Transport of Malic Acid and Other Dicarboxylic Acids Inthe Yeast

Transport of Malic Acid and Other Dicarboxylic Acids in the Yeast Hansenula anomala

MANUELA CORTE-REAL AND C. LEAO* Laboratory ofBiology, University of Minho, 4719 Braga Codex, Portugal Received 12 October 1989/Accepted 22 January 1990

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol -s- mg (dry weight)-l; Kn, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, a-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis- Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.

Recently, it was shown that the yeast Candida sphaerica the mid-exponential phase, centrifuged, washed twice with transports malate by a proton symport and that the symport ice-cold distilled water, and suspended in ice-cold distilled also accepts succinate, fumarate, oxaloacetate, and a-keto- water to a final concentration of about 25 mg (dry weight)/ml. glutarate (3). Earlier studies on the transport of malic acid in To estimate labeled malic or succinic acid uptake rates, 5-,u yeasts have provided evidence that the transport of this acid amounts of the yeast suspension were mixed in 10-ml conical is carrier mediated in Kluyveromyces lactis (13), Zygosac- centrifuge tubes with 35 ,ul of 0.1 M KH2PO4 buffer at pH charomyces bailii (1), and Schizosaccharomyces pombe (8) values between 3.0 and 5.5. After 2 min of incubation at 25°C and that it is not carrier mediated in Saccharomyces cerevi- in a water bath, the reaction was started by the addition of 10 siae (11). The subject, in addition to its academic interest, ,u of an aqueous solution of L-[U-_4C]malic acid or [2,3-14C] has a practical dimension, since L-malic acid and tartaric succinic acid at the desired concentration and stopped by acid are the principal organic acids in grape must and wine dilution with 5 ml of cold water. Sampling times for DL-malic (9) and since a microbiological deacidification process may acid-grown cells (active transport of dicarboxylate) were 0, include the use of yeasts which are able to degrade malic 5, and 10 s. Sampling times for glucose-grown cells (simple acid during wine fermentation. Hansenula anomala is one of diffusion of undissociated acid) were 15 and 30 s. The the yeast species which are often found on grapes and in reaction mixtures were filtered immediately through GF/C must; it is able to use L-malic acid and other acids of the filters (Whatman, Inc., Clifton, N.J.), washed on the filters Krebs cycle as carbon and energy sources. with 10 ml of ice-cold water, and counted in a scintillation fluid that contained 10% (wt/vol) naphthalene, 0.7% (wt/vol) MATERIALS AND METHODS 2,5-diphenyloxazole (PPO), and 0.3% (wt/vol) 1,4-bis-[2]-(5- Microorganism and growth conditions. H. anomala IGC phenyloxazolyl)benzene (POPOP) in 1,4-dioxane. Radioac- 4380 was maintained on a medium containing glucose (2%, tivity was measured with a liquid scintillation counter (Pack- wt/vol), peptone (1%, wt/vol), yeast extract (0.5%, wt/vol), ard Instrument Co., Inc., Rockville, Md.). and agar (2%, wt/vol). For growth under conditions of Uptake rates were also calculated from measurements of glucose repression, a mineral medium with vitamins and the proton uptake with a standard pH meter (PHM 62; 0.5% (wt/vol) glucose (12) was used at 25°C with mechanical Radiometer A/S, Copenhagen, Denmark) connected to a shaking. Derepressed conditions were obtained by substitut- flatbed Perkin-Elmer 024 recorder (The Perkin-Elmer Corp., ing 0.5% (wt/vol) DL-malic acid for glucose in the above Norwalk, Conn.). The pH electrode was immersed in a medium. water-jacketed chamber provided with magnetic stirring. To Measurements of uptake rates. Preliminary results showed the chamber were added 4.5 ml of 10 mM KH2PO4 and 0.5 that malic and succinic acids were accepted by the same ml of yeast suspension. The pH was adjusted to the desired carrier in DL-malic acid-grown cells. The uptake rates of value, and a baseline was obtained. The desired amount of dicarboxylates were measured by the use of L-[U-14C]malic dicarboxylic acid (adjusted to the experimental pH value) acid, [2,3-14C]succinic acid, or both; for economic reasons, was added, and the subsequent alkalinization was monitored the uptake of undissociated dicarboxylic acid was measured with the recorder. The initial uptake rate was calculated by the use of [2,3-14C]succinic acid. Cells were harvested in from the slope of the initial part of the pH trace. Calibration was performed with HCl. * Corresponding author Measurement of the intracellular volume. The intracellular 1109 1110 CORTE-REAL AND LEAO APPL. ENVIRON. MICROBIOL.

stirring. The reaction was started by the addition of 20 [L of 5.0 mM [2,3-'4C]succinic acid (about 2,000 cpm/nmol). At appropriate times, 10 pl was taken from the reaction mixture and filtered immediately through Whatman GF/C filters. The filters were washed three times with ice-cold water, and the cn radioactivity was counted as indicated above. The intracel- E lular concentration of succinic acid was calculated by using the value of the intracellular volume estimated as described 0 20 0l_--" above. E Estimation of amounts of glucose and L-malic acid. The 0.-~ amount of glucose was estimated by the glucose oxidase -> 10 ol_-0-, method (Test Combination; Boehringer GmbH, Mannheim, Federal Republic of Germany). The amount of L-malic acid was estimated by the enzymatic method previously de- 0 10 20 30 40 50 scribed (7). Calculations of concentrations. Concentrations of dicar- 1 ( mM)1 boxylates were calculated by the use of the Henderson- [Succina.te ] Hasselbach equation with the following pK values: succinic acid, pK1 = 4.18 and pK2 = 5.56; malic acid, pK1 = 3.50 and FIG. 1. Lineweaver-Burk plots of initial uptake rates of succinic pK2 = 5.05; fumaric acid, pK1 = 3.03 and pK2 = 4.54; acid and protons by malic acid-grown cells of H. anomala IGC 4380 oxaloacetic acid, pK1 = 2.56 and pK2 = 4.37; oa-ketoglutaric at pH 5.20 as a function of succinate concentration. Symbols: 0, acid, pK1 = 2.47 and pK2 = 4.68; citric acid, pK1 = 3.13, labeled succinic acid; 0, protons. pK2 = 4.76, and pK3 = 6.40; and isocitric acid, pK1 = 3.29, pK2 = 4.71, and pK3 = 6.40. volume was measured as previously described (4, 10). A RESULTS AND DISCUSSION value of 2.98 RI of intracellular water per mg (dry weight) of yeast was obtained for malic acid-grown cells. Characterization of the malate transport system of H. Measurement of succinic acid accumulation. DL-Malic acid- anomala. Proton signals were observed when malic or suc- grown cells (20 RI; 12 mg [dry weight]/ml) were added to 60 cinic acid was added to a suspension in weak buffer (pH 4.2) ,ul of 0.1 M KH2PO4 buffer (pH 5.0) and also to 60 ,ul of of cells that had been grown in DL-malic acid medium. buffer containing carbonyl cyanide m-chlorophenylhydra- Lineweaver-Burk plots of the initial rates of proton disap- zone (CCCP) and were incubated at 25°C with magnetic pearance calculated from the slopes of the proton signals, as well as plots of the initial rates of labeled malic or succinic acid, were linear as a function of the concentration of the acid. Figure 1 shows the results obtained for succinic acid 0 uptake. Similar results were obtained with L-malic acid. This indicates that the uptake mechanism obeyed Michaelis- Menten kinetics and suggests that the transport of either 100 l dicarboxylic acid was mediated by a saturable carrier,

808 /o 6.6 ~~~0 >1 50

CZ 40 D 60 E E _%% 30 6 40 E 20 C -> 10 20

10 20 30 40 50

20 40 60 80 nt1 (mM ) -1 1 (mM) [Succinatel [MALATE] FIG. 3. Lineweaver-Burk plots of initial uptake rates of labeled succinic acid at pH 5.50 as a function of succinate concentration. FIG. 2. Lineweaver-Burk plots of initial uptake rates of labeled Symbols: 0, absence of other dicarboxylic acids; O, presence of 0.9 malic acid at pH 5.0 as a function of malate concentration. Symbols: mM ox-ketoglutaric acid; O, presence of 0.9 mM D-malic acid; 0, *, absence of other dicarboxylic acids; 0, presence of 1 mM presence of 0.9 mM fumaric acid; A, presence of 5 mM oxaloacetic succinic acid. acid; *, presence of 0.9 mM L-malic acid. VOL. 56, 1990 MALIC ACID TRANSPORT IN H. ANOMALA 1111

A / 20 . o1 80

[Oxa(oacotate] ) 60 0.1 -.. 20 B 40 0 10 , / 0

xo~~~ -. E C) 20 (Ktogtutarato ] C$c-i 1*E 20 , C z /1 1 1 1 1 1 C5 0 10 80 _ B [ Fumarat]( Cl) 20 [ D 60 o0 ci 0 0 40 4j-) ---' [DMalate] (mMY) 20 / Qa Q 20 E 10 , 0

0 20 1.0 60 80 100 120 0 10 20 30 40 50 60

M )-1 Time (min) [L-Malate] (mM) FIG. 6. Accumulation of labeled succinic acid (O) by malic FIG. 4. Lineweaver-Burk plots of initial uptake rates of protons acid-grown cells of H. anomala IGC 4380 at pH 5.0. The initial by malic acid-grown cells of H. anomala IGC 4380 at pH 4.2 as a extracellular concentration of succinic acid was 1 mM. At the times function of dicarboxylate concentration. (A) Oxaloacetate; (B) indicated by arrows, samples of the suspension received the follow- a-ketoglutarate; (C) fumarate; (D) D-malate; (E) L-malate. ing: (A) cold succinic acid (0) and cold L-malic acid (0) to final concentrations of 10 mM; (B) CCCP (0) to a final concentration of 0.5 mM. In experiment B, CCCP was added (0) to the reaction possibly a proton symporter. Both dicarboxylates were mixture before the addition of labeled succinic acid. mutual competitive inhibitors (Fig. 2). From the plots of labeled acids, the following kinetic parameters were calculated: for malate, Vmax (pH 5.0), 0.2 ± 0.09 nmol * s-' mg (dry weight) of cells-'; Km (pH 5.0), 0.076 ± 0.006 mM; for succinate, Vmax (pH 5.0), 0.67 ± 0.1 nmol s-' mg (dry weight) of cells-1; Km (pH 5.0), 0.038 ± 0.005 mM. Results B are mean values ± standard deviations of three determina- 20 0 tions. L-Malic acid, D-malic acid, fumaric acid, oxaloacetic acid 10 and cx-ketoglutaric acid were competitive inhibitors of succinic acid transport at pH 5.50 (Fig. 3). This suggests that 40- these acids were transported by the same carrier that trans- 1 ported succinic and malic acid. Indeed, these dicarboxylates (M )-1 induced proton movements that followed Michaelis-Menten [ISOCITRATE] (mM) kinetics as a function of the concentrations of the acids (Fig. E 4). Maleic acid, malonic acid, oxalic acid, and L-tartaric acid 20 A o 0 TABLE 1. Michaelis constants of succinic acid transport as 10 a function of pH in H. anomala IGC 4380 for two H> .0o.-o hypothetical means of transport Km (mM) of transport of: 0 20 40 60 80 100 pH Anions Undissociatedacida

1 ( m m)~ 3.0 0.010 0.036 [CITRATE] 4.0 0.017 0.025 5.5 0.064 0.0024 FIG. 5. Lineweaver-Burk plots of initial uptake rates of protons 6.0 0.046 0.0023 by malic acid-grown cells of H. anomala IGC 4380 at pH 4.2 as a function of tricarboxylate concentration. (A) Citrate; (B) isocitrate. a Facilitated diffusion of undissociated acid. 1112 CORTE-REAL AND LEAO APPL. ENVIRON. MICROBIOL.

As reported earlier for C. sphaerica (3), the rapid efflux of - 1.0 r accumulated radioactive succinic acid that was observed in malic acid-grown cells after the addition of CCCP indicates 'rnL- that the dicarboxylic acid carrier of malic acid-grown cells of E H. anomala is a proton-dicarboxylate symporter that allows U- uphill transport and accumulation as a function of pH. To substantiate this hypothesis, estimates of the Michaelis 0 E Q5 constants of the transport of labeled succinic acid were obtained at several pH values (Table 1). The Km values ax between pH 3.0 and 6.0 varied by a factor of over 15 when calculated as the concentration of undissociated acid and by a factor of less than 6 when expressed as the concentration 0 .-- of succinate ions, suggesting that the negatively charged 0 .0 0 5.0 .0 7 .0 forms of the acid were transported and that therefore the 3w0 4.0 5.0 &0 7.0 observed proton movements represented proton symport activity. The capacity of the system, expressed as the Extracellular pH maximum transport velocity, was pH dependent (Fig. 7). It FIG. 7. Kinetic parameters, as a function of pH, of the proton- malate symport of H. anomala IGC 4380 with respect to succinic increased about twofold between pH 3.0 and 5.50, its opti- acid. Symbols: A, half-saturation constant (Kin); 0, transport ca- mum pH, and decreased steeply between pH 5.5 and 6.5. pacity (Vmax)- The affinity of the proton malate symport for succinate, expressed as Ki,, varied to the same extent. Cells of H. anomala grown in medium with either ethanol (0.5%, vol/ did not act as competitive inhibitors of succinic acid trans- vol) or glycerol (0.5% vol/vol) as the carbon source did not port, nor did they induce proton movements, indicating that transport succinic or L-malic acid, indicating that the dicar- these dicarboxylic acids were not transported by malic boxylic acid carrier was inducible. The growth ofthe yeast in acid-grown cells. Citric and isocitric acids also appeared to a medium containing glucose (0.1%, wt/vol) and DL-malic be accepted by a proton symport (Fig. 5). However, those acid (0.5%, wt/vol) was diauxic (Fig. 8). Transport of L-malic acids were not competitive inhibitors of succinic transport at acid accompanied by measurable proton movements devel- pH 5.50 by malic acid-grown cells, suggesting that citric and oped only after the glucose had been consumed, indicating isocitric acids were transported by a proton symport distinct that the system may be subject to glucose repression. As a from the succinic acid carrier. Transport of labeled succinic consequence, the malic acid uptake of H. anomala may be acid at pH 5.0 was accumulative (Fig. 6). After about 20 min, repressed during vinification because of high sugar concen- one-half of the accumulated radioactivity was nonmetabo- tration. At present, we are trying to isolate derepressed lized succinic acid, since added cold succinic or L-malic acid mutants of the yeast for the degradation of malic acid during induced counterflow to this extent (Fig. 6). This result is wine fermentation. another indication that malic acid and succinic acid used the Diffusion of undissociated acid. Glucose-grown cells lack- same camer. The accumulation ratio in terms of nonmetab- ing the malic acid carrier were still slightly permeable by the olized succinic acid was about 40. The protonophore CCCP acid. Plots of the initial uptake rates of succinic acid as a prevented accumulation and induced efflux of accumulated function of the concentration of undissociated acid were nonmetabolized succinic acid (Fig. 6). The observed accu- linear (Fig. 9), indicating simple, non-carrier-mediated diffu- mulation, while consistent with the hypothesis of the exist- sion. ence of a proton symport, does not constitute final proof, From the slopes of the linear plots obtained at various pH since simple or facilitated diffusion of undissociated dicar- values, estimates of the diffusion constant were obtained boxylic acid displays similar accumulation. (the diffusion constant as presented in Fig. 9 has the dimen-

10 100 awoo 5

.03 > 75 Q075

1050 * 1 02 - a005 ;- _ u Ci 0 li 0 25 o-" 1025 S ai-C1

0 - . _ 0 0 7 01 O a°

2 4 6 8 10 12 14 16 IS 20 22 24 rnme (h) FIG. 8. Growth of H. anomala ICG 4380 at pH 4.2 in a stirred medium with vitamins, glucose (0.1%, wt/vol) and DL-malic acid (0.5%, wt/vol). Symbols: 0, cell density (optical density at 640 nm); 0, glucose concentration (percent, wt/vol); A, L-malic acid concentration (percent, wt/vol); [1, relative activity of the proton malate symport measured at a saturating concentration of succinic acid (0.2 mM). VOL. 56, 1990 MALIC ACID TRANSPORT IN H. ANOMALA 1113

0.0 4 ° 10

,, 0.0 3 o*mI

E a 0.02 o 0 * 3.0 *.0 5.0 6.0 4.0 . * - / ~~~~~Extracellular pH > 0.0 I

0.0 2 0.0 4 0.0 6 0.08 0.1 0.12 [Undissociated Succinic acid ]( mM)

FIG. 9. (A) Initial uptake rates of undissociated succinic acid, as a function of its concentration, by glucose-repressed cells of H. anomala IGC 4380. Numbers at the ends of lines connecting datum points indicate pH values. (B) pH dependence of the diffusion constant, calculated from the slopes of the lines in panel A. sions of volume [microliters], reciprocal time [seconds-1], and other short-chain monocarboxylates in the yeast Saccharo- and reciprocal biomass [milligrams-1]). myces cerevisiae. Appl. Environ. Microbiol. 53:509-513. The values of the diffusion constants decreased steeply 3. Corte-Real, M., C. Lefio, and N. van Uden. 1989. Transport of with the extracellular proton concentration from a value of 9 L-malic acid and other dicarboxylic acids in the yeast Candida sphaerica. Appl. Microbiol. Biotechnol. 31:551-555. at pH 6.0 to a value of 0.1 at pH 3.0 (Fig. 9B). As a 4. De La Pefia, P., F. Barros, S. Gascon, P. S. Lazo, and S. Ramos. consequence, the passive diffusion of undissociated acid 1981. Effect of yeast killer toxin on sensitive cells of Saccharo- across the plasma membrane of H. anomala is subject to myces cerevisiae. J. Biol. Chem. 256:10420-10425. opposite pH influences: an increase due to the relative 5. Ledo, C., and N. van Uden. 1984. Effects of ethanol and other increase of undissociated acid with decreasing pH and a alkanols on passive proton influx in Saccharomyces cerevisiae. decrease due to decreasing permeability with decreasing pH. Biochim. Biophys. Acta 774:43-48. Similar behavior was observed earlier with respect to pas- 6. Ledo, C., and N. van Uden. 1986. Transport of lactate and other sive proton diffusion across the plasma membrane of S. short-chain monocarboxylates in the yeast Candida utilis. Appl. cerevisiae (5), passive diffusion of undissociated lactic acid Microbiol. Biotechnol. 23:389-393. across the plasma membranes of Candida ultilis and S. 7. McCloskey, L. P. 1980. Enzymatic assay for malic acid and cerevisiae (2, 6), and passive diffusion of undissociated malic malolactic fermentations. Am. J. Enol. Vitic. 31:212-215. 8. Osothsilp, C., and R. E. Subden. 1986. Malate transport in acid across the plasma membrane of C. sphaerica (3). Schizosaccharomyces pombe. J. Bacteriol. 168:1439-1443. 9. Radler, F. 1986. Microbial biochemistry. Experientia 42:884- ACKNOWLEDGMENTS 893. 10. Rottenberg, H. 1979. The measurement of membrane potential We thank N. van Uden for suggestions and discussions. and pH in cells, organelles and vesicles. Methods Enzymol. This work was supported by a research grant (contract 8719) from 55:547-569. the Junta Nacional de Investigacao Cientffica e Tecnol6gica, Lis- 11. Salmon, J. M. 1987. L-Malic acid permeation in resting cells of bon, Portugal, and grant DPE 5542-G-SS-8071-00 from the Agency anaerobically grown Saccharomyces cerevisiae. Biochim. for International Development, Washington, D.C. Biophys. Acta 901:30-34. 12. van Uden, N. 1967. Transport-limited fermentation and growth LITERATURE CITED of S. cerevisiae and its competitive inhibition. Arch. Microbiol. 1. Baranowski, K., and F. Radler. 1984. The glucose-dependent 58:155-168. transport of L-malate in Zygosaccharomyces bailii. Antonie van 13. Zmijewski, J. M., and A. M. MacQuillan. 1975. Dual effects of Leeuwenhoek J. Microbiol. 50:329-340. glucose on dicarboxylic acid transport in Kluyveromyces lactis. 2. Cassio, F., C. Leio, and N. van Uden. 1987. Transport of lactate Can. J. Microbiol. 21:473-480.