JOURNAL OF BACTERIOLOGY, Jan. 1972, p. 34-43Copyright © 1972 American Society for Microbiology

Vol. 109, No. 1Printed in U.S.A.

Genetic and Physiological Control of Serine andGlycine Biosynthesis in Saccharomyces'

RODNEY ULANE2 AND MAURICE OGURYeast Research Group, Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901

Received for publication 20 September 1971

Two of the three known metabolic pathways to serine and glycine have beenshown to be present in prototrophic yeast strains, i.e., the phosphorylatedpathway from glycolytic intermediates and the glyoxylate pathway from tricar-boxylic acid cycle intermediates. Two serine-glycine auxotrophs (serl andser2) were found to be blocked in the phosphoglycerate pathway. The serl gene

controls L-glutamate: phosphohydroxypyruvate transaminase biosynthesis, andthe ser2 gene controls phosphoserine phosphatase biosynthesis. The otherpathway to glycine, from isocitrate, is repressed by growth in glucose media,specifically, at isocitrate lyase and at the alanine: glyoxylate transaminase.This pathway is derepressed by growth to stationary phase in glucose mediayielding high activity of these enzymes. The phosphorylated pathway appearsto be the principal biosynthetic pathway to serine and glycine during growth onsugar media. Strains which are serine-glycine dependent in glucose media be-came capable of serine-glycine independent growth on acetate media. Theseresults describe a method of physiological control of a secondary metabolicpathway allowing a single lesion in the principal biosynthetic pathway to pro-duce auxotrophy. This may be termed conditional auxotrophy.

The existence of metabolites that can beformed by two or more pathways raised inter-esting questions for us concerning their regula-tion and the expression and suppression ofauxotrophic mutation. Thus, for example,three metabolic pathways to serine have beensuggested although they (necessarily) may notbe present simultaneously in any given cell.Two of these pathways are thought to derivefrom glycolysis via either phosphorylated ornonphosphorylated intermediates (6, 21). Thethird pathway is believed to originate from tri-carboxylic acid cycle intermediates and pro-duces serine via glyoxylate and glycine (13, 20).Mutant analysis has suggested the phospho-

rylated pathway as the principal biosyntheticpathway to serine in some bacteria (9, 12, 18),but the mechanism by which auxotrophy isexpressed, if other pathways to serine exist,requires additional clarification.

In Saccharomyces serine auxotrophs are' Presented in part at the 160th National Meeting of the

American Chemical Society, 14-18 September 1970. Takenfrom a dissertation submitted by Rodney Ulane in partialfulfillment of the requirements for the Ph.D. degree in Mi-crobiology, Southern Illinois University, 1971.

2 Present address: National Institute of Arthritis andMetabolic Diseases, National Institutes of Health, Bethesda,Md. 20014.

34

known, but neither the sites of their lesionsnor the principal biosynthetic pathway toserine has been clearly established. Biosyn-thesis of serine in Saccharomyces may occurby at least two of the pathways mentionedabove. One is suggested by indirect evidence(4) and perhaps originates from glycolysissomewhere above the level of pyruvate, pos-sibly via phosphoserine (15). The other isbased on more direct evidence (3) and appearsto originate in the tricarboxylic acid cycle atisocitrate proceeding via glyoxylate to glycineand serine (Fig. 1).The present study addresses itself to the fol-

lowing questions. (i) What are the pathways toserine and glycine present in Saccharomycesstrains? (ii) Is there a primary biosyntheticpathway and is serine normally a glycine pre-cursor or vice versa? (iii) At what enzymaticsteps are existing serine auxotrophs blocked,and how many lesions are necessary to produceauxotrophy, assuming multiple pathways? (iv)If a single genetic lesion produces auxotrophy,how are the other pathways regulated?We shall present evidence below indicating

that serine-independent strains, under certaincultural conditions, possess the phosphorylatedpathway to serine from glycolysis and the

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glyoxylate pathway from the tricarboxylic acidcycle. The serine-dependent strains studiedwill be shown to have lesions in the phospho-rylated pathway. Regulation of the phosphoryl-ated pathway by serine feedback inhibitionof 3-phospho-D-glyceric acid (PGA) dehy-drogenase and regulation of the glyoxyl-ate pathway by glucose repression of isocit-rate lyase (L.-isocitrate: glyoxylate-lyase, EC4.1.3.1) and L-alanine: glyoxylate transaminasewill be shown to occur. Partial release from theauxotrophic requirement by derepression ofthe secondary pathway will be demonstrated.

MATERIALS AND METHODSYeast strains. Bakers' yeast was obtained from

Anheuser-Busch, St. Louis, Mo. Other strains usedwere from the Carbondale collection: MO-11-48A a

SER, a serine-independent strain, and two serineauxotrophs, MO-171-11B a serl, derived from strain1453-2B a adl ga3 hi8 met2 thr4 p6 serl, and MO-170-22B a ser2, derived from strain 8256-52 a ser2adl.

Media. Media were (per liter) 1% glucose com-

plex [glucose, 10.0 g; peptone, 3.5 g; KH2PO4, 2.0 g;MgSO4-7H2O, 1.0 g; (NH4)2SO4, 2.0 g; Difco YeastExtract, 5.0 g] and 1% glucose minimal medium[glucose, 10.0 g; KH2PO4, 1.0 g; MgSO4*7H2O, 0.5g; (NH4)2SO4, 1.0 g; CaCl2, 0.3 g; KI, 10 mg; cholinechloride, 4 mg; inositol, 1 mg; nicotinic acid, 0.4 mg;calcium pantothenate, 0.4 mg; pyridoxine HCl, 0.4mg; thiamine HCl, 0.4 mg; p-aminobenzoic acid, 0.4mg; biotin, 0.002 mg; FeSO4*7H2O, 0.3 mg;MnSO4 4H20, 0.04 mg; (NH4)6Mo7O24 4H20, 0.018mg; Na2B4O7410H2O, 0.088 mg; CuSO4 5H20, 0.04mg; ZnSO4 7H20, 0.31 mg] with pH adjusted to 5.8with KOH. Acetate complex and acetate minimal-0.6% potassium acetate were substituted for glucosein the above media. Additions to the minimal mediumare specified in individual experiments.Growth of cultures. Cultures were incubated at

30 C on reciprocating shakers. Growth curve experi-ments were carried out either in screw-cappedmatched Klett tubes or in 250-ml side arm flaskswith turbidity measured in a Klett-Summerson pho-toelectric colorimeter by using a blue, 420-nm filter.

Reagents. Fine biochemicals were obtained com-

mercially either from Sigma Chemical Company, St.Louis, Mo., or Calbiochem, Los Angeles, Calif. Tet-rahydrofolic acid (FH4) was prepared from folic acidby low-pressure hydrogenation over platinum oxide(K & K Laboratories, Hollywood, Calif.) essentiallyby the method of Hatefi et al. (5) at neutral pH (1).

35

The FH4 was stored at -60 C under N2 atmospherein lyophilizing vials (VirTis Co., Inc., Gardiner,N.Y.) until used.Enzyme preparations. Stationary-phase cultures,

grown in 1% glucose complex medium, were usedunless otherwise noted. Cells were washed threetimes by centrifugation with distilled water, once inbuffer, and then suspended in the same buffer at a

concentration of 109 to 1010 cells per ml. The buffersused depended on the enzyme assay to be per-formed. For assays of the phosphorylated pathwayL-alanine: glyoxylate transaminase and isocitratelyase, 0.1 M potassium phosphate buffer (pH 7.5) was

used; for assays of serine transhydroxymethylase (L-serine: tetrahydrofolate-5, 10-hydroxymethyltrans-ferase; EC 2.1.2.1), 0.05 M tris(hydroxymethyl)methyl-aminopropane sulfonic acid (TAPS) buffer, pH 8.0,was used.The cell suspension (12 ml at 109 to 1010 cells per

ml) was disrupted by sonic treatment at 0 to 4 C witha model S110 Branson Sonifier for 4 min at a power

setting of 7, tuned for maximum amperes. All subse-quent operations were carried out at 0 to 4 C. Thedisrupted cell suspension was centrifuged at 48,000x g in a Sorvall RC2B high-speed centrifuge for 30min. The supematant fluid was saved and the pelletwas discarded. For enzyme assays of the phosphoryl-ated pathway L-alanine: glyoxylate transaminaseand isocitrate lyase, the supernatant fluid was dia-lyzed overnight against three 1-liter changes of 0.1 Mpotassium phosphate buffer, pH 7.5. The resultingdialysate (yeast enzyme preparation) was used forthe enzyme assays. In some cases, the supernatantfluid of the disrupted cell suspension was fraction-ated by ammonium sulfate precipitation. Fractiona-tion was carried out by the addition of the appro-priate amount of a cold, saturated, ammonium sul-fate solution (pH 7.5), allowing the mixture to standwith gentle stirring for 20 min, and then centrifugingfor 40 min at 48,000 x g. The resulting pellet wassuspended in 2 ml of 0.1 M potassium phosphatebuffer (pH 7.5) and dialyzed overnight against three1-liter changes of the same buffer. For enzyme as-

says of serine transhydroxymethylase, the superna-

tant fluid of the disrupted cell suspension was

passed through a Sephadex G-25 column and as-

sayed immediately. Protein was determined by themethod of Waddell (19).Assay procedures. All incubations with radioac-

tive substrates were carried out in plastic microtiterplates (Cooke Engineering Co., Alexandria, Va.).Incubation mixtures contained the following: (i) phos-phorylated pathway-yeast enzyme preparation (2.5to 3.0 mg of protein per ml), 5 gliters; Na-3-phospho-D-glycerate-U-14C (specific activity 28 mCi

TWO PATHWAYS OF SERINE BIOSYNTHESIS

HAD NADHM-N GLUTAMATE .-KETOGLUTARATE Pi FH.N!N'l-,I14 n-FH4

11It PYRUVATE ALANINE

3-PHOSPHOGLYCERIC ACID PPHOSPHOHYDROXYPYRUVATE PPHOSPHOSERINE SERINE GLYCINE t GLYOXYLATEr ISOCITRATE

SUCCFNATE

FIG. 1. Pathways to serine in Saccharomyces.

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per mmole, Calbiochem), 0.007 Mmoles; nicotin-amide adenine dinucleotide (NAD), 0.5 Amoles; Naglutamate, 0.5 Amoles; Mg2+, 0.5 Mmoles; potassiumphosphate buffer (pH 7.5), 10.0 gmoles in a totalvolume of 100 uliters; (ii) phosphoserine (P-serine)phosphatase-yeast enzyme preparation (2.5 to 3.0 mgof protein per ml), 5 pliters; P-DL-serine-U-l4C (spe-cific activity 4.68 mCi per mmole; Tracerlab, Walt-ham, Mass.), 0.110 jAmoles; Mg2+, 0.5 qmoles; tris(hydroxymethyl)aminomethane (Tris)-maleate buffer(pH 7.5), 10.0 Amoles in a total volume of 100 gliters;(iii) serine transhydroxymethylase-yeast -enzymepreparation (1.2 to 2.0 mg of protein per ml), 10jLliters; glycine-2-14C (specific activity 17.5 mCi permmole; Calbiochem), 0.028 Mmoles; FH4, 0.2 umoles;pyridoxal phosphate (PALP), 0.2 umoles; formalde-hyde, 0.2 ,moles; TAPS buffer (pH 8.5), 5.1 Amoles ina total volume of 120 Aliters; (iv) L-alanine: glyox-ylate transaminase-yeast enzyme preparation (1.5to 2.0 mg of protein per ml), 5 Aliters; sodium gly-oxylate-1,2-'4C (4.62 mCi per mmole; Calbiochem),0.005 timoles; L-alanine, 0.5 umoles; PALP, 0.5,qmoles; potassium phosphate buffer (pH 7.5), 5.0Amoles in a total volume of 50 Atliters. The above mix-tures were incubated at 30 C in an air incubator. Atthe elapsed times shown in the figures, 5-Mliter sam-ples of the mixtures were spotted for chromatographyon paper strips (Schleicher and Schuell, no. 589Orange Ribbon-C). Isocitrate lyase was assayed in thedirection of glyoxylate production by the method ofOlson (8). Absorbance at 252 nm of the glyoxylatesemicarbazone formed was monitored continuouslywith a Gilford 2000 recorder and a Beckman DU spec-trophotometer. Specific activity was defined as nano-moles of glyoxylate formed per minute per milligramof protein, using the molecular extinction coefficientof 12,400 for the glyoxylic semicarbazone.Paper chromatography. Incubation mixtures and

appropriate standards spotted on paper strips wereresolved and compared after descending chromatog-raphy in a number of solvent systems. RF values forphosphohydroxypyruvate (P-OH-pyruvate) are com-pared with those of other better known intermedi-ates in the solvent systems used in the present studyin Table 1. RF values of unknown peaks and rele-vant standards generally differed by less than 0.02units. When P-serine, serine, and PGA were to beresolved simultaneously, the chromatograms weredeveloped twice in the same direction with differentsolvent systems. The P-OH-pyruvate standard wasdetected by spraying the chromatogram with a solu-tion of 0.05% o-phenylenediamine in 10% aqueoustrichloroacetic acid and heating for 2 min at 100 C.Under ultraviolet illumination, the P-OH-pyruvateappeared as a yellow-green fluorescing spot. Radio-active regions on chromatograms were located andestimated with a Nuclear Chicago actigraph III radi-ochromatogram strip scanner and a model 8735 dig-ital integrator set at the following parameters: slitwidth, 3 mm (12 mm with integrator); scan speed, 60cm per hr; high voltage, 960 v; time constant, 20 sec.

RESULTSGrowth experiments. Figure 2A describes

the growth of a serl mutant strain on variousglucose media. This strain grew only whenserine or glycine was added to the minimalmedium. Most other serl strains tested re-quired a one-carbon source (formate) in addi-tion to glycine for growth. The latter strainswill be considered later. Glyoxylate failed tosubstitute for serine or glycine as did adenine.Figure 2B describes the growth of a leaky ser2mutant strain. The strain grew well on serineor glycine and almost as well on adenine, al-though the latter effect is probably due to asparing action by adenine. The addition ofglyoxylate failed to yield better growth thanobserved on minimal medium alone. The addi-tion of threonine (not shown) to the minimalmedium failed to yield better growth with ei-ther strain.Phosphorylated pathway. With the nutri-

tional requirements of the two yeast strainsestablished, an investigation into the enzy-matic lesions responsible for auxotrophy wasundertaken.The existence of the phosphorylated

pathway to serine in Saccharomyces was dem-onstrated in cell-free preparations from com-mercial bakers' yeast and from a haploid,serine-independent strain of the Carbondalecollection (MO-11-48A SER).

Figure 3 summarizes several experiments inwhich yeast enzyme preparations from SER,serl, and ser2 strains were incubated withPGA-U-14C, NAD, and glutamate. The mix-tures were sampled at the indicated time in-tervals, chromatographed by two successivemigrations in different solvent systems, andscanned on a radiochromatogram stripscanner.The first scan represents a 3-hr incubation

of the complete system and reveals partial dis-appearance of the substrate PGA as well as theappearance of two new product peaks identi-fied as P-serine and serine in seven solventsystems.The fourth scan represents the complete

system plus sodium fluoride. It shows the ac-cumulation of P-serine, with very little serineformed in the presence of F- ion. The latterinhibits the phosphoglycerate pathway in Sac-charomyces at phosphoserine phosphatase in amanner similar to that already reported byinvestigators of mammalian and bacterial sys-tems (10, 11, 18). The degree of inhibition in-creased with increasing F- concentration asdid the rate of disappearance of PGA. Thelatter observation suggested a feedback inhibi-tion by serine relieved by the F- ion inhibitionof serine formation.

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BIOSYNTHESIS IN SACCHAROMYCES

TABLE 1. RF values of compounds under study in various solvent systems

Solvent systemsbCompounda

I II III IV V VI VII VIII IX X XI

Glycine ........ 0.32 0.28 0.32 0.66 0.31 0.38 0.38Glyoxylate .. .. 0.42 0.32 0.35 0.30PGA ........... 0.02 0.03 0.67 0.09 0.27 0.13 0.32 0.32 0.14 0.03P-OH-pyruvate 0.03 0.03 0.05 0.67 0.16 0.14 0.24P-serine ........ 0.03 0.05 0.34 0.54 0.10 0.09 0.06 0.18 0.16 0.15 0.04Serine .......... 0.24 0.42 0.30 0.64 0.30 0.33 0.21 0.37 0.34 0.27 0.12

a Abbreviations: PGA, 3-phospho-D-glyceric acid; P-OH-pyruvate, phosphohydroxypyruvate; P-serine,phosphoserine.

b Composition of solvent systems: solvent I-phenol (80%): isopropanol:water, 25/5/5 (v/v/v) (add 1 ml of14% ammonia and 15 mg of 8-hydroxyquinoline per 100 ml of solvent); solvent II-n-butanol: acetone: water:diethylamine, 20/20/10/3 (v/v/v/v); solvent III-ethanol (100%): water, 40/1 (v/v); solvent IV-t-butanol:water: methyl ethyl ketone: diethylamine, 10/10/5/2 (v/v/v/v); solvent V-n-butanol: pyridine: water, 1/1/1(v/v/v); solvent VI-ethyl acetate: formic acid: water, 10/3/2 (v/v/v); solvent VII-n-butanol: acetic acid:water, 4/1/1 (v/v/v); solvent VIII-t-butanol: formic acid: water, 70/15/15 (v/v/v); solvent IX-t-butanol:methyl ethyl ketone: formic acid: water, 40/30/15/15 (v/v/v/v); solvent X-t-butanol: methyl ethyl ketone:0.1 M phosphate buffer, pH 7.5: water, 80/80/30/30 (v/v/v/v); solvent XI-n-propanol: acetic acid:tris(hydroxymethyl)aminomethane-hydrochloride: water, 70/10/0.41/10 (v/v/w/v).

The amine donor and cofactor requirementsfor the phosphorylated pathway are repre-sented in Table 2. Omitting NAD or glutamatereduced the activity of the system to lowlevels. Although partial restoration of activitycould be demonstrated by substituting aspar-tate or alanine for glutamate and NAD phos-phate (NADP) for NAD, the significance ofthese substitutions will have to await moreextensive purification of the enzymes involved.The second scan in Fig. 3 represents an in-

cubation mixture containing an enzyme prepa-ration from a serl mutant (MO-171-11B) andreveals the absence of P-serine and serine butthe accumulation of a new labeled peak con-firmed as P-OH-pyruvate in seven solvent sys-tems. No P-OH-pyruvate peak was formedwhen NAD was omitted. This suggested thatthe serl mutant possessed PGA dehydrogenaseactivity but was blocked after P-OH-pyruvate.We were able to demonstrate P-serine phos-phatase activity in the serl mutant compa-rable to that in SER strains. Thus, for exam-ple, in one experiment the enzyme preparationfrom a SER strain exhibited P-serine phospha-tase activity of 2,326 counts per min of serineformed per 30 min compared to 2,516 countsper min of serine formed per 30 min by a serlenzyme preparation at comparable proteinconcentration (2.5 mg of protein/ml). There-fore, it is possible to conclude that the serlmutant was blocked at L-glutamate: P-OH-pyruvate transaminase and accumulated P-OH-pyruvate from PGA.

Reconstruction experiments combining serland SER enzyme preparations, or ammonium

sulfate fractions thereof, indicated that theserl extracts did not contain an inhibitor ofthe phosphorylated pathway and that the P-OH-pyruvate accumulated in serl enzyme in-cubations was metabolized to P-serine by saltfractions from SER strains incapable of con-verting PGA to P-serine.The third scan describes an incubation mix-

ture containing an enzyme preparation from aleaky ser2 mutant (MO-170-22B) and reveals amarked accumulation of P-serine in the ab-sence of F- ion, comparable to that in the SERstrain in the presence of F- ion. This sug-gested a lesion in P-serine phosphatase in ser2.The leakiness of this ser2 mutant is indicatedby slow growth on minimal medium notedabove. Slow serine production may be due ei-ther to an altered enzyme or to nonspecificphosphatase activity.The accumulation of P-OH-pyruvate from

PGA by serl preparations presented an oppor-tunity for testing PGA dehydrogenase as thepossible site for serine feedback inhibition.Table 3 shows that the addition of increasingamounts of serine to incubation mixtures con-taining PGA and serl enzyme preparations diddecrease the rate of P-OH-pyruvate produc-tion.Attempts to demonstrate a nonphosphor-

ylated pathway from glycerate to serine.Similar experiments with labeled glyceratefailed to detect any evidence for a nonphos-phorylated pathway from D-glycerate to L-serine in the serine independent Saccharo-myces strains tested.TCA cycle pathway to glyoxylate, gly-

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I-

J

In

LJ-i

HOURS

0 5 10 15 20HOURS

FIG. 2. (A) Growth response of strain MO-171-JIB, serl. Symbols: A, glucose minimal medium; *,glucose minimal medium plus 300 mg of L-serineper liter; 0, glucose minimal medium plus 30 mg ofglycine per liter; *, glucose minimal medium plus300 mg of sodium glyoxylate per liter; 0, glucoseminimal plus 40 mg of adenine sulfate per liter. (B)Growth response of strain MO-170-22B, ser2. Sym-bols are as described above.

cine, and serine. SER, serl, and ser2 culturesharvested in stationary phase from glucosecomplex media were used for the production ofyeast enzyme preparations and assayed forserine transhydroxymethylase activity. In viewof the reported lability of this enzyme (2),

preparations were freed of small molecules byrapid, Sephadex G-25 column filtration in thecold, rather than by slow dialysis. Incubationof the enzyme preparation with 14C-glycineproduced 14C-serine with complete, disappear-ance of substrate glycine in 1 hr and the ap-pearance of product serine. The system wasdependent on the presence of FH4, PALP, andformaldehyde as shown in Table 4. It wasfound that SER, serl, and ser2 strains yieldedcomparable activities for serine transhydrox-ymethylase and that the mutants were notlacking this enzyme. The presence of this en-zyme in the mutants had already been inferredfrom the ability of glycine to substitute forserine in the growth experiments described.

Similar yeast enzyme preparations from sta-tionary-phase cultures grown on glucose com-plex medium also were found to exhibitalanine: glyoxylate transaminase activity, con-verting 14C-glyoxylate to 14C-glycine. Table 5represents an experiment with an enzymepreparation from a SER strain. Alanine wasfound to be the best amino donor, with gluta-mate, glutamine, aspartate, and asparagineyielding very low activity by comparison. Thisdemonstration, the first in yeast, differs fromthe situation in other organisms where, in ad-dition to alanine:glyoxylate transaminases,glutamate: glyoxylate transaminases have beendescribed (7, 17). These transaminases, like theone in yeast, appear to be irreversible.Tested in the same manner as the SER

strain, the serl and ser2 enzyme preparationsyielded comparable transaminase activitiesand no evidence, therefore, of a mutant lesionin this enzyme system.The conversion of isocitrate to glyoxylate by

isocitrate lyase was also demonstrated in thethree strains. A comparison of the specific ac-tivities of isocitrate lyase in yeast enzymepreparations from SER, serl, and ser2 culturesgrown under various conditions is representedin Table 6. No evidence of a mutant lesion af-fecting isocitrate lyase was found in the serl orser2 strains, but the earlier report of glucoserepression of isocitrate lyase (22) was con-firmed in these experiments.

Since there was no evidence found for amutant lesion in the glyoxylate pathway in theserl or ser2 mutant, the glucose repression ofisocitrate lyase seemed to provide a possiblemechanism for the necessary physiologicalregulation of the secondary pathway, if a blockin the principal pathway were to produce aux-otrophy.The fact that the serl strain failed to grow

on a glucose minimal medium plus glyoxylate,

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VOL. 109, 1972 BIOSYNTHESIS IN SACCHAROMYCES 39

SER ENZYME PREPARATIoNCOMPLETE' -

- * s . * _.3HOURS

*I PGA

- - * t *SER1I4E

P-SERINE | - -|

ENZYNW PREPARATION COMPLETE

3 HOURS

PGAP-OH- PYRUVATE

- - see2 ENZYMEPREPARATION COMPLETE......... W_ A 3-.

H3OURS

* .| P-SERINE _ . ._

a *\ *5 _\ t ~PGA

... .** _ s _ * SER ENZYME PREPARATION COMPLETE PLUS NSaFLUORIDE *

5 .. 5*\_. * _ 3 HOURS, *_ *

P-SERINE- -. .,

MSELN8E SOLVENT FRONT

FIG. 3. Phosphorylated pathway. A comparison of radiochromatogram scans showing the metabolism of 3-phospho-D-glycerate-U-'4C by enzyme preparations from SER, serl, and ser2 yeast strains. The bottom scanis that of the complete system plus 20 Amoles of sodium fluoride per 100 piliters. Chromatograms were devel-oped in solvent IX and subsequently in solvent L

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TABLE 2. Conversion of 14C-phosphoglycerate to14C-phosphoserine

PhosphoserineIncubation mixturea formed in 2 hr

(counts per min)

Complete (+ sodium fluoride) 4,260- NAD5 <50- Glutamate 549- Glutamate + alanine 1,106- Glutamate + aspartate 1,921- Glutamate + isoleucine 590- Glutamate + phenylalanine 368- Glutamate + leucine 544- Glutamate + valine 478- Glutamate + lysine 530- Glutamate + arginine 503

a Complete system (Materials and Methods) con-taining sodium fluoride was incubated at 30 C for 2hr. Chromatograms were spotted with 5 uliters ofeach incubation mixture, developed in solvent IX(Table 1), and analyzed in the radiochromatogramstrip scanner with the digital integrator.

b Nicotinamide adenine dinucleotide.

TABLE 3. Effects of increasing amounts of serine onthe formation of 14C-phosphohydroxypyruvate (14C-P-OH-pyruvate) from 1 4C-3-phospho- D-glyceric acida

% InhibitionRadioactivity in of P-OH-

Concn of L-serine '4C-P-OH- pyruvate(molar) pyruvate peak production in

(counts per min) the presenceof serine

0 1,345 05.0 x 10-4 1,148 152.0 x 10-s 1,022 254.0 x 10-1 696 498.0 x 10-i <50 100

a Chromatograms were spotted after 2 hr of in-cubation with 5 gliters of the complete F--inhibitedassay system for the phosphorylated pathway plusthe indicated amounts of unlabeled L-serine. Chro-matograms were developed in solvent IX andscanned on the actigraph III employing the digitalintegrator.

TABLE 4. Serine transhydroxymethylase activity

"C-Serine formedIncubation mixturea per hr

(counts per min)

Complete 3,520- FH4 <150- Formaldehyde < 150- PALP <150- Formaldehyde + formate < 150

a Abbreviations: FH4, tetrahydrofolic acid; PALP,pyridoxal phosphate.

TABLE 5. Conversion of 1 4C-glyoxylate to '4C-glycine in the presence of different amino donorsa

Glycine formedIncubation mixture per 40 min

(counts per min)

Complete 3,638- L-Alanine 75- L-Alanine + L-glutamate 352- L-Alanine + L-glutamine 780- L-Alanine + L-aspartate 116- L-Alanine + L-asparagine 218

a Chromatograms were spotted with 5 pliters ofeach incubation mixture (Materials and Methods)after 40 min of incubation, developed in solvent VI,and analyzed with a radiochromatogram stripscanner with a digital integrator.

TABLE 6. Specific activities of isocitrate lyase inyeast enzyme preparations from various strains and

growth conditions

Specific activities(nmoles of glyoxylate

per min per mgof protein)

Geno- Strain Growth conditionstype

Station- Log-ary- phase Lgphase glu- phaseglucose cose acetate

SER Budweiser cake yeast 45.68aSER MO-11-48A 42.89 1.07 78.00serl MO-171-11B 53.05 0.66 92.00ser2 MO-170-22B 75.05

a This enzyme preparation was made directly from acommercial sample of the cake yeast.

however, suggested that pathway regulation byisocitrate lyase shown above was an inade-quate explanation for serine auxotrophy andthat still another site of control might exist inthe secondary pathway beyond glyoxylate. Forthis reason, the glucose repressibility of thealanine: glyoxylate transaminase was alsotested.Enzyme preparations from both logarithmic

and stationary-phase cultures of the serl mu-tant grown in a glucose complex medium weretested for alanine: glyoxylate transaminase ac-tivity. The results in Table 7 show that therewas no measurable transaminase activity inpreparations from the glucose logarithmicphase culture, whereas activity was restored inthe stationary phase enzyme preparation. Thisdemonstrated that the alanine: glyoxylatetransaminase, in addition to isocitrate lyase,was glucose repressed.

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What had been uncovered, therefore, weretwo sites of control of the glyoxylate pathwayto glycine and serine. It should thus be pos-sible for a serine auxotroph, blocked in thephosphorylated pathway, to become proto-trophic if cultured on a medium containing aderepressing carbon source. Results of such anexperiment are illustrated in Fig. 4. The serlstrain, cultured in the presence of acetate assole carbon source, became serine-independentand grew slowly on a minimal medium. Thisstrain grew more rapidly on an acetate min-imal medium supplemented with glyoxylate.When 0.1% glucose was added to the acetateminimal or the acetate minimal plus glyoxy-late medium, however, the strain behaved asan auxotroph and did not grow. Plating testsfor reversion of growing cultures were negative.A yeast enzyme preparation from a serl

strain grown under derepressed conditions onacetate minimal plus glyoxylate medium con-verted 14C-PGA to 14C-P-OH-pyruvate butproduced no P-serine or serine. It is thus ap-parent that, under derepressed growth condi-tions in acetate media, serine is being synthe-sized via the glyoxylate pathway rather thanby a possible derepression of any step in thephosphorylated pathway.

DISCUSSIONThe present study has clarified some aspects

of serine biosynthesis in Saccharomyces. Al-though it was suggested in another fungal spe-cies, Neurospora crassa (16), that both phos-phorylated and nonphosphorylated routes fromglycolysis led to serine, the situation in Sac-charomyces was uncertain because only indi-rect evidence was available (4), suggesting thatsome glycolytic route beginning above pyru-vate led to serine. We have shown that thephosphorylated route is present but could findno evidence for the nonphosphorylated routein two serine-independent yeast strains.The present study has also revealed a phe-

riomenon of conditional auxotrophy. This maybe defined at the functional level as exhibitinga growth requirement under one set of condi-tions but not under another set of conditions.The term conditional auxotroph is suggestedby analogy to the terms conditional lethal andconditional sensitive, already well establishedin microbial genetic usage. We have, in addi-tion, revealed one mechanism for conditionalauxotrophy, i.e., derepression of enzymes in asecondary metabolic pathway where the pri-mary biosynthetic pathway is blocked by agenetic lesion. Although the role of other path-

TABLE 7. Alanine:glyoxylate transaminase activityin enzyme preparations from log- and stationary-

phase cultures ofa serl straina

Radioactivity in glycineafter 1 hr of incubation

Incubation mixture (counts per min)StationaryLog phase phase

Complete ................. < 50 3,540- Enzyme, PALP <50- L-Alanine 120

a Five Mliters of the indicated incubation mixtureswas spotted after a 1 hr of incubation and chromato-graphed in solvent II. Radiochromatograms werescanned on the actigraph III and peak heights weremeasured with the digital integrator. After incuba-tions longer than 2 hr in the absence of pyridoxalphosphate (PALP) and enzyme, a small amount ofglycine was formed nonenzymatically.

160

140

120 -

100

so

60_ /o60~~~~

40

20

0 10 20 30 40 S0

HOURS

FIG. 4. Growth response of strain MO-171-11B,serl, on a derepressing carbon source. Symbols: 0,acetate minimal medium plus 300 mg of L serine perliter; 0, acetate minimal medium plus 4.0 g of so-dium glyoxylate per liter; 0, acetate minimal me-dium; U, acetate minimal medium plus 4.0 g of so-dium glyoxylate per liter plus 1.0 g glucose per liter.

ways to a given metabolite has been appre-ciated for some time as providing a possibleescape from the limitation imposed by a muta-tion in the primary biosynthetic pathway, ithas normally been assumed that this escaperequired a second genetic event restoring anenzyme of the secondary pathway to functionalactivity. Against this background, conditional

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auxotrophy becomes the circumvention of ablock in a primary biosynthetic pathway byrestoring a secondary pathway to functionwithout an intervening genetic event. In thecase at hand, conditional serine auxotrophs inyeast, serl and ser2, were shown to be blockedin the primary biosynthetic pathway, fromglycolysis to serine and glycine, based on ge-netic lesions in L-glutamate: P-OH-pyruvatetransaminase and P-serine phosphatase bio-synthesis, respectively. These lesions are ex-pressed as a growth requirement for serine orglycine in glucose media since the otherpathway to glycine and serine from the tricar-boxylic acid cycle is repressed by glucose atisocitrate lyase and alanine:glyoxylate trans-aminase so that this pathway is nonfunctionalin glucose media. Under derepressed condi-tions (i.e., acetate media), the secondarypathway becomes functional and slow growthof the mutant without added glycine or serineis demonstrated.The recognition of conditional auxotrophy in

no way contradicts the existence of pathwaysuppressors (i.e., the circumvention of a ge-netic lesion in a primary biosynthetic pathwayby a second genetic event restoring a sec-ondary metabolic pathway to activity).

Since the second genetic event might in-volve either structural or regulatory genes, it iseven possible to imagine the genetic eventleading to the pathway suppressor as involvinga mutation to a state of conditional auxo-trophy.From this vantage point, it is interesting to

view the primacy of one or another metabolicpathway to a given intermediate in more rela-tivistic fashion than has hitherto been cus-tomary. For example, mutant analysis, alreadyapplied to Escherichia coli, Salmonella typhi-murium, and Haemophilus influenzae (9, 12,18), has suggested that the phosphorylatedpathway is the principal biosynthetic route inyeast to serine, which, therefore, is the "nor-mal" precursor of glycine rather than viceversa. Thus, if a genetic lesion in one of severalmetabolic pathways to a given intermediateproduces auxotrophy, it is assumed to be theprimary biosynthetic pathway. The question ofprimacy may however depend on regulatorymechanisms and growth conditions. It is con-ceivable that conditional auxotrophs in theglyoxylate pathway might be induced and iso-lated based on a requirement for glycine andserine if there were growth conditions whichrepressed the phosphorylated pathway toserine. If such mutants could be obtained, onecould argue with comparable logic for the pri-macy of the glyoxylate pathway to serine when

acetate is the principal carbon source, withglycine serving as the "normal" serine pre-cursor.

In the current study, we have not clarifiedthe nature of the system yielding the one-carbon fragment when the serl mutant grewon glycine. A number of independent serl iso-lates recently obtained by ethylmethane-sulfonate treatment and also blocked at L-glu-tamate: P-OH-pyruvate transaminase grew onglycine plus formate but not on glycine alone.Our records indicate that the serl strain usedprimarily in the current study had become al-tered over years in the Carbondale stock cul-ture collection developing the ability to gen-erate the one-carbon fragment endogenously,presumably either from the carbon source orfrom glycine by the glycine cleavage reactionalready reported (14). Cell-free preparations,even of the latter strain, however, did not con-vert '4C-glycine to '4C-serine unless formalde-hyde was added. It seems possible that theglycine cleavage system may have been labileto the techniques used to produce yeast en-zyme preparations. It should be noted, how-ever, that a methionine auxotroph in Saccharo-myces (met5), known to lack serine transhy-droxymethylase, requires methionine becauseof an apparent lack of an available one-carbonfragment for methionine biosynthesis. We havefound that glycine will not substitute for me-thionine in met5 strains, and one must inferthat the glycine cleavage system is either ab-sent or nonfunctional in the met5 strains aswell as in the newly induced serl strains wehave examined. Pizer has reported (11) that inan E. coli mutant blocked at serine transhy-droxymethylase (requiring glycine), the glycinealpha carbons provided 30 to 40% of the one-carbon fragments needed by the cell. Still an-other view of this E. coli mutant has been ex-pressed recently by Newman (Bacteriol. Proc.,p. 133, 1971). It is apparent that some clarifi-cation is still needed of the source and controlof one-carbon fragments in serine and methio-nine biosynthesis.

ACKNOWLEDGMENTSWe thank the many people who provided the strains on

which the present Carbondale collections are based.This investigation was aided by the Graduate School,

Southern Illinois University, and by grants from the Amer-ican Cancer Society and the National Institute of Arthritisand Metabolic Diseases, National Institutes of Health.

LITERATURE CITED

1. Blakley, R. L. 1957. The interconversion of serine andglycine: preparation and properties of catalytic deriv-atives of pteroyl-glutamic acid. Biochem. J. 65:331-342.

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2. Botsford, J. L., Jr., and L. W. Parks. 1969. Serine trans-hydroxymethylase in methionine biosynthesis inSaccharomyces cerevisiae. J. Bacteriol. 97:1176-1183.

3. DeBoiso, J. F., and A. 0. M. Stoppani. 1967. Metabo-lism of serine and glycine in baker's yeast. Biochim.Biophys. Acta 148:48-59.

4. Gilvarg, C., and K. Bloch. 1951. The utilization of aceticacid for amino acid synthesis in yeast. J. Biol. Chem.193:339-346.

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6. Ichihara, A., and D. M. Greenberg. 1957. Further studieson the pathway of serine formation from carbohy-drate. J. Biol. Chem. 224:331-340.

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8. Olson, J. A. 1959. The purification and properties ofyeast isocitric lyase. J. Biol. Chem. 234:5-10.

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10. Pizer, L. I. 1964. Enzymology and regulation of serinebiosynthesis in cultured human cells. J. Biol. Chem.239:4219-4226.

11. Pizer, L. I. 1965. Glycine synthesis and metabolism inEscherichia coli. J. Bacteriol. 89:1145-1150.

12. Pizer, L. I., M. Ponce-De-Leon, and J. Michalaka. 1969.Serine biosynthesis and regulation in Haemophilusinfluenzae. J. Bacteriol. 97:1357-1361.

13. Rabson, R., N. E. Tolbert, and P. C. Kearney. 1962.

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Formation of serine and glyceric acid by the glycolatepathway. Arch. Biochem. Biophys. 98:154-163.

14. Sagers, R. D., and I. C. Gunsalus. 1961. Intermediarymetabolism of Diplococcus glycinophilus. I. Glycinecleavage and one carbon interconversions. J. Bac-teriol. 81:541-549.

15. Schramm, M. 1958. o-Phosphoserine phosphatase frombaker's yeast. J. Biol. Chem. 233:1169-1171.

16. Sojka, G. A., and H. R. Garner. 1967. The serine biosyn-thetic pathway in Neurospora crassa. Biochim. Bio-phys. Acta 148:42-47.

17. Thompson, J. S., and K. E. Richardson. 1966. Isolationand characterization of a glutamate-glycine transami-nase from human liver. Arch. Biochem. Biophys. 117:599-603.

18. Umbarger, H. E., M. A. Umbarger, and P. M. L. Siu.1963. Biosynthesis of serine in Escherichia coli andSalmonella typhimurium. J. Bacteriol. 85:1431-1439.

19. Waddell, W. J. 1956. A simple ultraviolet spectropho-tometric method for the determination of protein. J.Lab. Clin. Med. 48:311-314.

20. Wang, D., and E. R. Waygood. 1962. Carbon metabolismof C '-labelled amino acids in wheat leaves. I. A path-way of glyoxylate-serine metabolism. Plant Physiol.37:826-832.

21. Willis, J. E., and H. J. Sallach. 1962. Evidence for a

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Alkoholdehydrogenase, Malatdehydrogenase, Isocit-ratlyase, und Malatsynthase in Hefe durch Glucose.Biochim. Biophys. Acta 118:522-537.

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