Urea Transport in Saccharomyces cerevisiae

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JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 571-576 Vol. 121, No. 2 Copyright i 1975 American Society for Microbiology Printed in U.S.A. Urea Transport in Saccharomyces cerevisiae TERRANCE G.

1 JOURNAL OF BACTERIOLOGY, Feb. 1975, p Vol. 121, No. 2 Copyright i 1975 American Society for Microbiology Printed in U.S.A. Urea Transport in Saccharomyces cerevisiae TERRANCE G. COOPER* AND ROBERTA SUMRADA Department of Biochemistry-FAS, University of Pittsburgh, Pittsburgh, Pennsylvania Received for publication 11 October 1974 Urea transport in Saccharomyces cerevisiae occurs by to pathays. The first mode of uptake is via an active transport system hich: (i) has an apparent Km value of 14,M, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-groth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake hich occurs at external urea concentrations in excess of.5 mm is via either passive or facilitated diffusion. Saccharomyces cerevisiae can utilize urea as sole nitrogen source by degrading it in to steps to ammonia and CO2 (1). The enzymes responsible for this degradation are urea carboxylase and allophanate hydrolase (11). Their production is contingent upon the presence of allophanic acid (2, 13) or the gratuitous inducer oxaluric acid (9) and is subject to nitrogen repression hen cells are gron on readily utilized amino acids (1). Although there is reasonable understanding of the biochemistry, genetics, and physiology of the urea-degradative enzymes, there is no information concerning ho urea is taken into the cell. Domnas reported (3) that during groth on allantoin the urea generated appears in the medium suggesting its exit from the cell, but no further data are given. In vie of this deficiency e have investigated the mechanisms by hich urea is accumulated and report that to modes of uptake are operative. MATERIALS AND METHODS Strains and media. To strains defective in urea metabolism ere used in this ork. M-62 lacks urea carboxylase and M-64 lacks allophanate hydrolase activity (11). These strains ere used in place of the ild-type strain because urea taken into ild-type cells is rapidly metabolized to CO2 hich is then released into the medium (12). The medium used here as that described earlier (2). Except here indicated otherise the nitrogen source as.1% ammonium sulfate. Urea uptake assay. For assay of urea uptake cultures ere gron at 3 C to a cell density of 1.3 x 17 cells per ml (45 Klett units). At zero time an 8.5-ml portion of the culture as transferred to a flask containing ['IC lurea (specific activity, 4.7 ;Ci/4mol). Incubation as carried out at 3 C in a shaking ater bath under conditions identical to those used during groth. At appropriate times 1.-ml samples ere transferred to nitrocellulose membrane filters (.45 gm pore size) (Millipore Corp.) and unless indicated otherise ashed four times ith 8 ml of cold medium containing 1 mm urea. Washed filters ere placed in 5 ml of Aquasol (Ne England Nuclear Corp.) scintillation fluid and counted 16 to 24 h later. The incubation time in Aquasol as needed to allo the filters to become transparent. Failure to do this results in unevenly quenched samples and loss of assay precision. The report by Leder and Perry (Fed. Proc. 26:394) that ashing ith cold medium leads to a rapid loss of accumulated substrates prompted evaluation of our ashing procedures. The losses reported by these investigators do not occur in Saccharomyces treated ith cold medium (Table 1). Note also that only 8 ml of medium are required to completely remove remaining external urea; 32 ml as routinely used, hoever, as a precaution. Unless otherise indicated, all data are expressed as amounts of radioactive urea observed per milliliter of the original culture. RESULTS A urea carboxylase-minus strain of S. cerevisiae (M-62) cannot accumulate ["C lurea hen gron on minimal ammonia medium (closed circles) (Fig. 1A). Hoever, if oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, is present during groth this strain is capable of urea accumulation. A quite different behavior is observed hen this, experiment is repeated using an allophanate hydrolase-minus strain (M-64). [TC ]urea is accumulated in this strain even in the absence of exogenous gratuitous inducer (Fig. 1B). To decide hether oxaluric acid is functioning here as a urea or allophanate analogue the experiment in Fig. 1A as repeated using arginine as sole nitrogen source in place of ammonia. This assures a high intracellular concentration of urea during cell groth. The presence of large quantities of urea ithin cells did not significantly increase their ability to accumulate urea from the medium. 71 Donloaded from on July 8, 218 by guest

2 572 COOPER AND SUMRADA J. BACTERIOL. TABLE 1. Washing procedures for assay of urea uptake nmol of urea/ml Wash solutiona Temp (C) of culture 2 min 16 min Minimal medium Minimal medium mm urea Minimal medium min 16 min Minimal medium (8 ml) Minimal medium (16 ml) Minimal medium (24 ml) Minimal medium (34 ml) a Volume of ash solution as 32 ml unless otherise indicated. 36 A M OXLU / --OXLU * / 2.4 o,1.8 -j -j uj 1.2_ -C a. B M-64 a Z 85 -J FIG. 1. Uptake of urea in urea carboxylase, M-62 (A) and allophanate hydrolase, M-64 (B) minus strains of Saccharomyces. Cultures of each strain ere gron on ammonia minimal medium in the presence () or absence (-) of.5 mm oxaluric acid. Urea uptake assays ere performed as indicated in Materials and Methods. External radioactive urea concentration as.36 mm. Hoever, this (Fig. 2) ability as observed if oxaluric acid as present during groth. It should be noted that the difference beteen the levels of urea accumulated in cells gron on ammonia and arginine likely results from isotope dilution hich arises from catabolism of arginine to urea. These data closely parallel those observed in studies demonstrating the contingency of allantoin degradative enzyme production upon the presence of allophanic acid. If indeed these data reflect an ability to transport urea into cells it should be possible to "chase" radioactive urea out of the cells by addition of a large excess of nonradioactive urea to the culture. This as observed for both strains (Fig. 3). Addition of excess nonradioactive urea to cultures actively accumulating [4C ]urea resulted in abrupt loss of radioactivity from the cells. The half-life of this loss as 9. o 2.8 AMMONIA + OXLU O 2.4 ai. C,) z Cn 2. 1_j ( / ARGININE + OXLU 1.2 C) AGNNE ALONE C FIG. 2. Effect of intracellular urea upon production of the urea uptake system. Three cultures of strain M-62 ere gron respectively on minimal ammonia medium plus oxaluric acid (), minimal arginine medium plus oxaluric acid (a) and minimal arginine medium alone (U). At a cell density of 45 Klett units each culture as harvested by filtration and resuspended in fresh, prearmed, pre-aerated, minimal ammonia medium. Those cultures previously gron in the presence of oxaluric acid also received oxaluric acid after resuspension in fresh medium. The three cultures ere incubated in this medium for 3 min to permit urea ithin the cells to leave. The time required for this to occur as determined from the data shon in Fig. 4. At the end of this incubation period (zero time in the figure) urea uptake as assayed as described in Materials and Methods. Failure to remove urea from cells gron on arginine using these procedures greatly decreased their ability to accumulate added radioactive urea. This is due to a steady state amount already being present ithin the cells at the time of radioactive urea addition to the medium. Donloaded from on July 8, 218 by guest

VOL. 121, 1975 UREA TRANSPORT 573 2 ~~~~~~M-64 IL 6 O ~~~~~~M-62 5 a r EXCESS _j4 UREA _j 4- /ADDED.5 3 2 3.2 D 2.4 a 2 1.6 U,.8 8 16 24 32 4 48 56 64 FIG. 3. Reversibility of urea uptake in Saccharomyces.

3 VOL. 121, 1975 UREA TRANSPORT ~~~~~~M-64 IL 6 O ~~~~~~M-62 5 a r EXCESS _j4 UREA _j 4- /ADDED D 2.4 a U, FIG. 3. Reversibility of urea uptake in Saccharomyces. The conditions of this experiment are identical to those used for the induced cultures of Fig. 1A. Hoever, at 16.5 min nonradioactive urea as added to the assay mixture to a final concentration of 15 mm. and 14.5 min for strains M-62 and M-64, respectively. This suggests that urea is capable of both entering and leaving yeast cells. To demonstrate the exit of urea directly, to parallel cultures of each strain (M-62 and M-64) ere alloed to accumulate [14C]urea for 12 min. At this time the four cell samples ere harvested by filtration, ashed ith prearmed, preaerated medium, and resuspended in their original volume of fresh medium. At various times after resuspension samples ere removed from the cultures and assayed either for the amount of radioactivity remaining ithin the cells (Fig. 4) or appearing in the medium. There is a reciprocal relationship beteen the gradual loss of urea from cells and its time-dependent accumulation in the medium (Fig. 4). Radioactive material observed in the cells and medium as demonstrated to be urea by its sensitivity to highly purified urease (Table 2). The data in Fig. 1 and 2 raise the possibility that production of a urea transport system is regulated in a fashion similar to that of the allantoin degradative system (2). The analogy beteen these to systems extends to their mutual repression hen cultures are gron in the presence of a readily utilized amino acid (Fig. 5). Urea accumulation is greatest in cultures gron on proline as sole nitrogen source; they progressively lose this ability hen gron on ammonia, aspartate, serine, or asparagine, respectively. It should also be noted (Fig. 5) that production of the transport system in a urea carboxylase-minus strain (M-62) is more FIG. 4. Loss of previously accumulated urea into the medium. Induced cultures (to from each strain) of strainsm-6 M-64 ere incubated for 12 mm in the presence of.36 mm urea. At this time, 1 ml of each culture ere harvested, ashed ith approximately 1 ml of prearmed pre-aerated medium, and resuspended in their original volume of fresh medium. At the indicated times a sample of the cells (a) or medium () ere obtained for each strain. It should be emphasized that cells ere received from one culture and medium from an identical culture. A portion of the medium as counted directly in Aquasol. The cells ere ashed as described in Materials and Methods before being placed in Aquasol. The data are expressed as nanomoles of [14CJurea per milliliter of cells (original culture) or medium. sensitive to the repressive effects of aspartate than is an allophanate hydrolase-minus strain (M-64). The reason behind this difference, hoever, is not presently knon. The distinguishing characteristic of active transport against a concentration gradient as opposed to passive or facilitated diffusion is the energy requirement of the former process. To decide hether urea accumulation as the result of active transport or one of the to types of diffusion, the effects of various energy metabolism inhibitors upon this process ere ascertained. Urea accumulation could be eliminated by arsenate, dinitrophenol, cyanide, and fluoride, clearly attesting the energy requirement of urea transport (Fig. 6). Not only is energy required to accumulate urea, but it is Donloaded from on July 8, 218 by guest

4 574 TABLE 2. Urea accumulation and loss from Saccharomyces cerevisiaea Source assayed COOPER AND SUMRADA 13 counts/min per ml of culture M-62 M-64 Urea incorporated into cells Urea lost from cells into the medium Urease-sensitive material in the cells Urease-sensitive material in the medium asamples of cells and medium similar to the 4-min points of Fig. 3 ere treated ith 1 mg of highly purified urease for 3 min. CO2 released upon acidification as then collected and its radioactivity content determined Ḻ i2.4 SF-ILAb-? A M-62 PROLINE ~~~~AMMONIA 6.8_AS PARTATE _ a. z I-~~~~~~~~~~~~~~~~~~~~~~~~~ W U, W ~~~~~~~~~~~~~A SPA RTAT E FIG. 5. Sensitivity of urea uptake to nitrogen repression. These experiments ere performed in a manner similar to that used for the induced culture in Fig. 1A and the uninduced culture in Fig. lb except that in place of ammonia the nitrogen source used as that indicated at a concentration of.1%. The radioactive urea concentration as.36 mm. also required to retain urea ithin the cell. This as shon (Fig. 7) by permitting a culture to accumulate urea for 16 min. At that time one-half of the culture as transferred to a second incubation vessel containing dinitrophe FIG. 6. Sensitivity of urea uptake to inhibitors of energy metabolism. This experiment as performed as described in Fig. 1 or 5. Hoever, 2 min before beginning the assay an inhibitor as added. Inhibitors ere used at the folloing final concentrations: arsenate, 5 mm; dinitrophenol, I mm; cyanide, 1 mm; and fluoride, 5 mm. -j WL 3. ce,*2. J. BACTERIOL FIG. 7. Requirement of energy to retain accumulated urea ithin the cell. This experiment as performed as described for the induced culture of Fig. JA except that dinitrophenol (1 mm final concentration) as added at 16.5 min. nol (1 mm final concentration). As depicted in this figure addition of dinitrophenol resulted in the immediate exit of urea that had been previously accumulated. A Lineeaver-Burk plot of a urea concentration curve as used to determine the Km value Donloaded from on July 8, 218 by guest

VOL. 121, 1975 UREA TRANSPORT 575 V V.4 oo4/ 4 2 _ 4 8 12 16 2 24 28 32 36 FIG. 8. Lineeaver-Burk plot of a urea contcentration curve.

5 VOL. 121, 1975 UREA TRANSPORT 575 V V.4 oo4/ 4 2 _ FIG. 8. Lineeaver-Burk plot of a urea contcentration curve. An induced culture of strain M-62 as used at a cell density of 45 Klett units. At zero time a 2.-ml portion of the culture as added to an appropriate amount of radioactive urea (.25 to 5. mm at a specific activity of 6.2 uci/pmol). Incubation as carried out for 4 min and 1.-ml samples ere transferred to a membrane filter (Millipore Corp.) and ashed ith 32 ml of cold medium containing 2 mm urea. The K. value of 14 um is the average of three experiments (12, 16, and 15 MM). of the urea transport system. This plot is biphasic, consisting of one linear portion yielding an apparent Km value of 14,uM and a second linear portion hich begins at approximately.5 mm. At concentrations in excess of.5 mm there is a much greater rate of entry than expected. This likely results from the existence of to systems participating in urea uptake: an energy-dependent, lo Km active transport system and an energy-independent, passive or facilitated diffusion system. Table 3 depicts a limited preliminary characterization of the urea uptake process occurring at an external concentration of 1 mm. At this concentration urea uptake is rapid, independent of induction by oxaluric acid, and insensitive to both repression and energy metabolism inhibitors. DISCUSSION These data provide evidence for to pathays of urea uptake in S. cerevisiae. The first mode of uptake exhibits a Km value of 14 MM. Like the active transport systems of Escherichia coli, urea transport at lo concentration is absolutely energy dependent (5) as is urea retainment ithin the cell (4). Production of the urea-active transport system appears to be subject to the same control exerted on the other enzymes of allantoin and urea metabolism; it is induced by allophanate and subject to repression by readily utilized amino acids. A second mode of uptake becomes apparent at external S urea concentrations above.5 mm. This uptake system acts rapidly reaching equilibrium in under 2 min. It does not require energy or pregroth in the presence of inducer and is not sensitive to nitrogen repression. Hoever, a note of caution is necessary because the accuracy and precision of the data in Table 3 are considerably loer than those reported in Fig. 1 to 8. The major problem, resulting in scatter of the data, is that urea taken up into the cells can rapidly diffuse out again during the ash procedure. This problem has been alleviated to an extent by the inclusion of urea (2 mm) in the ashing medium, but until this problem is completely resolved the data in Table 3 must be considered as preliminary. The demonstration of 'urea rapidly entering the cell hen present at high (1 mm) external concentrations may offer an explanation for differences observed in the induction kinetics of allophanate hydrolase and a-glucosidase. Lather and Cooper (7) reported that 3 min elapse beteen addition of 1 mm urea and appearance of allophanate hydrolase activity. On the other hand, Kuo et al. (6) observe a lag of 1 to 2 min beteen addition of the inducer, maltose, and appearance of a-glucosidase activity. If maltose cannot enter the cell by facilitated or passive diffusion then a significant portion of the 1- to 2-min lag observed by the TABLE 3. Characteristics of urea uptake at 1 mm external concentrationa Groth conditiṅ Time of sample (min) ['4Cjurea uptake (nmol) per milliliter of culture. Uninduced Uninduced Induced Induced on asparagine Uninduced on asparagine Induced and DNP added before assay athe values reported here are from the 14-min point of experiments similar to those depicted in Fig. 1, 5, and 6. A single value is reported instead of the entire curve in the interest of brevity. Hoever, it should be emphasized that the data are not as precise as those depicted in the above figures; the error is likely ± 1%. In these experiments the concentration of urea in the assay mixture as 1 mm at a specific activity of 1.2 ;Ci/umol. Also the harvested cells ere ashed ith 8 ml of minimal medium containing 2 mm urea instead of 32 ml of 1 mm urea medium. This alteratiorn in ashing procedure significantly improved the quality of the observed data but it is still not totally satisfactory. Donloaded from on July 8, 218 by guest

576 COOPER AND SUMRADA J. BACTERIOL. latter orkers may elapse before the internal cellular concentration of maltose reaches the threshold value required to bring about induction.

6 576 COOPER AND SUMRADA J. BACTERIOL. latter orkers may elapse before the internal cellular concentration of maltose reaches the threshold value required to bring about induction. Consistent ith this is the fact that maltose is transported by a specific inducible uptake system (8). The inducibility and sensitivity to nitrogen repression of the urea active transport system in S. cerevisiae should prove valuable in studies addressing the sequence of events occurring beteen synthesis of the presumed transport protein and its incorporation into the membrane of the cell. It is likely that these to events can be temporally separated using the methods previously developed to study allophanate hydrolase induction (Lather and Cooper, J. Bacteriol., in press). If this can be done, an important tool ill become available hich in concert ith genetic approaches may be brought to bear on this problem. ACKNOWLEDGMENTS We express gratitude to Ronald Kaback and Richard Abrams for their helpful comments. This ork as supported by Public Health Research grants GM and GM-2693 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bossinger, J., R. P. Lather, and T. G. Cooper Nitrogen repression of the allantoin degradative enzymes in Saccharomyces cerevisiae. J. Bacteriol. 118: Cooper, T. G., and R. P. Lather Induction of the allantoin degradative enzymes in Saccharormyces cerevisiae by the last intermediate of the pathay. Proc. Nat. Acad. Sci. U.S.A. 7: Domnas, A Amide metabolism in yeasts. The uptake of amide and amide-like compounds by yeast. J. Biochem. 52: Fields, K. L., and S. E. Luria Effects of colicins El and K on transport systems. J. Bacteriol. 97: Kennedy, E. P. and G. A. Scarborough Mechanism of hydrolysis of o-nitropheny,8-galactoside in Staphylococcus aureus and its significance for theories of sugar transport. Proc. Nat. Acad. Sci. U.S.A. 58: Kuo, S. C., F. R. Cano, and J.. Lampen Lomofungin, an inhibitor of ribonucleic acid synthesis in yeast protoplasts: its effect on enzyme formation. Antimicrob. Agents Chemother. 3: Lather, R. P., and T. G. Cooper Effects of inducer addition and removal upon the level of allophanate hydrolase in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 55: Sols, A., C. Gancedo, and G. Delafuente Energyyielding metabolism in yeasts, p. 29. In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 2. Academic Press Inc., Ne York. 9. Sumrada, R., and T. G. Cooper, Oxaluric acid: a non-metabolizable inducer of the allantoin degradative enzymes in Saccharomyces cerevisiae. J. Bacteriol. 117: Whitney, P. A., and T. G. Cooper Urea carboxylase and allophanate hydrolase: to components of a multienzyme complex in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 49: Whitney, P. A., and T. G. Cooper Urea carboxylase and allophanate hydrolase: to components of ATP: urea amido-lyase in Saccharomyces cerevisiae. J. Biol. Chem. 247: Whitney, P. A., and T. G. Cooper Requirement for HCO, by ATP:urea amido-lyase in yeast. Biochem. Biophys. Res. Commun. 4: Whitney, P. A., T. G. Cooper, and B. Magasanik The induction of urea carboxylase and allophanate hydrolase in Saccharomyces cerevisiae. J. Biol. Chem. 248: Donloaded from on July 8, 218 by guest

Saccharomyces cerevisiae?

Saccharomyces cerevisiae? JOURNAL OF BACTERIOLOGY, Aug. 1983, p. 623-627 21-9193/83/8623-5$2.O/ Copyright 1983, American Society for Microbiology Vol. 155, No. 2 What Is the Function of Nitrogen Catabolite Repression in Saccharomyces