S-ADENOSYLMETHIONINE IN THE VACUOLE OF CANDIDA UTILIS

G. SVIHLA AND F. SCHLENK1Division of Biological and Medical Research, Argonne National Laboratory, Lemont, Illinois

Received for publication October 19, 1959

Recently, it has been demonstrated by ultra-violet microscopy that S-adenosylmethionine isaccumulated in the vacuole of Candida utilis(Svihla and Schlenk, 1959). The exceptionalconcentration of this compound under certaincultural conditions, and the availability of simpleanalytical procedures permit a correlation ofoptical and biochemical studies. The opportunityof observing a well-defined chemical compoundin the vacuole of viable cells without staining orpretreatment should yield information on thestructure and function of this part of the yeastcell, especially during the growth cycle. Thepresent report deals with some of these problems.

MATERIALS AND METHODS

Candida utilis strain ATCC 9950 was used,and the technical details of culture and analysiswere the same as outlined earlier (Schlenk andDePalma, 1957; Svihla and Schlenk, 1959).Yields of yeast are reported on a dry weightbasis. The culture medium had the followingcomposition per L: KH2PO4, 2 g; K2HPO4, 1 g;(NH4)2SO4, 2 g; trisodium citrate, 0.3 g; MgCl2-6H20, 0.1 g; MnSO4 7H20, 0.1 g; CaC12, 0.1 g;ZnSO4 7 H20, 0.1 g; glucose (sterilized sepa-rately), 15 g. Various supplements were added asindicated in the experiments. This medium istermed "nitrogen-containing medium," whereas"nitrogen-free medium" had the same composi-tion except for substitution of K2SO4 for(NH4)2SO4.

In previous experiments (Svihla and Schlenk,1959), L-methionine or a combination of L-homocysteine and L-methylmethionine was usedas the organic sulfur supplement to stimulate theproduction of S-adenosylmethionine. In thepresent experiments, a combination of all threecompounds was used. The concentration of S-adenosylmethionine attained by this procedureis higher than that reported in our earlier experi-ments. A concentration of 5 ,umoles per ml of

1 Research performed under the auspices of theU. S. Atomic Energy Commission.

each sulfur amino acid was tolerated withoutundue decrease of cell yield. Apparently, theorganic sulfur compounds enter the yeast cellswithout mutual interference or competition,whereas the equivalent supplement of 15 ,umolesof L-methionine per ml causes serious inhibitionof growth. L-Homocysteine was prepared fromcommercial L-homocysteine thiolactone hydro-chloride by dissolving the latter in 1.5 ml of1 N NaOH per mmole; after 5 min at 25 C, oneadds 3.0 ml of 0.5 M KH2PO4 for neutralization.The presence of L-homocysteine in the yeast

culture medium leads to the formation of S-adenosylhomocysteine under special circum-stances (Schlenk, unpublished data). This sub-stance has the same ultraviolet absorptionspectrum as S-adenosylmethionine, and it wouldbe difficult to distinguish the two compounds inultraviolet microscopy. Chromatography of per-chloric acid extracts of the yeast used in thisstudy did not reveal more than 1 mimole ofS-adenosylhomocysteine per gram of cells;therefore, it does not interfere in the experimentsreported here.

S-Adenosylmethionine and 5'-methylthio-adenosine, used as supplements, were producedby incubation of commercial, dried Saccharo-myces cerevisiae in large quantities of the mediumspecified above with a supplement of 5 imolesper ml of L-methionine (Schlenk et al. 1959).S-Adenosyl-L-homocysteine was synthesized fromadenosine and L-homocysteine by liver enzyme(de la Haba and Cantoni, 1959). These com-pounds were used in highly purified form.Microscopy with the UV91 color-translating

ultraviolet microscope was the same as describedearlier (Svihla and Schlenk, 1959) with thefollowing exceptions: A 1OX Bausch and Lombocular replaced the 6 X ocular. The wave lengthsused were changed from 260, 350, and 280 m,uto 275, 350, and 265 m,. This does not change thecontrast of the resulting picture noticeably butdoes simplify operation. Energy from the A-H6lamp is greater at 265 m,u than at 260 m,u, anid

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by lowering the operating voltage from 600 v toabout 550 v the line emission at 265 m,u is in-creased as observed on the photocell exposurecontrol meter. Utilization of the 265 m,u linemade wave length calibration checks easy andreduced exposure time. The 275 m,u wave lengthgave approximately the same energy as the 265m,u line as shown by the control meter. Figure 1illustrates the results obtained at the differentwave lengths with control material and enrichedcells.A Radio Corporation of America image con-

verter tube no. 7404 in a suitable case, used inconjunction with the microscope, permitteddirect observation of the living cells under il-lumination with any wave length of light pro-duced by the lamp, passed by the microscope,and received by the tube. The useful range was235 to 620 ms.

RESULTS

Stability of S-adenosylmethionine in the vacuole.Earlier experiments (Svihla and Schlenk, 1959)have shown that S-adenosylmethionine is formedrapidly, and that its concentration remains al-most unchanged after the maximum level hasbeen attained. It is not utilized after the nutri-ents of the culture medium are exhausted, de-spite starvation of the cells by continued aera-tion. Further information on the persistence ofS-adenosylmethionine was desired as a pre-requisite to ultraviolet optical studies. For thispurpose, cells containing S-adenosylmethioninewere transferred into nitrogen-free medium andaerated at 30 C by agitation in the presence ofglucose (table 1). An increase in the amount ofyeast was observed which corresponded to aboutthe maximum expected if all S-adenosylmethio-nine in the vacuoles had been used as a source ofnitrogen. However, extraction of the cells,chromatography, and spectrophotometric analy-sis showed that the sulfonium compound wasnearly untouched. Nitrogen reserves other thanS-adceoslimethionine must have been used forthe limited growth of the culture.The possibility remained that the utilization

of S-adenosylmethionine is linked with growthancl metabolism of a type or intensity not occur-ring under the conditions of the experiment de-scribed in table 1. To test this possibility, cellscontainiing S-adenosylmethionine were trans-ferred into medium containing ammonium saltan(l glucose. Table 2 shows that growth of the

S-adenosylmethionine-containing cells resemblesthat of control cells. The total amount of sul-fonium compound, although declining per unitof weight, stays rather constant as calculatedfor the entire culture. By repeated transfer intomedium devoid of organic sulfur supplement,the low level of endogenous S-adenosylmethio-nine content of the control cultures is approachedeventually. It is apparent from these experi-ments that the sulfur adenine compound iscatabolized very slowly. This observation offereda basis for further ulraviolet optical studies andfor extended biochemical research.

Optical experiments. The persistence of S-adenosylmethionine permits optical studies of itsdistribution in growing cultures. Obviously, thereare the following alternatives: the mother cellsmay retain all of the sulfonium compound andproduce buds, leading to daughter cells whosevacuoles do not contain any of the substanceand are translucent to ultraviolet light at 265 mlt.Mother and daugher cells should be easily dis-tinguished on this basis. On the other hand,S-adenosylmethionine may be shared by thedaughter cells. This would result in a cell popula-tion with diminished but rather uniform ultra-violet density of the vacuoles.

These alternatives were put to a test by ultra-violet photomicrography of consecutive samplesfrom a culture of cells that contained S-adeno-sylmethionine. The experimental conditions cor-responded closely to those listed in table 2. Thecells were transferred for continued growth intomedium which contained ammonium salt, butno organic sulfur supplement. Thus, furthersynthesis of S-adenosylmethionine remained atthe low endogenous level, and the ultraviolet-absorbing material visualized in figure 2, A toC, was largely derived from the initial enrichedparent cells. Each row, A to C, represents selectedtypical cells from one sample of the culture.The material was withdrawn from the cultureflask at time intervals that, as closely as possible,provided for doubling the cells. For comparison,row D shows corresponding material from a con-trol culture. It is apparent from figure 2, A to C,that the mother cells share the vacuolar S-adeno-sylmethionine with the daughter cells. As thecells multiply, the concentration of the compoundis gradually reduced to a level at which distinc-tion from control cultures is no longer possibleby our present technique.

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TABLE 1Growth limitation of Candida utilis in the absence

of available nitrogen

Time of Culture Yeast Present in Total S-Adeno-Medium sylmethionine

hr gIl/O ml ,umoles

0 0.40 23.04.5 0.44 20.27.0 0.58 21.2

21.5 0.56 19.1

TABLE 2Fate of intravacuolar S-adenosylmethionine during

growth of Candida utilis

S-Adenosylmethionine- Control Cellscontaining Cells

Time ofCulture Amt of Amt of Conc of S-

cells in Conc of S-Adeno- cells in adenosyl-culture sylmethionine culture methioninemedium medium

hr g/100 mnl yeasi totalloo gIlO/ ml jAmoleslg yeast

0 0.10 83.0 8.3 0.10 2.1 + 0.83 0.22 43.8 8.7 0.21 2.1 4± 0.85 0.41 19.4 7.9 0.41 2.1 4± 0.812 0.60 9.6 5.8 0.58 2.1 i 0.8

S-Adenosylmethionine-containing cells werecultivated in the presence of organic sulfursupplement; control cells were obtained underidentical conditions except for the omission of thesupplement. After 24 hr of culture at 30 C, thecells were centrifuged and resuspended for con-tinued growth without organic sulfur supplementin medium containing ammonium salt and glucose.Samples were withdrawn and analyzed at thespecified times.

This result raises questions concerning themechanism of transfer of the vacuolar materialinto the bud and daughter cells. Again, twopossibilities are at hand: the vacuole may pro-duce a temporary extension into the bud forpartial transfer of its contents. Alternatively, thecompounds of the vacuole may be released andspread into the cytoplasm at the time of budding.After transfer into the bud and separation of thelatter from the mother cell, they would be re-assembled in a new vacuole of the daughter cell.Our best pictures of a selected series from one

culture (figure 3) do not show signs of a protru-sion of the vacuole into the bud. The alternative,release of the material from the vacuole into the

cytoplasm, finds occasional support in the ultra-violet photographs of some cells taken at theonset of budding.

Biochemical experiments. The observation thatS-adenosylmethionine in the vacuole of C. utilispersists through several divisions raises furtherquestions. It would be important to find outwhether the confinement in the vacuole is re-sponsible for the metabolic inertness of the sul-fonium compound, or whether C. utilis lacks theenzymes for conversion of the material intonitrogenous fragments which can be assimilated.Biochemical experiments were carried out toobtain information on these points.

S-Adenosylmethionine was incorporated intothe culture medium as the sole exogenous sourceof nitrogen. For comparison, the same supple-ment was provided in a medium which containedthe usual amount of ammonium salt. The con-centration of the latter in the medium was 30,umoles of NH4+ per ml. Assuming that all ofthe six nitrogen atoms of S-adenosylmethioninemay be utilized, the latter was supplied in aconcentration of 5 ,umoles per ml to match thetotal amount of nitrogen which is usually avail-able. After a culture period of 26 hr at 30 C, thecells were centrifuged, washed twice with water,and analyzed. It is apparent from table 3 thatS-adenosylmethionine in the culture medium isnot a good source of nitrogen, but its presencedoes not interfere with the utilization of am-monium salt. There is moderate accumulation ofS-adenosylmethionine in the cells; this is some-what higher in the absence of ammonium salt.In all instances, 60 to 80 per cent of the sul-fonium compound was found to be left in themedium after the culture period.

It would seem possible that the cell wall andmembrane are obstacles to the uptake of S-adenosylmethionine from the culture medium,or that the cellular enzymes are unable to de.grade the compound to more readily availablefragments. To clarify this point, the ability ofthe principal components to serve as nitrogensources was tested (table 4). Analysis of the re-sulting yeast for S-adenosylmethionine wasincluded to see which of the structural unitsstimulates the accumulation of the sulfoniumcompound in the cells. There have been earlier ex-periments by DiCarlo et al. (1951) and byChantrenne and Devreux (1956) on the utiliza-tion of adenine and some of its derivatives.

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A

j?j.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*OSI. ..

Figure 2. Distribution of S-adenosylmethionine in growing cells of Candida utilis, photographed at265 m,u. The experimental conditions are explained in the text.

Schultz and Pomper (1948) have examined perimental conditions. In our experiments,methionine as a nitrogen source for C. utilis. adenine and adenosine as well as ammoniumOur results are in agreement; differences in the salt were used as nitrogen sources, while 5'-degree of response may be explained by the ex- methylthioadenosine, L mnethionine, and DL-

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Figure S. Details of bud formation in cells containing S-adenosylmethionine after transfer intomedium without organic sulfur supplement. Photomicrography at 265 m/A.

TABLE 3Utilization of exogenous S-adenosylmethionine by

Candida utilis

S-Adenosyl-Nitrogen Supplement Conc in Yield of methionine

in Medium Medium Cells Extractedfrom Cells

pmoles/ml g/100 ml ,umolesig

None 0.20 2.5NH4+ 30 0.86 6.0S-Adenosylmeth- 5 0.37 30.5

ionineS-Adenosylmeth- 1 5

ionine + 0.98 12.0NH4+ J 30

The initial amount of yeast was 0.15 g; the cul-ture period was 26 hr at 30 C.

FragmentsTABLE 4

of S-adenosylmethionine assources for Candida utilis

Nitrogen Source Conc in

None .............Adenine ..........Adenosine.........5'-Methylthio-adenosine .......

L-Methionine ......S-Adenosyl-L-homocysteine .

DL-Homoserine ....NH4+.............

,untoles/mli

66

630

53030

Yield ofYeast

g/100 mtl0.170.620.66

0.340.39

0.450.490.67

S-Adenosyl-methionineContent of

Yeast

pmoles/g<3<3<3

36.0<3<3

The initial amount of yeast was 0.15 g; the cul-ttire period was 40 hr at 30 C.

homoserine were significantly inferior. Of thelatter compound only the racemic form was

available to us. Methionine not only served as asource of nitrogen, but also stimulated the for-mation of S-adenosylmethionine. S-Adenosyl-L-homocysteine permitted moderate growth, andpart of it was converted into S-adenosylmethio-nine. 5'-Methylthioadenosine exerted a weakeffect in both respects. In all other instances,there was no increase in the production of S-adenosylmethionine beyond the endogenouslevel.From the effective utilization of adenine and

adenosine as contrasted with the limited effectof S-adenosylmethionine one may conclude thatthe latter is not split readily into these fragmentsby yeast enzymes. Removal of the methyl group,perhaps by transmethylation, leads to S-adeno-sylhomocysteine which is a better source ofnitrogen than S-adenosylmethionine. The limitedeffect of the latter in promoting the growth ofyeast cells may be explained in this way. Theonly other enzymatic degradation which hasbeen studied in some detail leads to 5'-methyl-thioadenosine, homoserine lactone, and homo-serine (Shapiro and Mather, 1958; Mudd, 1959).In yeast cells this is a slow reaction and, as seenfrom table 4, the products themselves are notefficient as nitrogen sources. The irreversibilityof the biosynthesis of the sulfonium compoundfrom adenosine triphosphate and methioninehad been noted before (Mudd and Cantoni,1958).

DISCUSSION

The limited ability of the enzymes of C. utilisto degrade S-adenosylmethionine may account

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in part for the persistence of this compound inthe cells. In addition, storage in the vacuole maybe suspected of being responsible for the slowcatabolism. However, the present experimentsshow that this confinement is not the main reason,because an extracellular supply of the sulfoniumcompound likewise is not utilized to any appre-ciable extent. Regardless of the reasons, it isclear that S-adenosylmethionine, formed in ex-cessive amounts in the presence of methionine,is of little avail to the yeast cell as a source ofnitrogen. To the biochemist, overproduction andaccumulation open an easy preparative route tothis still scarce substance. From a cytologicalviewpoint, special interest is stimulated by theaccumulation and permanence of the compoundin the vacuole. Presumably, the present experi-ments constitute the first instance of demonstrat-ing a well-defined chemical substance in thevacuole without fixing, staining, or other pre-paratory manipulation of the cells. Thanks tothe slow catabolism of the compound, its where-abouts can be followed through two or threedivisions of the cells. The present ultravioletoptical experiments furnish proof that S-adeno-sylmethionine is transferred into the bud andconcentrated in a new vacuole which becomesvisible even before the bud parts from the mothercell.

Despite obvious advantages of S-adenosyl-methionine as an "optical tracer substance," ithas not yet been possible to explain the mecha-nism of this transfer. Yuasa and Lindegren(5959) state: "The first step (in budding) is theformation of a long tube leading from the vacu-ole to the periphery of the cell." Our presenttechnique has not permitted observation of aconnecting structure between the vacuoles of themother and daughter cells, but such detail maybe beyond the resolution of our system. Nor havewe been able to confirm the other extreme, re-ported, for example, by Narayana (1956), thaton transfer to fresh medium the vacuoles disap-pear temporarily with the onset of budding.Observation of a great many cells by ultravioletphotomicrography in the course of our work hasshown the vacuole to be present constantly inC. utilis. It varies somewhat in shape and size;occasionally it is lobed. A few cells have beenseen in an early stage of budding which appearedto lack a vacuole and the cytoplasm showedsomewhat greater ultraviolet absorption thanthat of other cells in the field. However, most

cells in the same stage of budding show a well-defined vacuole. Abolition of the vacuole wouldhave to be a transitory process of very shortduration as judged by the infrequency of cells inwhich the ultraviolet density appears to be con-tinuous. It is possible and probable, however,that at this stage of cellular development part ofthe vacuolar S-adenosylmethionine is dissemi-nated into the cytoplasm and thence finds itsway into the bud. This process would involvegreat dilution. An approximate calculation, con-sidering for simplicity both the cell and thevacuole as spherical bodies, with a diameter of6 ,u and 2 ,u, respectively, shows that uniformdistribution of the material from the vacuole intoall parts of the cell would result in 27-fold dilu-tion. Even with a much larger vacuole, the dilu-tion would be such that the material couldbarely be detected by our technique. Further-more, it appears from our ultraviolet photomicro-graphs that a concentration gradient betweenthe vacuole and its surroundings is nearly alwaysmaintained. It is not surprising, therefore, thatthe transfer into the bud is not visible.

ACKNOWLEDGMENT

The technical assistance of Mrs. Aldona Al-menas and Miss Julia L. Dainko is gratefullyacknowledged.

SUMMARY

S-Adenosylmethionine can be accumulated inthe vacuoles of Candida utilis by culture in asimple medium supplemented with L-methionineand related sulfur amino acids. Continued cul-ture in the absence of sulfur supplement showsthat S-adenosylmethionine in the vacuole is notreadily catabolized. Ultraviolet photomicrog-raphy reveals that part of the material is trans-ferred from the vacuoles of the mother cells intothe vacuoles of the buds and daughter cells.The possible mechanism of this transfer and someproperties of the vacuole are discussed.

REFERENCES

CHANTRENNE, H. AND DEVREUX, S. 1956 As-similation de l'adenine par la levure. Bio-chim. et Biophys. Acta, 21, 385-387.

DE LA HABA, G. AND CANTONI, G. L. 1959 Theenzymatic synthesis of S-adenosyl-L-homo-cysteine from adenosine and homoeysteine.J. Biol. Chem., 234, 603-608.

DICARLO, F. J., SCHULTZ, A. S., AND MCMANUS,D. K. 1951 The assimilation of nucleic

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acid derivatives and related compounds byyeasts. J. Biol. Chem., 189, 151-157.

MUDD, S. H. 1959 Enzymatic cleavage of S-adenosylmethionine. J. Biol. Chem., 234,87-92.

MUDD, S. H. AND CANTONI, G. L. 1958 Activa-tion of methionine for transmethylation.III. The methionine-activating enzyme ofbakers' yeast. J. Biol. Chem., 231, 481-492.

NARAYANA, N. V. A. 1956 The vacuole in yeast.Proc. Indian Acad. Sci., B, 43, 314-324.

SCHLENK, F. AND DEPALMA, R. E. 1957 Theformation of S-adenosylmethionine in yeast.J. Biol. Chem., 229, 1037-1050.

SCHLENK, F., DAINKO, J. L. AND STANFORD,S. M. 1959 Improved procedure for theisolation of S-adenosylmethionine and S-

adenosylethionine. Arch. Biochem. Biophys.83, 28-34.

SCHULTZ, A. S. AND POMPER, S. 1948 Aminoacids as nitrogen source for the growth ofyeasts. Arch. Biochem. Biophys., 19, 184-192.

SHAPIRO, S. K. AND MATHER, A. N. 1958 Theenzymatic decomposition of S-adenosyl-L-methionine. J. Biol. Chem., 233, 631-633.

SVIHLA, G. AND SCHLENK, F. 1959 Localizationof S-adenosylmethionine in Candida utilisby ultraviolet microscopy. J. Bacteriol.,78, 500-505.

YUASA, A. AND LINDEGREN, C. C. 1959 Theintegrity of the centriole in Saccharomyces.Antonie van Leeuwenhoek. J. Microbiol.Serol., 25, 73-87.

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