Proc. Nati. Acad. Sci. USAVol. 83, pp. 2378-2382, April 1986Biochemistry

Sporulation of the yeast Saccharomyces cerevisiae is accompaniedby synthesis of adenosine 5'-tetraphosphate andadenosine 5'-pentaphosphate

(two-dimensional TLC/in vivo 32P-labeling/signal nucleotides/nitrogen starvation/development)

HIERONIM JAKUBOWSKI*Department of Microbiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103

Communicated by Bruce N. Ames, December 13, 1985

ABSTRACT Two-dimensional TLC analysis of 32P-labelednucleotides extracted from the yeast Saccharomyces cerevisiaereveals that two highly phosphorylated nucleotides are synthe-sized during sporulation. These nucleotides have been identi-fied as adenosine 5'-tetraphosphate (ppppA) and adenosine5' pentaphosphate (pppppA). The synthesis of ppppA andpppppA commences late in sporulation and follows formationof ascospores. The maximum concentration of ppppA andpppppA in sporulating yeast cultures was 2% and 1.5%,respectively, that of ATP. Adenosine 5'-tetraphosphate and5'-pentaphosphate are unique to this stage of yeast develop-ment and are absent in vegetative yeast cells. Since thesenucleotides are also absent in asporogenous a/a and a/a cells,it is reasonable to propose that they are signal nucleotidesmarking one of the stages ofyeast development-i.e., ascosporeformation.

Upon nutrient deprivation, many bacilli and fungi undergo aseries of complex biochemical, genetic, and morphologicalchanges that lead ultimately to the formation of spores.Sporulation of the yeast Saccharomyces cerevisiae providesa relatively simple model system for the study of eukaryoticcell differentiation. Many studies have defined some bio-chemical, genetic, and morphological events during sporula-tion of S. cerevisiae (1). Profound changes in gene expressionduring sporulation have been observed (2-6). However, themolecular details of mechanisms responsible for these effectsare still obscure. Conceivably, the molecular mechanismsunderlying yeast sporulation may involve some yet uniden-tified low molecular weight nucleotides as effectors. CyclicAMP and ppGpp have already become textbook examples oflow molecular weight compounds involved in transcriptionalregulation of gene expression in bacteria (e.g., see refs. 7 and8), but analogous mechanisms are unknown in eukaryotes (9).Diadenosine tetraphosphate (AppppA), a ubiquitous dinucle-otide (10, 11) whose concentration in mammalian cells bearsa positive relationship to DNA synthesis and mitosis, hasbeen proposed to be a pleiotropic effector or signal nucleotide(12).The work reported in this paper was motivated by a search

for AppppA and related compounds in yeast cultures atvarious times during the course of sporulation. Formic acidextracts of [32P]phosphate-labeled yeast cultures were ana-lyzed by two-dimensional TLC on PEI-cellulose plates. NoAppppA was detected in yeast during the course of sporula-tion. Instead, two sporulation-specific 32P-labeled nucleo-tides, identified as adenosine 5'-tetraphosphate (ppppA) andadenosine 5'-pentaphosphate (pppppA) and appearing late insporulation, were detected.

MATERIALS AND METHODSYeast Strains. Several strains of S. cerevisiae were used.

The diploid strain AP3 has the genotypea adel ade2 gall tyrl lys2 lys7 ural + + +

a + ade2 + + + + + ura3 can) cyh2+ CSP+

leul CSP+The corresponding asporogenous cell types, a/a and a/a, areisogenic to AP3 except at the MAT locus, where they arehomozygous. The diploid LM1 has the genotype

a adel ade2 + CANS gaII-4 his7-1a + ade2 adeS canR + his7-2

+ lys2-2 + + tyrl-2 ural +leul-12 lys2-1 metl3d trpSd tyri-l + ura3-13

Diploid SK1, - was also examined.a HO

Growth, Sporulation, and [32P]Phosphate-Labeling Condi-tions. Cells were grown on 1% (wt/vol) yeast extract(Difco)/2% (wt/vol) bactopeptone (Difco)/2% (wt/vol) glu-cose (YEPD) at 30°C in a New Brunswick (New Brunswick,NJ) gyratory shaker (at 250 rpm). Approximately 12 hr afterthe end of exponential growth (12-14 hr after reaching 200klett units), cells were harvested by centrifugation (4000 rpmfor S min at 20°C), washed once with supplemented sporula-tion medium [1% (wt/vol) KOAc, pH 6.5/0.05% (wt/vol)glucose/0.1% (wt/vol) yeast extract], and resuspended (in125-ml Erlenmeyer flasks) in 5 ml of supplemented sporula-tion medium containing carrier-free [32P]phosphate at 0.1mCi/ml (1 Ci = 37 GBq) (Amersham). Final cell density was2.5 x 108 cells per ml. Sporulation cultures were maintainedat 30°C with vigorous aeration in a New Brunswick rotaryshaker set at 250 rpm.

Alternatively, the cells were grown and 32Pi-labeled inlow-phosphate YEPD medium and sporulated in 1% KOAc(pH 6.5). The low-phosphate medium was prepared fromYEPD medium by precipitation of phosphate at MgNH4PO4(13). The cultures were inoculated at 5 x 105 cells per ml in5 ml of low-phosphate YEPD containing carrier-free[32P]phosphate (0.1 mCi/ml) and grown as before. Station-ary-phase cultures were harvested by centrifugation, washedonce with 1% KOAc (pH 6.5), resuspended in 1% KOAc (pH6.5) at a cell density of 2.5 x 108 cells per ml and maintainedat 30°C with vigorous aeration.

Preparation of [32P]Phosphate-Labeled Nucleotide Extracts.At specified time intervals, aliquots (200 ,ul) of the 32p;-labeled yeast cultures were removed, extracted with 8 Al of

*Visiting Scientist from the Institute of Biochemistry, University ofAgriculture, Poznan, Poland.

2378

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Proc. Nadl. Acad. Sci. USA 83 (1986) 2379

24 M formic acid for 30 min at 0WC and then frozen overnightat -20'C. After thawing, the extracts were clarified bycentrifugation in an Eppendorf centrifuge for 5 min at 40C,and the clear formic acid extracts were analyzed immediatelyor stored at -20'C. To assess whether any nucleotides areexcreted by yeast cells during sporulation, aliquots (200 jdl)of 32Pi-labeled yeast cultures were centrifuged in the Ep-pendorf centrifuge for 2-3 min at room temperature, and clearsupernatants were collected and either analyzed immediatelyor stored at -20'C. All samples from any single experimentwere processed within 10-14 days. Storage at -20'C did notaffect the stability of any of the nucleotides analyzed.Two-Dimensional TLC Analysis of 32P-Labeled Nucleotides

in Yeast Extracts. The procedures used were as described byBochner and Ames (14) with some modifications. Extracts(10-20 ,s1) were applied as a spot on PEI-cellulose plates (20x 20 cm; Merck or Machery & Nagel, Cologne, F.R.G.).Either 0.85 M (with Merck TLC plates) or 1.2 M LiCl (withMachery & Nagel TLC plates) was used as the first-dimen-sion solvent, and 3 M (NH4)2SO4/2% disodium EDTA wasused as the second-dimension solvent. This solvent systemgives resolution similar to that of the system used by Bochnerand Ames (14) for separation of nucleotides with highnegative charge. Usually two to four standard nucleotides,applied as 1-2 ul of 10 mM solution on the plate, werechromatographed with the 32P-labeled samples. The stan-dards were located under UV light. 32P-labeled compoundswere visualized by autoradiography at -80'C using KodakXAR-5 film and cassettes with intensifying screens. Forquantitation purposes, the 32p spots were cut out from thechromatogram, placed in a toluene-based scintillation fluid,and counted in a scintillation counter.Enzymatic Synthesis of ppppA and pppppA. Inorganic

tripolyphosphate and tetrapolyphosphate were adenylylatedin reactions catalyzed by aminoacyl-tRNA synthetases.ppppA has been synthesized in an analogous reaction cat-alyzed by lysyl-tRNA synthetase (15). The reaction mixture(100 ,ul) contained 50 mM Hepes (pH 8.0), 20 mM MgCl2, 2.5mM ATP, 5 mM Na5P30io or (NH4)6P4013, 0.1 mM eachalanine, lysine, phenylalanine, and proline, 0.4mM ZnCl2, 2,gof yeast inorganic pyrophosphatase (Sigma) and 50 ,ug ofEscherichia coli aminoacyl-tRNA synthetases preparation [en-riched in activities for alanine, lysine, phenylalanine, andproline by chromatography on aminohexyl-Sepharose (16) andseparated from diadenosine tetraphosphatase (17) by SephacrylG-200 gel filtration]. The reaction was completed after 15min at30°C as determined by disappearance of ATP in aliquots (2,ul)of the reaction mixture taken at time intervals and chro-matographed on PEI-cellulose (Merck) with 0.85 M LiCl assolvent. Only two products in roughly equimolar quantitieswere formed in each reaction: ADP and either ppppA orpppppA. After all ATP was used up, the reaction mixtures wereheated for 2 min at 100°C to precipitate protein, clarified bycentrifugation, and stored at -20°C.

Purification of Sporulation-Specific 32P-Labeled Nucleo-tides. A 5-ml culture of S. cerevisiae strain AP3 a/a wasgrown, labeled with [32P]phosphate, and sporulated as de-scribed above. After 40-48 hr in sporulation medium, theculture was centrifuged at room temperature for 5 min at 4000rpm, and the supernatant was collected and transferred onice. About 150 mg of acid-washed charcoal (Sigma) wasadded, and after 1 hr at 0°C the mixture was centrifuged andthe supernatant was discarded. The charcoal was washedthree times with 0.25 M HCOOH, once with water, and theadsorbed nucleotides were eluted with four portions of 1%ammonia in 50% ethanol (0.4 ml each). The eluates werecombined, lyophilized, and dissolved in 100 /dl of H20. Fiftymicroliters of the preparation was applied on PEI-cellulose(Machery & Nagel) plate, and the plate was developed in thefirst dimension with 1.2 M LiCl and in the second dimension

with 3 M (NH4)2SO4/2% disodium EDTA. After location ofthe relevant compounds by autoradiography, the correspond-ing areas were scraped from the chromatogram. Each phos-phorylated nucleotide was eluted with two 0.15-ml portionsof 0.25 M NH4HCO3 for 1 hr at 0C. The PEI-cellulose waspelleted in an Eppendorf centrifuge for 5 min at 40C. The twoeluates were combined and used for analyses.

RESULTSSporulating Yeast Cells Synthesize Two Highly Phosphoryl-

ated Compounds. Diploid yeast strain AP3 a/a was sporu-lated in supplemented sporulation medium containing 32P,.First asci appeared at 15 hr; at 30 hr, 60% of cells sporulated,and the maximum 65% of sporulated cells were observedfrom 42 hr on. Within the first hour, 95% of 32P, was taken upby yeast cells, and after a few hours, the 32P, content in themedium began to increase, reaching -50% of the originalinput at 42 hr. Two nonsporulating diploid strains, AP3 a/aand AP3 a/a, were grown and transferred to nitrogen-defi-cient sporulation medium in an identical manner.

After 42 hr in the supplemented sporulation mediumcontaining 32Pi, the 32P-labeled nucleotides were extractedfrom the three yeast strains and resolved by two-dimensionalTLC on PEI-cellulose plates. Autoradiograms exposed fromtwo ofthese chromatograms are presented in Fig. 1. Two new32p spots appeared in the AP3 a/a strain (spots 1 and 2 in Fig.1A) but not in asporogenous AP3 a/a (Fig. 1B) and AP3 a/astrains (not shown). One of these spots comigrated withauthentic ppppA (spot 1) and the other comigrated withpppppA (spot 2). Usually, the two-dimensional resolutionof the 32P-labeled nucleotides from formic acid extractsprepared from yeast maintained in sporulation medium wasrather poor because of overloading of the plates, which wasrequired for the detection of the new spots. However, thisproblem was overcome by the observation that the 32p_labeled nucleotides are excreted by yeast cells maintained insporulation medium. 32P-labeled nucleotides present in cell-free medium prepared from AP3 a/a, a/a, and a/a cultureswere resolved by two-dimensional TLC on PEI-celluloseplates. Fig. 2A shows a schematic diagram of the resolution.Fig. 2 B-D are autoradiograms of 32P,-labeled cell-free me-dium prepared from the yeast cultures that have beenmaintained for 42 hr in sporulation medium containing 32P1. Itis apparent that two clearly separated 32p spots (labeled 1 and2 in Fig. 2B) appeared in the sporulating a/a culture, but theywere absent in asporogenous a/a and a/a cultures. Again,these spots comigrated with authentic ppppA and pppppA

B

FIG. 1. Two-dimensional TLC separation of nucleotides fromyeast cultures maintained in sporulation medium. LiCl (1.2 M) wasused for the first-dimension solvent, and 3 M (NH4)2SO4/2% diso-dium EDTA was used for the second-dimension solvent. Autoradi-ograms exposed from the two-dimensional separations of formic acidextracts from 32P,-labeled diploid yeast AP3 a/a (A) and AP3 a/a (B)cultures maintained in sporulation medium for 42 hr are shown. Thetwo 32p spots appearing in a/a but not in a/a cultures are indicatedwith numbers 1 and 2.

Biochemistry: Jakubowski

Proc. Natl. Acad. Sci. USA 83 (1986)

A NADP| O~~C(IMPGDP-maYe (ZCMP

ApppA /AMP

ADPr~ '- GMPAppppAT n QGDP

3'5iADPCC0,ATP cGi1 ppppA'GtP

pppppA

2nd__

C

I--

4..r

FIG. 2. Two-dimensional TLC separation of nucleotides excret-ed by yeast cells maintained in sporulation medium. First dimension,1.2 M LiCl; second dimension, 3 M (NH4)2SO4/2% disodium EDTA.(A) Identities ofthe nucleotides based on comigration with standards.Also shown are chromatographic locations of ApppA and AppppAstandards (broken lines). Autoradiograms exposed from the two-dimensional separations of cell-free medium from 32P,-labeled diploidyeast AP3 a/a (B), AP3 a/a (C), and AP3 a/a (D), culturesmaintained in sporulation medium for 42 hr, are shown. Numbers 1and 2 indicate the two spots appearing in a/a but not in a/a and a/acultures.

standards, respectively. No other significant differences innucleotide pools in sporulating and nonsporulating yeastwere seen on the autoradiograms.As can be seen from the autoradiograms presented in Figs.

1 and 2, there are no 32p spots that comigrate with AppppAand ApppA standards. Actually, I did not detect AppppA orApppA at any time during sporulation in the two yeast strainsexamined (AP3 a/a and SK1). Both AppppA and ApppAwould have been detected ifpresent at concentrations at least0.1% that of ATP. AppppA has been detected in logarithmic-phase yeast cultures and its content was 3.6 pmol per gg ofprotein (11), which is -0.02% of the ATP content (19). Themaximal increase, if there is any, in AppppA concentrationin sporulating yeast could not be more than by a factor of 5over its concentration in vegetative yeast cells, which wouldbe much less dramatic than changes in AppppA content inrapidly proliferating mammalian cells (12).

Characterization of the Two New Compounds Synthesizedby Sporulating Yeast. Each of the two new compounds waspurified from cell-free medium of sporulating AP3 a/a cellsby charcoal adsorption and elution followed by two-dimen-sional TLC on PEI-cellulose as described in Materials andMethods. From the method of purification, it follows that thetwo 32P-labeled compounds are nucleotides, since they areadsorbed on charcoal. As already mentioned in the previoussection, one of the 32p spots (spot 1) comigrated withauthentic ppppA and the other (spot 2) comigrated withauthentic pppppA standards. It should be emphasized thatthe nucleotide patterns on two-dimensional PEI-celluloseplates do vary somewhat from one run to the other, but in allruns the standards comigrated exactly with the two new 32pspots.

To further prove their identities, the two purified 32p spotswere subjected to several other tests. Fig. 3 shows resolutionby TLC on PEI-cellulose ofthe products ofmild acid hydrolysisof the two 32p spots. The 32p spot that comigrates with ppppAyields four 32P-labeled products that comigrate with unlabeledphosphate, AMP, ADP, and ATP. The pattern of hydrolysis isthe same as that of the original ppppA standard (20). The 32pspot that comigrates with pppppA yields five 32P-labeled prod-ucts upon mild acid hydrolysis and subsequent resolution byTLC. These products comigrate with phosphate, AMP, ADP,ATP, and ppppA standards. Again, original pppppA yieldsidentical products of mild acid hydrolysis. The apparent simi-larity of the mild acid hydrolysis patterns of the two newcompounds indicates their close structural relationship.

Next, each of the two 32p spots was digested by phospha-tase and by venom phosphodiesterase. The products ofdigestions were analyzed by two one-dimensional TLCsystems that resolve nucleotides based on different principlesof separation (i.e., number of negative phosphate groups andtheir content of nucleobases, respectively). Patterns of theenzymatic digestions of both 32p spots were similar, andthose for spot 1 are shown in Fig. 4. All 32P radioactivity isrecovered as 32Pi after digestion of both spots with phospha-tase. Digestion of 32P-labeled spots 1 and 2 with phosphodi-esterase yields two 32p products. One of them comigrateswith AMP and the other comigrates with polyphosphatestandards. Ratio of 32P radioactivity recovered as AMP afterdigestion to that in spots 1 and 2 before digestion was 1:3.9and 1:4.85, respectively. Based on these criteria, one of thetwo compounds synthesized by sporulating yeast (spot 1) wasidentified as ppppA, and the other (spot 2) was identified aspppppA.Time Course of Synthesis of ppppA and pppppA by

Sporulating Yeast. Fig. 5 depicts the kinetics of synthesis ofppppA and pppppA in relation to kinetics of spore formationby the diploid AP3 a/a. Detectable quantities of the twoadenosine 5'-polyphosphates appear after 22 hr-i.e., afterspores had been formed-and reach maximal level after42-49 hr, which is maintained until the end of the experimentat 72 hr. Maximal concentration of ppppA formed during

...t -AMP

-ADP

-ATP

-ppppA--- pppppA

1 2 3 4

FIG. 3. One-dimensional resolution of the products of mild acidhydrolysis of spots 1 and 2. LiCl (1.2 M) was used as solvent. Mildacid hydrolysis was performed at 100'C for 1 hr in a solution (40 j1)of either purified 32P-labeled spot 1, 0.1 mM ppppA or purified32P-labeled spot 2, 0.1 mM pppppA in 1 mM HCl (pH 4.5). Afterhydrolysis, the solutions were lyophilized, taken up in 5-10 ul ofH20, and applied on a PEI-cellulose TLC plate. Autoradiogramexposed from the one-dimensional separations is shown. 32P-labeledspot 1 before (lane 1) and after (lane 2) hydrolysis. 32P-labeled spot2 before (lane 4) and after (lane 3) hydrolysis.

i

2380 Biochemistry: Jakubowski

Proc. Natl. Acad. Sci. USA 83 (1986) 2381

A B

9l

ppp-- IP.

AMP

ppppA- I

AMP

Origin s Origin

1 2 3 1 2 3

FIG. 4. One-dimensional resolution of enzymatic digestions ofspot 1. Solvents were 3 M (NH4)2SO4/2% disodium EDTA (A) and1.2 M LiCl (B). Digestions were performed overnight with eithervenom phosphodiesterase (0.1 mg/ml) or bacterial alkaline phospha-tase (5 pg/ml) in 0.25 M NH4HCO3 (pH 8.0) at 30°C. 32P-labeled spot1 was run before (lane 1) and after digestion with venom

phosphodiesterase (lane 2) or alkaline phosphatase (lane 3). In B,ppppA and ppp remain at the origin.

sporulation was 2% that of ATP. pppppA reached a some-what low maximal concentration (equal to 1.5% the concen-

tration of ATP). However, since most of the adenosine5'-polyphosphates are excreted into sporulation medium,actual cellular concentration of ppppA and pppppA may beat least one order of magnitude lower. Again, no ppppA or

pppppA was detected at any time in asporogenous a/a anda/a yeast cultures maintained in sporulation media for up to72 hr.ppppA and pppppA Are Synthesized Exclusively During

Sporulation of Yeast. As described above, ppppA and pp-pppA are made late during sporulation of AP3 a/a, whereasasporogenous a/a and a/a strains maintained under the sameconditions do not make them. To determine how general isthe ability to form ppppA and pppppA by yeast duning that

E 0.025 100-

a: 0.020 - O 0- 80

0.~~~~~~~~~~~~~~~~~~~~~~c< 0.010 60 x

Q coQL0. 0 10 20 3440007

0

a-< 0.005-2

CLC OO"0 ~10 20 30 40 50 60 70L

Time in sporulation medium, hr

FIG. 5. Sporulation and synthesis of ppppA and pppppA indiploid AP3 a/a. Cells pregrown in YEPD medium were washed withwater and transferred at time 0 to supplemented sporulation mediumcontaining 32P, (0.1 mCi/ml). Sporulation was detected with aphase-contrast microscope. At time intervals, aliquots of cell-freemedium were analyzed for ppppA and pppppA by two-dimensionalTLC on PEI-cellulose. 32P-labeled nucleotides in the culture wereextracted with formic acid and resolved in the same manner. Spotscontaining radioactive ATP, ppppA, and pppppA were counted on aliquid scintillation counter and molar ratios of ppppA/ATP (o) andpppppA/ATP (o) were calculated and plotted as a function of timein sporulation medium.

particular stage of development, I have tested two othersporulating yeast strains, SKi and LM1. In addition, toassess whether the two highly phosphorylated nucleotidesare sporulation specific or present also during other stages ofyeast development, I have attempted to detect ppppA andpppppA in diploid strains SKi and AP3 during various stagesof vegetative growth.The diploids SKi and LM1 were grown and transferred to

sporulation medium containing 32P;. After 48 hr, sporulatingcells were spun down and 32P-labeled nucleotides in clearcell-free medium were resolved by two-dimensional TLC onPEI-cellulose plates. The patterns of nucleotides from SKiand LM1 cultures were similar to that originally observedwith AP3 a/a culture (Fig. 2B) and, more significantly,ppppA and pppppA were detected also in these two cultures(not shown).

In another set of experiments, diploid AP3 a/a was grownin low-phosphate YEPD medium containing 32P1. At a celldensity of 2.5 x 107 cells per ml and 3 x 108 cells per ml, 89%and 98% of 32Pi, respectively, were taken up by the yeastcells. Formic acid extracts were prepared from cells duringlogarithmic growth (at a cell density of 2.5 x 107 cells per ml)and during late stationary phase (at a cell density of 3 x 108cells per ml). 32P-labeled nucleotides in the extracts wereresolved by two-dimensional TLC on PEI-cellulose plates.Autoradiograms exposed from these chromatograms weresimilar to that depicted in Fig. 1B. No ppppA and/or pppppAwas detected in vegetative diploid AP3 a/a strain eitherduring logarithmic growth or during stationary phase. Whenyeast cells from the same 32P1-labeled vegetative cultureswere transferred to phosphate-free sporulating medium (1%KOAc, pH 6.5) and 32P-labeled nucleotides were analyzed bytwo-dimensional TLC, ppppA and pppppA were detected.Amounts of the two adenosine 5'-polyphosphates and kinet-ics of their synthesis were similar to those presented in Fig.5. Similar results were obtained in analogous experimentswith diploid SK1. Taken together, these results indicate thatneither vegetative (logarithmic or stationary) nor asporoge-nous (a/a and a/a) yeast cells are able to synthesize ppppAand pppppA; hence, the synthesis of ppppA and pppppA issporulation specific.

DISCUSSIONIn this investigation, I attempted to identify nucleotide signalmolecules that may have a function in eukaryotic celldifferentiation. For that purpose, two-dimensional TLC onPEI-cellulose (14) was applied for resolution of nucleotidesextracted from 32Pi-labeled yeast cultures during variousstages of their life cycle. Analysis of the 32P-labeled nucleo-tides in various cultures of diploid yeast S. cerevisiaedemonstrates that highly phosphorylated nucleotides aresynthesized exclusively during one of the stages of yeastdevelopment-i.e., during sporulation. Characterization ofthese two compounds after purification by various enzymat-ic, chemical, and chromatographic methods has establishedtheir identity as ppppA and pppppA.The adenosine 5'-polyphosphates have not been detected

before in yeast. However, ppppA was reported to constituteas much as 33% ofATP preparations from ox muscle (21, 22).Commercial ATP from yeast was also found to contain 8%ppppA and 1% pppppA (23). In addition, ppppA has beenreported in horse muscle (20), rabbit muscle (24), in rat liverslices incubated with [14C]adenine (15), and in isolated ratliver mitochondria incubated with 32p; (25). ppppA has beenreported to constitute 0.035% of the adenosine mononucle-otide content of rabbit muscle (24). As determined in thisstudy, ppppA and pppppA constitute 2% and 1.5%, respec-tively, of the ATP content of sporulating yeast cultures, while

Biochemistry: Jakubowski

Proc. Natl. Acad. Sci. USA 83 (1986)

in vegetatively growing cells or early during sporulation thecontent of ppppA and pppppA is <0.1% that of ATP.The origin ofppppA and pppppA formed in yeast spores is

unknown. However, in vitro ppppA can be synthesized fromtripolyphosphate and ATP in a back-reaction of aminoacyl-tRNA synthetases (ref. 15; this work) and from ATP and1,3-diphosphoglycerate in a reaction catalyzed by yeast3-phosphoglycerate kinase (24). As demonstrated in thiswork, pppppA can also be synthesized in vitro fromtetrapolyphosphate and ATP in a back-reaction ofaminoacyl-tRNA synthetases. Tri- and tetrapolyphosphates are the mostabundant (26) ofthe polyphosphates accumulated in vacuolesof yeast (27). Their concentration approaches and evenexceeds the concentration of ATP during growth (26). How-ever, during the growth of yeast cells in low-phosphatemedium, the polyphosphates do not accumulate (13, 18).Since ppppA and pppppA are synthesized by sporulatingyeast cells pregrown in both high-phosphate and low-phos-phate media, it does not seem likely that polyphosphatesaccumulated during vegetative growth are substrates for thesynthesis of the two highly phosphorylated nucleotides.Alternatively, the polyphosphates may be synthesized duringsporulation in phosphate-free or low-phosphate medium.This notion is supported by the observation that >50% of the32p; taken up by yeast cells at the beginning of sporulation isfinally excreted by asci. Apparently, the amount of phos-phate in vegetative cells, even in those grown in low-phosphate medium, is much in excess over the needs ofsporulating cells. The notion that tripolyphosphate andtetrapolyphosphate may be the precursors of ppppA andpppppA, respectively, is strengthened by direct measure-ments of acid-soluble polyphosphate in sporogenous andasporogenous yeast maintained in sporulation media. Thereis a substantial increase in levels of the polyphosphate duringsporulation of AP3 a/a but not in asporogenous strains AP3a/a and AP3 a/a maintained under identical conditions (notshown). Ifinorganic polyphosphates are precursors ofppppAand pppppA, there has to be some specific mechanism ofrelease of the polyphosphates from the vacuoles. Althoughvegetative yeast cells growing logarithmically contain highconcentrations of polyphosphates, the polyphosphates arenot available for synthesis of ppppA and pppppA. Theputative mechanism may not operate during vegetativegrowth (ppppA and pppppA are not detected during this stageof the yeast life cycle) but may be activated during differen-tiation.The finding that ppppA and pppppA are synthesized

exclusively in yeast cells that differentiated into asci but notduring other stages of yeast life cycle or in asporogenous a/aand a/a yeast cultures suggests that the function ofthose twohighly phosphorylated adenine nucleotides is associated withjust one specific stage of yeast development. I propose thatppppA and pppppA are signal nucleotides marking comple-tion of sporulation in yeast.

I am grateful to David Kaback and Steve Kurtz for yeast strainsused in this work. This work was supported by National Institutes ofHealth Grant GM27711 to Emanuel Goldman and in part by Foun-dation of the University of Medicine and Dentistry of New JerseyGrant 14-86.

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