ClaI fragment hybridized to the S. cerevisiae probe, a size-selected ClaI library was prepared. It was screened with a probe consisting of a 0.7-kb were analyzed in a Porton Instruments PI 2090E gas phase EcoRV-EcoRI fragment from clone 1-10 which was expected microsequencer. Nucleotide sequence accession number. The nucleotide to be adjacent to the missing region. Six clones containing all sequence of the C. albicans PMAI gene has been submitted of the expected restriction fragments were obtained. The to GenBank and will appear under accession no. M74075. putative intact Candida PMAI gene, contained within the 12-kb ClaI fragment, was subjected to DNA sequence analas described in Materials and Methods. ysis RESULTS DNA sequence and predicted protein sequence. A partial restriction map for the C. albicans PMAI gene is shown in Cloning Candida PMAI. A 2.1-kb KpnI fragment from S. Fig. 1. The complete nucleotide sequence is shown in Fig. 2, cerevisiae PMAI was used as a probe in a Southern analysis together with the predicted amino acid sequence of the of genomic DNA from C. albicans ATCC 10261. Under longest open reading frame (2,685 bp), which encodes a normal-stringency conditions (see Materials and Methods), protein of 895 amino acids. The coding region initiates within the probe strongly hybridized to a single 12-kb ClaI fragment a typical eukaryotic ATG start and includes an adenosine at and two EcoRV fragments of 5.0 and 1.9 kb, respectively. Restriction digests of C. albicans genomic DNA produced nucleotide -3, a purine at +4, and a thymidine at +6 (1, 20). an approximate restriction map similar to that of the S. Termination occurs at a TAA stop codon, and within the first
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6830
J. BACTERIOL.
MONK ET AL.
FIG.
Isolation of the C. albicans 97-kDa
plasma membrane octylglucoside from deoxycholate-extracted plasma membranes and separated on a glycerol step gradient, and the ATPase was identified by SDS-PAGE analysis as described in Materials and Methods. Lanes: 1, plasma membranes; 2, octylglucoside-extracted deoxycholate-treated plasma membranes; 3, octylglucoside extract; 4 to 14, glycerol gradient fractions starting from the top of the gradient. Approximately 10 jgg of protein was loaded in lanes 1 and 2, while 5 g±l of each fraction 3.
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indicates the location of the
ATPase.
100 nucleotides downstream of this
site,
an
S.
cerevisiae
transcriptional termination site (TTTTTATA), a putative transcription termination site (IZIXTTATIAI AATT=), and a putative polyadenylation signal site (AA consensus
TAAT)
are
(2, 17, 55). In addition,
seen
region containing
a
28-nucleotide
24 nucleotides of
dyad symmetry (A[ GA-CAAIT) capable of producing a secondary structure is located between and slightly overlapping the putative transcription termination and poly(A) signals. The protein uses 42 of the 61 possible codons, giving a codon bias quantitatively similar to that of the highly expressed tubulin gene of C. albicans (50). The gene is 83%
mUGA
identical to the S. cerevisiae PMAJ when
maximally aligned
deletions and
an
gene at the DNA level
to take into account a set of three
insertion
near
the
beginning of
the
coding
region (see Discussion). A slightly lower level of identity (76%) was recorded with the S. cerevisiae PMA2 gene (43), a related gene of unknown function. At the protein level, the C. albicans and S. cerevisiae PMAI genes encode sequences that are 83% identical in the region of overlap and 92% similar when conservative substitutions are taken into account. Secondary-structure (12) and hydropathy (24) analyses beyond the extensively modified amino-terminal region produced profiles very similar to those observed for the S. cerevisiae PMAI gene (data not shown). Protein chemical analysis of the 100-kDa ATPase. Protein microsequencing was used to confirm that the cloned DNA encodes the highly abundant H+-ATPase enzyme found in C. albicans plasma membranes. The H+-ATPase was purified to greater than 90% purity by detergent extraction and glycerol gradient centrifugation (Fig. 3). Several attempts to sequence the intact purified 100-kDa enzyme (see Materials and Methods) following electrotransfer to polyvinylidene difluoride membranes were unsuccessful and raised the possibility that the N terminus of this protein is blocked. Since the amino terminus did not appear to be accessible to routine sequence analysis, internal sequence information was obtained after partial tryptic digestion, enrichment for hydrophobic fragments, separation on low-molecular-weight resolving SDS-polyacrylamide gels, and electrotransfer to polyvinylidene difluoride. The principal fragments were 21, 17, 12, and 7 kDa in size and were comparable to fragments obtained by partial digestion of the S. cerevisiae enzyme with trypsin (data not shown). The results of gas phase microsequence analysis are presented in Table 1. The 21kDa band gave a single major sequence of 21 residues corresponding to amino acids S-637 to K-657 of the predicted protein sequence and amino terminal to putative transmembrane segment 5 (Fig. 2). The 12-kDa band contained a major sequence of 9 amino acid residues (A-249 to S-257) and a minor sequence of 12 amino acid residues (Y-77 to N-88) which are expected to be amino terminal to transmembrane segments 3 and 1, respectively. The fragments of 7 and 17 kDa both yielded major amino acid sequences of 24 amino acid residues (Y-77 to G-100) that reiterated and extended the region of the amino acid sequence given by a 12-kDa minor sequence. The sequences in these regions agreed precisely with the predicted amino acid sequence of the
TABLE 1. Amino acid sequences of internal tryptic fragments from C. albicans plasma membrane ATPasea Polypeptide size (kDa)
Sequence
21
Major sequence: SAADIVFLAPGLSAIIDALKT S. cerevisiae PMAI-PMA2: SAADIVFLAPGLSAIIDALKT
12
Major sequence: AALVNKA (S?) S. cerevisiae PMAI-PMA2: AALVNKAS Minor sequence: YGLNQMAEEQEN
7 17
Major sequence: YGLNQMAEEQENLVLKFVMFFV (G?)P Major sequence: YGLNQMAEEQENLVLKFVMFFVGP S. cerevisiae PMAI: YGLNQMADEKESLVVKFVMFFVGP S. cerevisiae PMA2: YGLNQMAEENESLIVKFLMFFVGP *******^*#* -*+ -**+******
a Internal tryptic fragments (see Materials and Methods) of pulified C. albicans H+-ATPase were recovered in a 0.75 M ammonium bicarbonate-insoluble fraction. The fragments were separated by SDS-PAGE, electrotransferred to polyvinylidene difluoride, and analyzed by gas phase microsequencing. Relationships to PMA genes of S. cerevisiae: *, identical to PMAI and PMA2; +, identical to PMAI and similar to PMA2; -, similar to PMAI and PMA2; similar to PMAI and identical to PMA2; #, similar to PMA2; -, dissimilar to both PMAI and PMA2.
CLONING AND CHARACTERIZATION OF CANDIDA H+-ATPase
VOL. 173, 1991
1 2 3 4
A.
5 6 7
6831
2 3 4 5 6
A T G C
U! ' B. 4000nt
RGLU..
26S-
U
c
F
GR CC
18S
-*
AR R RU
CRU
n CC
UC
RH
CU
abh
ir
C C '
RCRRFU
U
U
RR
A
R
U
CU
1200nt--* 900nt---> _
C
UU R
UR
co
U
R
RU
*n -
LU U
UR C
U
p
R
CR
a..
CCRRC
R
U
_,
U
UR
_
F;ENUodd UR
~
u ju
RU R
CU
UJRRUC U
R
A U
U
FIG. 4. Northern blot analysis of C. albicans poly(A) mRNA. Total mRNA and poly(A) mRNA were isolated, separated, and analyzed as described in Materials and Methods. Vector pJAM25 was digested to completion with EcoRV and AvaI and treated in parallel with the RNA fraction for electrophoresis. The pJAM25 digest was in lanes 1, 4, and 6; total mRNA was in lane 3; and poly(A) mRNA was in lanes 2, 5, and 7. The migration of 18S RNA (1,789 nucleotides; Rubsov et al. [42]) and 26S RNA (3,393 nucleotides; Veldman et al. [54]) in the total RNA fraction from C. albicans is as indicated. Molecular weights are estimated relative to the migration of X DNA markers and to S. cerevisiae 18S and 26S rRNAs. The following probes were applied: lanes 1, 2, and 3, oligonucleotide probe 1048/1064; lanes 4 and 5, oligonucleotide probe 175/191; and lanes 6 and 7, 1,200-nucleotide EcoRV-AvaI fragment from pJAM25.
Cancdid
UCARUR
URR
RNR
i
v
L;
U
U
;
U
U
CU R
U
A
CGU
R4
U
RC4U
U. HpR UrRU
c
cR U
R
CU
UU
U242
+ - ^ a C . UG C c
F'
~ C ~
cloned C. albicans PMAI gene and included three amino residues (Q-85, N-87, and L-91) that are not found in the S. cerevisiae PMAI and PMA2 sequences (Table 1). These data strongly support the contention that the cloned PMAJ gene of C. albicans is the dominant expressed form of the plasma membrane ATPase in this organism. Northern (RNA) analysis. Total mRNA and poly(A) RNA from strain B311 were subjected to Northern blot analysis (Fig. 4). The RNA blot was probed with 32P-labelled oligonucleotide probes complementary to nucleotides 175 to 191 (probe 175/191) and 1048 to 1064 (probe 1048/1064), which lie within the N-terminal region and highly conserved active (phosphorylation)-site region, respectively. In addition, we used a randomly primed stringent probe which consisted of an AvaI-EcoRV fragment that included the N-terminal portion of the C. albicans PMA1 gene and a small amount of plasmid pJAM25 sequence. This probe contained nucleotides encoding the first 176 amino acids of the coding region plus about 700 nucleotides 5' of the ATG start site and was chosen because it represents a region that is highly divergent among different PMAJ genes. Probes 175/191 and 1048/1064 hybridized most strongly, as expected, to the homologous fragments, respectively, from an AvaI-EcoRV digest of plasmid pJAM25 (Fig. 4). The observed olfset between the probes results from their different sequence specificities. The AvaI-EcoRV fragment probe gave a reactivity profile identical to that of probe 173/191. All of the probes detected
A
CC RU
GC
5.
FIG.
~
~
~
RU u C
u U~~~~24 cu
~
~
CU
Analysis of the leader in C. albicans
PMAI
mRNA. (A)
Identification of the mRNA start site was performed by primer extension analysis as described in
MIaterials
and Methods. The DNA
sequencing profile obtained with probe 175/191, equivalent to the sequence for the negative strand of pJAM25, is given on the left. Lanes 1 to 3 contained
0.5, 1.0, and 2.0
,ul,
respectively, of the
untreated primer extension mixture; lanes 4 to 6 contained 0.5, 1.0, and 2.0
,ul
of the RNase A-treated primer extension mixture. (B)
Folding of leader sequences, as described by Zuker (56), for fungal
PMAI
genes from C. albicans and S. cerevisiae.
a single transcript of about 4,000 nucleotides in the poly(A) RNA fraction. No other homologous poly(A) RNA species were detected by any of these probes. The apparently long mRNA tail (>800 nucleotides) is interesting, but its significance remains unclear. Primer extension. late-log-phase extension
C.
using
The
5'
end
albicans B311 probe
175/191.
of RNA extracted
from
was analyzed by primer
This
procedure
gave
an
RNase-resistant cDNA which had a mobility consistent with termination at 242 nucleotides upstream from the proposed initiation codon (Fig. 5A). The presence of a leader sequence of this size in the Candida 233- to 239-nucleotide
PMAI
gene is analogous to the
leader found for the S.
cerevisiae
PMAl gene (6). A TATA-like region is located 95 nucleo-
6832
J. BACTERIOL.
MONK ET AL.
%~ ..~
OR IR ~
t
,l
4~
9
FIG. 6. Chromosomal location of the C. albicans PMAJ gene. C. albicans chromosomes were separated by the contour-clamped homogeneous electric field method, transferred to Nytran, and probed as described in Materials and Methods. The following probes were employed: lane 2, 1.2-kb EcoRV-AvaI fragment of pJAM25; lane 3, 4-kb EcoRV-XbaI fragment of p1129 which includes the ARGS7 gene of C. albicans; lane 4, 2.5-kb EcoRV fragment of pJAM9 which includes the entire coding region of the ADE2 gene of C. albicans. Lane 1 shows the ethidium bromide profile of the separated C. albicans chromosomes.
tides upstream of the Candida message initiation site, and an RNA polymerase CAAT start site (TCAAT) begins 6 nucleotides upstream of the message start. The region between the message start and the TATA box includes a pyrimidinerich sequence similar to that found by Capieaux et al. (6) in the S. cerevisiae PMAI gene. This region in the coding strand of the Candida PMAI gene contains the sequence TTTTC repeated five times and may be a CT block, as found in many highly transcribed genes (9, 33). A hypothetical folding of the Candida PMAI leader in comparison with its Saccharomyces equivalent is given in Fig. 5B. This figure suggests that the two leaders fold extensively but dissimilarly. Chromosomal localization of the Candida PMAI gene. Separation of strain ATCC 10261 chromosomes by the contourclamped homogeneous electric field method gave the expected pattern of eight ethidium-stained bands (Fig. 6, lane 1) corresponding to the chromosomes designated by Magee et al. (27). Probing of the blot with the highly specific 1,200-bp AvaI-EcoRV fragment shows that the Candida PMAJ gene hybridizes with chromosome III (Fig. 6, lane 2). This chromosome, as expected, is recognized by chromosome III-specific probe ADE2 (Fig. 6, lane 4) but not by chromosome 7-specific probe Arg57 (lane 3). The HindIII fragment containing the entire S. cerevisiae PMAJ gene also hybridized with chromosome III (data not shown). Immunochemical properties of the Candida ATPase. Plasma membranes prepared from both S. cerevisiae and C. albicans are dominated by a major band with an Mr of -100,000. This band comprises 20 to 40% of the total protein seen in Coomassie blue-stained SDS-PAGE gels. In the C. albicans preparations, the level of the -100-kDa band increased as the cells went from the log to the stationary phase (Fig. 7A). The -100-kDa band in both the Candida and Saccharomyces plasma membranes bound prepared affinity-purified antibodies to the intact S. cerevisiae enzyme and its carboxylterminal sequence (Fig. 7B). The latter antibody requires the 18 carboxyl-terminal amino acids of S. cerevisiae ATPase
h#.
^*M
FIG. 7. Electrophoretic and immunochemical analysis of the C. albicans plasma membrane ATPase. (A) Growth curve and composition of plasma membranes during growth of C. albicans B311 on glucose medium. Plasma membranes (2-,ug aliquots) isolated after growth for the indicated times were separated by SDS-PAGE as described in Materials and Methods and stained with Coomassie blue. Lanes: 1, 15.7 h; 2, 17.6 h; 3, 19.5 h; 4, 22 h; 5, molecular size standards (29, 45, 68, 97, and 205 kDa). (B) Plasma membranes from S. cerevisiae diploid wild-type strain Y55 and C. albicans wild-type strains ATCC 10261 and B311 were separated by SDS-PAGE and stained with Coomassie blue (i) and then subjected to Western blot analysis with affinity-purified antibodies to the S. cerevisiae whole ATPase (ii) or a carboxyl-terminal portion (iii), as described in Materials and Methods. Aliquots of 2 ,ug of plasma membrane protein were separated in each lane; in mixed samples, 1 ,ug of each plasma membrane type was used. Lanes: 1, B311; 2, B311-Y55; 3, Y55; 4, Y55-ATCC 10261; 5, ATCC 10261; 6, molecular weight markers.
for binding (31). The reaction of the two antibodies with ATPase from C. albicans was 5- to 10-fold weaker than that with the S. cerevisiae enzyme. The antigenic band from C. albicans appeared to have a slightly greater mobility during SDS-PAGE than the species from S. cerevisiae (Fig. 7B). This was confirmed by mixing plasma membranes from S. cerevisiae Y55 with C. albicans wild-type strains ATCC 10261 and B311. The differential mobilities of the two ATPase bands were visualized by Coomassie staining of SDS-PAGE profiles and by Western blot analysis using affinity-purified whole-ATPase and carboxyl-terminal antibodies. The ATPases from both wild-type C. albicans strains have electrophoretic mobilities indicating relative masses about 3 kDa lower than that of their S. cerevisiae counterpart, which is in agreement with the predicted molecular weight of 97,398 based on the amino acid sequence. Effect of glucose metabolism-starvation on ATPase. Several recent studies indicate that the S. cerevisiae plasma membrane ATPase is regulated by the presence of glucose (36). Modification of the S. cerevisiae enzyme due to removal of
VOL. 173, 1991
CLONING AND CHARACTERIZATION OF CANDIDA H+-ATPase
TABLE 2. Kinetic properties of S. cerevisiae and C. albicans plasma membrane ATPases as a function of glucose metabolism-starvation' Cell type
S. cerevisiae
Glucose metabolism
(Mm)
Yes No
0.8 4.0
K
m
Vmax (,umol mg`1 min- 1)
vanadate
(>M)
pH 6.5/5.2 ratio of ATPase
4.0 0.5
6 15
1.48 0.64
Ki,
6833
A
S.c. P1MA2 S.c.PIA1 C.a.PMA1 S.p. P1181 N.c.PMA1
MSSTEAKQYKEKPSKEYLHASDGDDPANNSAASSSSSSSTSTSASSSAAAVPRKAAAAS 59 MTDT--------------------------SSSSSSSSASSVSANQPTQEKPAKTYDD MS-----------------------------------------ATEPTNEKVDKIVSD MADN--------------------------AGEYNDAEKNAPEQQAPPPQQPAHAAAP MA -------------------------Scp STNTESGKFDEKAAEAAAY AMINO TERMINAL EXTENSION
32 17 32 31
---AADDSDSDEDIDQLIDELQSNYGEGDESGEEEVRTDGVHAGQRVVPEKDLSTDPAYGLTSDEVARRRKKY 129 ---AASESSDDDDIDALIEELQSNHGVDDED--SDNDGPVAAGEARPVPEEYLQTDPSYGLTSDEVLKRRKKY 100 ------D--EDEEIDQLVADLQSNPGAGDEEEEEENDSSF-----KAVPEELLQTDPRVGLTDDEVTKRRKRY 77 --- -AQDDEPDDDIDALIEELFSEDVQEEQEDNDDA--P-AAGEAKAVPEELLQTDMNTGLTMSEVEERRKKY
98 OPK 1P0V0DDEDIDALIEDLESHDGHDAEEEEEEA-TPGG---RWPEDMLQTDTRVGLTSEEWQRRRKY 100 DELETION INSERTION DELETION
(TRYPSIN SENSITIVE TURN) a
C. albicans
Yes No
2.2 2.9
3.9 3.9
9 14
2.64 1.42
Conditions for glucose metabolism-starvation are described in Materials and Methods.
medium glucose results in a lowered Vmax, an increased Km for MgATP, reduced sensitivity to inhibition by vanadate, and an altered pH optimum (Table 2). The plasma membrane ATPase from C. albicans showed few of the kinetic properties attributed to the in vivo effects of glucose on the S. cerevisiae enzyme. For example, removal of glucose by washing Candida cells prior to plasma membrane isolation had no effect on the Vmax of the enzyme at pH 6.5. Its apparent Ki for vanadate was increased only 55%, and the Km for MgATP was not appreciably altered under these conditions (Table 2). These data suggest that the C. albicans enzyme responds weakly to glucose metabolism. The pH profile for the Candida enzyme was displaced in the alkaline pH range relative to the S. cerevisiae enzyme for cells undergoing either glucose metabolism or starvation (Table 2). Interestingly, the activity of the C. albicans enzyme continued to increase at pH 8.0, showing pH 8.0/pH 5.2 ratios of 3.62 for glucose-metabolizing cells and 1.71 for glucose-starved cells. This enhanced activity at pH 8.0 was insensitive to the mitochondrial F1-ATPase inhibitors azide (1 mM) and oligomycin (100 ,uM) but was nearly completely abolished by the plasma membrane ATPase inhibitors vanadate (100 ,uM) and diethylstilbestrol (100 ,uM). DISCUSSION
A C. albicans PMAI gene. A PMAJ gene from C. albicans which encodes the dominant expressed form of the plasma membrane proton pump was cloned from a genomic library, sequenced, and characterized. The gene specifies a major poly(A) mRNA of about 4,000 nucleotides, including a leader structure of 242 nucleotides. The gene encodes an 895-amino-acid protein of 97,398 Da. The Candida H+ATPase is 83% identical to its S. cerevisiae homolog and 92% similar when conservative substitutions are taken into account. These results suggest that both enzymes have very comparable secondary structures and should fold similarly. The major regions of amino acid diversity between the two sequences occur in the amino-terminal domain prior to transmembrane segment 1 (Fig. 8A), prior to a highly conserved region involved in ATP binding (38), and in the carboxyl-terminal domain after the final transmembrane segment of the enzyme. The two ATPases also differ in the predicted cell surface extracytoplasmic loops, particularly between transmembrane segments 3 and 4. There are no changes in residues conserved among the P-type ATPase family (44), and all other changes in the regions thought to be important for ATPase catalytic function are conservative. The S. cerevisiae and C. albicans PMA1 genes are both highly expressed. They encode major components of the
S.cerevIslae PMA1 (Amino acids 1-22) M T D T S S S S S S S S A S S V S A H Q P T ATGACTGATACATCATCCTCTTCATCATCCTCTTCAGCITCTTCTGTTT-AGCTCATCAGCCAACT ATGAGT GCTACTGAACCAACC M S A T E P T
C.albicans Pt141 (Amino acids 1-7)
C S.cerevlslae P0481 (amino acids 28-40) K T Y D D A A S E S S D D AAGACTTACGATGA GCTGCATCTGAATCTTTGACGAT TGA TGAAGAC AAAATCGTCTCCGA K I V S D D E D C.albicans P0A1 (amino acids 13-20) D
S.cerevlslae PMAI1 (Amino acids 53-64) N
H
G
V
D
D
E
D
S
D
N
D
AATCACGGTGTCUj^;GAAGACAGGATAACGAT AACTACGGTGAGGGTGAATCTGGTGGTACGTACT N
Y
G
E
G
D
E
S
G
E
E
E
V
R
T
S.cerevlslae Pf482 (amino acids 80-94)
AATCACGGTGTCi^:GAAGAC.i&uGATAACGAT N H G V G D E D E E E D N D Modified P14}1 product of reciprocal and non-reciprocal recombination AACCCAGGTGCTGGTGATGAAGAAGAAGAGGAGGAAAATGAC N P G A G D E E E E E E C.albicans PMA1 (amino acids 33-46)
N
D
FIG. 8. Amino-terminal region of C. albicans plasma membrane ATPase. (A) Comparison of amino-terminal sequences predicted from fungal PMA genes. Regions discussed in the text are underlined and identified. (B) Deletion in S. cerevisiae (S.c.) PMAI which accounts for the shortened amino-terminal extension in C. albicans (C.a.). (C) Double deletion mechanism in S. cerevisiae PMAI which accounts for an amino-terminal deletion in C. albicans. (D) Possible ancestral double recombination between S. cerevisiae PMAI and PMA2. S.p., S. pombe; N.c., N. crassa.
plasma membrane, have codon biases typical of highly expressed genes, and contain comparable upstream regulatory features, including a CT block. Further experimentation will be required to determine whether the Candida PMAI gene is regulated like its Saccharomyces counterpart by the UASRpc region that binds TUF (6). The downstream sequences of the C. albicans PMAI gene contain structures consistent with normal S. cerevisiae transcriptional termination and polyadenylation and include a structure with dyad symmetry that could cause pausing of polymerase II prior to transcription termination. However, the leader sequences of the poly(A) mRNAs specified by the two PMAI genes appear to fold differently, and this aspect may provide an explanation for the difficulties encountered in expressing the C. albicans PMAI gene in S. cerevisiae (23). Homology with related PMA genes and possible evolutionary relationships. It is somewhat surprising that such a high degree of DNA sequence conservation (83% homology in the region of overlap) is observed between the PMAI genes of C. albicans'and S. cerevisiae, since they represent ancient lineages that contain many highly divergent genes. While introns have been found in several other Candida genes and in the otherwise highly homologous Neurospora crassa PMAI gene, neither the Candida nor the Saccharomyces PMAI gene contains introns. It is tempting to speculate that maintenance of the C. albicans PMAI gene may be promoted by the conservative effects of a diploid genome and
6834
J. BACTERIOL.
MONK ET AL.
the lack of a sexual cycle in C. albicans. Deleterious mutations of the plasma membrane ATPase are strongly selected against in S. cerevisiae, and mitotic recombination within the diploid genome of C. albicans could well promote stability of this essential gene. Recent studies suggest that reciprocal recombination between PMAI genes in S. cerevisiae diploids also provide a suitable protective mechanism (16). In the haploid genome of S. cerevisiae, ectopic recombination between the PMAJ and PMA2 genes has been shown to conserve a functional PMAJ gene (16). Data obtained in the present study are consistent with the idea that there has been an exchange between ancestral PMAI and PMA2 alleles (see below). We do not know whether the C. albicans PMAJ alleles are naturally heterozygous or whether C. albicans carries a PMA2 gene. Amino-terminal deletions and insertions. The 5' coding region of the C. albicans PMAI gene has three deletions and an insertion relative to the S. cerevisiae PMAI gene (Fig. 8A). The PMAI genes of other fungal ATPases show major sequence differences at the deleted sites, including both deletions and additions (Fig. 8A). Deletion of the first 27 amino acids of the S. cerevisiae ATPase has no effect on enzyme activity, while deletion of the first 60 amino acids gives a product which is not targeted to the plasma membrane (36). The sequences deleted from the Candida enzyme are clearly nonessential to the targeting process. The first deletion in the C. albicans ATPase sequence, which occurs after amino acid 2, removes a serine-rich sequence of 15 amino acids. This deletion from the amino-terminal extension region is most simply accounted for by a single in-frame deletion of 45 nucleotides (Fig. 8B) or an equivalent out-offrame deletion commencing at an additional nucleotide downstream. A second deletion occurs in the C. albicans ATPase sequence (after amino acid residue 17), immediately prior to a highly conserved acidic stretch of residues (equivalent to amino acids 39 to 47 in the S. cerevisiae ATPase). Deletion of this further serine-rich sequence and the accompanying amino acid modification (C. albicans D18) can be accommodated by two short out-of-frame deletions of nine and six nucleotides in the S. cerevisiae PMAJ gene rather than by a single event (Fig. 8C). We are unable to invoke a simple genetic mechanism to explain the third deletion in C. albicans PMAI (residues 68 to 72 in S. cerevisiae PMAI ). At the biochemical level, the deletion converts a putative loop of 11 amino acid residues bounded by two prolines in the S. cerevisiae ATPase to a shorter turn of 6 amino acids in C. albicans which includes only one boundary proline. The PMA2 gene of S. cerevisiae maintains the length of the loop by using a dissimilar sequence to PMAI, dispensing with one of the bordering proline residues. Both the Schizosaccharomyces pombe and N. crassa ATPases retain the boundary prolines, but the loops are reduced by one and three residues, respectively. Conservation of net charge in the amino-terminal domain to transmembrane segment 1 may be an important feature of the S. cerevisiae and C. albicans ATPases. The S. cerevisiae PMAI gene encodes 27 acidic groups (D and E), 10 basic groups (K and R), and 2 histidine residues in this 115-aminoacid region. The S. cerevisiae PMA2 gene encodes 30 acidic residues, 13 basic residues, and 2 histidine residues within 143 amino acids (including a 26-amino-acid amino-terminal extension), while the C. albicans PMA1 gene encodes 28 acidic and 10 basic groups and no histidines in the equivalent 92-amino-acid region. A feature consistent with net charge maintenance is insertion of a pair of glutamic residues (E-41 and E-42) into the Candida PMAI sequence relative to
Saccharomyces PMAJ. A speculative mechanism that provides a suitable precursor to the present Candida PMAI gene is a pair of homologous recombination events involving one identical (six nucleotides) exchange and one nonidentical (nine nucleotides replace three nucleotides) exchange of genetic information between PMAI and PMA2 of S. cerevisiae (Fig. 8D). Interestingly, both the S. pombe and the N. crassa ATPases include acidic insertions at the analogous site. These sequences could have arisen in a similar fashion or, alternatively, the S. cerevisiae H+-ATPase sequence is derived from a common ancestor from which some acidic residues have been deleted. Regulation of the plasma membrane ATPase. The kinetics for ATP hydrolysis by the S. cerevisiae plasma membrane ATPase were significantly altered when glucose was removed from the cell medium. When similarly treated, the C. albicans ATPase was less responsive to altered metabolic flux than was the S. cerevisiae enzyme (Table 2). The carboxyl-terminal domain of the Saccharomyces ATPase is conformationally active (31) and is thought to interact directly with the active site (36). Several explanations may be advanced to account for the differential responses of the two enzymes to glucose metabolism. The regulatory mechanisms which govern the levels of protein phosphorylation, and are presumed to modify the carboxyl-terminal domain (38), may be less sensitive to metabolic changes in C. albicans than in S. cerevisiae. Alternatively, the differential responses of the two fungal enzymes may be governed by specific primary sequence differences which alter secondary structure in neighboring conserved catalytic elements. An understanding of the response of the C. albicans ATPase to metabolic change is an important preliminary to experimental analysis of the role of the ATPase in germ tube formation. In such studies, cells are usually experimentally starved for carbon and then induced to form germ tubes upon carbon source addition. Under these conditions, ATPase activity transients may provide or reflect morphogenic signals (14, 51). Disruption of a single homolog of Candida PMAJ is an important step towards assessing the role of the ATPase in growth and morphogenesis. The disrupted strains are more hygromycin B resistant (23), which is consistent with diminished ATPase function (35, 53). These experiments support the view that the plasma membrane ATPase of C. albicans has potential as a target for the development of antifungal agents. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant GM 38225 to D.S.P. and a grant from Merck Sharp & Dohme Research Laboratories to B.C.M. and D.S.P. We acknowledge the PHRI Microchemistry Facility and NSF grant DIR-8910043, which supported this facility. We thank T. Fulton for the Candida contour-clamped homogeneous electric field gel and F. Foor for providing instruction in DNA sequence analysis. REFERENCES 1. Ammerer, G., R. Hitzeman, F. Hagie, A. Barta, and B. D. Hail. 1981. Recombinant DNA, p. 185-197. In A. G. Walton (ed.),
Proceedings of the Third Cleveland Symposium on Macromolecules. Elsevier Scientific Publishing Co., Amsterdam. 2. Bennetzen, J. L., and B. D. Hail. 1982. The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase I. J. Biol. Chem. 257:3018-3025. 3. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen.
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CLONING AND CHARACTERIZATION OF CANDIDA H+-ATPase
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