Synthesis of ppGpp by mouse embryonic ribosomes

Cell, Vol. 3, 249-253,

November

1974,

Copyright

0 1974 by MIT

Synthesis of ppGpp by Mouse Embryonic Ribosomes J. D. Irr*, M. S. Kaulenast, and B. R. Unsworth Department of Biology Marquette University Milwaukee, Wisconsin 53233

Summary The unusual nucleotide guanosine tetraphosphate (ppGpp) is synthesized in vitro by ribosomes isolated from mouse embryos, but is not formed by ribosomes isolated from adults. Analysis of specific organs shows a developmental change in detectable ppGpp-forming ability. Eleven day embryonic liver ribosomes synthesize ppGpp, but this ability is essentially lost by 14 day embryonic liver and adult liver. Eleven day embryonic brain ribosomes also synthesize ppGpp at a level comparable to that observed using E. coli ribosomes. The synthesis of ppGpp appears to be associated with the early stages of differentiation, when cell proliferation is rapid and specialized protein synthesis is low or absent. The potential role of ppGpp as an effector molecule or regulator in the eucaryotes is discussed. Introduction Amino acid starvation of re/+ bacterial species causes the accumulation of the two nucleotides, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), (Cashel and Gallant, 1969; Gallant and Margason, 1972). These two nucleotides are synthesized by bacterial ribosomes (Haseltine et al., 1972) as an “idling reaction” under conditions which do not favor extensive protein synthesis (Cashel and Gallant, 1969). Recently, a number of groups have attempted to demonstrate production of ppGpp and pppGpp in eucaryotic cells without success (Fan, Fisher, and Edlin, 1973; Buckel and B&k, 1973; Alberghina et al., 1973; Mamont et al., 1972). However, Klein (1974) found that during the differentiative phase of the cellular slime mold Dictyostelium discoideum, induced by starvation of amoebae in axenic culture conditions, ppGpp accumulated to some extent. Since ppGpp is thought to be a negative effector of ribosomal RNA synthesis (Travers, 1973), Klein’s discovery can be correlated with the work of Kessin (1973) which shows inhibition of ribosomal RNA synthetic activity during this phase of development. The results of Klein’s study prompted us to investigate the production of ppGpp and pppGpp in a dif*Present address: Genetics Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. + On sabbatical leave from: Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01002.

ferentiating system. Richter (1973) has demonstrated that some mammalian cell ribosomes are incapable of forming these nucleotides. In some embryonic mouse tissues, however, we find that ribosomes, extracted from early developmental stages, direct ppGpp synthesis at rates almost equivalent to those observed with purified Escherichia coli ribosomes. Results Ribosomes were purified from eleven day mouse embryos, adult mouse livers, and a re/+ strain of E. coli. After establishing optimal conditions of the assay with the E. coli ribosomes, the mouse ribosome preparations were tested for the production of ppGpp or pppGpp. These assays were carried out in Buffer C with 0.2 mM ATP, 0.1 mM 3H-GTP (specific activity 27.5 pCi/pmole), and ribosomes at 20 A&ml in a total volume of 50 ~1. After 1 hr incubation at 37”C, reactions were terminated by the addition of 5 pl of 8 M formic acid. The adult liver preparation yielded virtually no activity, whereas considerable synthesis was detected in the reaction mixture containing embryonic ribosomes (Table 1, column I). An acidified mixture containing E. coli ribosomes was also run as a blank control. Omission of ATP reduced activity to virtually zero for both the embryonic ribosomes and for E. coli ribosomes. This initial observation suggests that ribosomes extracted from the developing embryo have an acTable

1. Identification

of ppGpp

by Thin

Chromatographic I II Ribosome

Source

11 Day Embryos Adult

Liver

E. coli Acid

Blank

3H DPM 3635

Layer

System

Chromatography * III

3H DPM3*P

DPM

3H DPM32P

2429

1507

3242

1087

NT

NT

NT

NT

2887

1485

1715

2217

1053

324

201

1675

47

1218

368

DPM

ji Solvent Systems: (I) 1.5 M KH2P04 as described in Experimental Procedures. (II) A one dimensional separation on 1% PEI cellulose in 4.0 M Na fomate (pH 3.4). Samples were dried on the plate, which was then soaked in methanol prior to ascending chromatography to 12 cm above the origin. (Ill) A two dimensional separation on 1% PEI cellulose called the “deoxy” separation. After spotting and drying of the samples, plates were soaked in methanol prior to the first dimension. First dimension: 1 .O M LiCI-0.5 M HAc to 6 cm above the origin, followed immediately by 1.25 M LiCI-0.5 M HAc to the top of the plate. Another methanol soak was used prior to the second dimension, which was 16 cm in 3.0 M ammonium acetate + 4.3% Boric acid. Tritium-labeled ppGpp was the product of various in vitro reactions. The SZP-labeled ppGpp was extracted from an amino acid starved culture of a stringent strain of E. coli. NT: Not Tested

Cell 250

tivity which is missing from the adult liver and which is similar to the procaryotic ribosomes of E. coli. By virtue of the tritium label, the compound produced must be a guanine derivative. It must also have at least one phosphate group derived from GTP (See below where the compound is labeled with ol-32P-GTP.). It is therefore a guanine nucleotide, and because of its chromatographic mobility on PEI cellulose, it must possess more phosphate groups than do GTP. Identity of ppGpp Synthesized by Embryonic Mouse Ribosomes Two additional chromatographic systems were used to establish the co-identity of the putative compound produced by the embryonic ribosomes with the ppGpp formed by E. coli. Portions of the reaction mixture were co-chromatographed with 32P-labeled ppGpp extracted from an amino acidstarved culture of E. coli strain CP78 that had been incubated with 3*Pi during the starvation period (Irr, 1972). Sufficient quantities of carrier ribonucleoside triphosphates and unlabeled ppGpp were also spotted with the samples to allow detection on the chromatography plates under UV light. For controls, the same tests were performed on two reaction mixtures containing E. coli ribosomes. One of these was acidified with 5 pl of 8 M formic acid before the incubation period. Table 1 illustrates results obtained with the standard chromatographic procedure which uses 1.5 M KH2P04 as the solvent in a one dimensional system, and describes the additional solvent systems which clearly separate ppGpp and pppGpp from GTP and other known nucleotides. The third system is actually a two dimensional chromatogram that lowers the acid blank counts to near background. When products of the reaction mixture containing ribosomes from the mouse embryos were separated from GTP in these three systems, there was a significant amount of tritium label found in the regions of the chromatograms where authentic ppGpp was located. The same results were obtained with the E. coli ribosome reaction mixture. Until greater quantities of the compound produced by the mouse embryonic ribosomes can be obtained and subjected to further analyses, we feel justified in concluding that these ribosomes produce a compound which is identical to the ppGpp formed by E. coli. We therefore shall refer to it as ppGpp. In vitro Production of 32P-ppGpp by Embryonic Mouse Ribosomes The production of ppGpp by ribosomes isolated from 11 day mouse embryos was verified by the use of o(-32P-GTP as a substrate. This label allowed autoradiographic detection of ppGpp on the thin layer

chromatograms. In addition to the 11 day embryonic ribosome preparation, we tested ribosomes extracted from IO day embryos and 11 day embryonic brain. Adult mouse kidney and liver ribosomes were checked also for the ability to make ppGpp (Table

2). The adult liver and adult kidney ribosomes showed insignificant activities, less than 7% of the E. coli ribosome control. On the other hand, all of the embryonic ribosomes demonstrated significant levels of ppGpp biosynthetic activity, the 11 day brain ribosomes being the most active species, with an activity equal to 69% of that recorded with E. coli ribosomes. The total 11 day and 10 day embryonic ribosomes, although not quite as active as the embryonic brain ribosomes, displayed significant levels of ppGpp biosynthetic capacity. Formation of ppGpp in Developing Liver Ribosomes were extracted from 11 day embryonic liver rudiments, 14 day embryonic livers, and adult livers, and assayed for ppGpp biosynthesis (Table 3). As controls, a new preparation of 11 day total embryonic ribosomes and another batch of E. coli ribosomes were tested together in the same system. During incubations of as long as 3 hr, the adult mouse liver ribosomes produced only slight amounts of ppGpp when compared with the E. coli ribosomes. The 11 day liver ribosomes had activities comparable with the total 11 day embryo ribosomes. This rate of ppGpp synthesis was nearly one half the rate seen with the E. coli preparation, and was strikingly greater than the diminutive rate found with adult liver ribosomes. Of possibly even greater interest is the observation that ribosomes extracted from livers of 14 day embryos essentially lack Table

2. Ribosomal

Ribosomal

conversion

of a-32P-GTP

Source

to DPGPP DPM/Ax~~~/H~”

E. coli

4922

10 Day Mouse

Embryo

1810

11 Day Mouse

Embryo

1873

11 Day Mouse

Embryonic

Brain

3382

Adult

Mouse

Kidney

128

Adult

Mouse

Liver

337

Adult

Mouse

Liver

(Salt Washed)

*

232

a%The values equal the average of two samples of each ribosome preparation, one being twice the concentration of ribosomes than the other. In all cases the amount of radioactivity approximately doubled with a 2 fold increase in ribosome concentration. * Ribosomes were salt washed by centrifugation through a sucrose cushion containing 0.5 M KCI. Ribosomes from the indicated sources were incubated in reaction mixtures containing 0.2 mM ATP, 0.1 mM GTP and 1 @Ci a-32P-GTP (Amersham Searle), and sufficient buffer C to make a final volume of 50 UI.

ppGpp 251

Synthesis

by Mouse

Ribosomes

ppGpp biosynthetic activity. There are marked morphological and biochemical differences between 11 day liver and 14 day liver (Nagel, 1968), and a change in the capacity of embryonic liver ribosomes to form ppGpp appears to be correlated with the developmental changes. None of the mouse preparations showed any signs of pppGpp production, yet the assay conditions favored the formation of this compound in the E. coli system. The significance of this observation is not immediately apparent. Discussion The unusual nucleotide guanosine tetraphosphate (ppGpp) is synthesized by mammalian ribosomes. Ribosomes isolated from various embryonic mouse tissues were able to form ppGpp at 37-69% of the rate observed using E. coli ribosomes under the same reaction conditions. The inability of other workers to demonstrate ppGpp synthesis in many eucaryote systems may be related to the fact that the chromatographic assay for ppGpp was frequently carried out following in vivo incubation with radioactive phosphate (Fan et al., 1973; Buckel and Bock, 1973), although, Table 3. Conversion ribosomes

of a-3*P-GTP

to ppGpp

and pppGpp

by

Time Ribosome

Source

11 day embryo liver

14 day embryo liver

II day embryo total

Adult

E. coli

liver

0-W

PPGPP

PPPGPP

1

24.0


2

178.0


1

0.1

to.1

2

0.1


3

1.2


1

56.0


2

112.0


1

0.6

10.1

2

2.5


3

30.5


1

154.0

2

430.0

336.0 830.0

3

732.0

1206.0

Ribosomes were incubated for the indicated times in reaction mixtures comparable with those given in the legend to Table 2, but of greater volume. At the indicated times, 50 pl samples were removed and mixed with 5 ~1 of 8 M formic acid and assayed as described in Experimental Procedures. Data were converted to picomoles by comparison of radioactivity with that of a 5 pl standard taken from one of the reaction mixtures. Results are expressed as oicomoles of nucleotide formed per 1 .O Anao of ribosomes.

in the case of mouse 3T3 cells, degradation of ppGpp was not responsible for the negative results, since 85% of added 3*P-labeled bacterial ppGpp was recovered following disruption of the cells (Mamont et al., 1972). Our use of purified ribosomes in an in vitro incubation system also cannot entirely explain the positive results, since ribosomes from yeast cytoplasm, reticulocytes, and calf brain failed to synthesize ppGpp in vitro (Richter, 1973). Perhaps the most pertinent feature of our experimental system is the source of ribosomes. The ribosomes most active in the synthesis of ppGpp were isolated from embryonic tissue, and more particularly, from the early embryonic tissue. Ribosomes isolated from 11 day liver actively synthesized ppGpp, but this ability was essentially absent in ribosomes prepared from 14 day liver. Ribosomes prepared from embryonic 11 day mouse brain have about 69% of the ppGpp synthesizing ability of E. coli ribosomes (Table 2), but this ability is lost by adult brain ribosomes (not illustrated). The mouse brain shows its greatest rate of growth postnatally (Himwich, 1962; Hafemann and Unsworth, 1973), and embryonic brain is an extremely rapidly dividing tissue (Sung, 1971). Embryonic brain cells proliferate rapidly but do not synthesize specific proteins at a rate comparable with the postnatal brain; even myelination, which is a process restricted to nervous tissue, is mainly a postnatal event (Uzman and Rumley, 1958). Our results support the idea that ppGpp synthesis may be related to the state of differentiation of the cells. Prior to extensive specialized product formation, the cells have the capacity to form this unusual nucleotide. In cells synthesizing specialized products, this ability appears to be lost. If ppGpp performs a regulatory role in mammalian cells similar to that proposed by Cashel and Gallant (1969) for bacteria-that is, inhibition of stable RNA synthesis which is linked to an “idling” reaction of the ribosomes under conditions of shortage of substrate for protein synthesis-the results are not those which could be readily anticipated. Organ rudiments undergo rapid cellular proliferation, and this stage of development is hardly one in which conditions for growth restriction would be expected to occur. If ppGpp indeed plays a regulatory role during organogenesis, it may mean that a switch to specialized protein synthesis involves a change in control mechanisms, and in the early stages of development there is a need for the type of control in which this nucleotide plays a part. Such a requirement may be interpreted in at least two ways. (a) In situations where there is considerable formation of new ribosomes, but limited synthesis of cell-specific proteins (as in the early stages of organogenesis), ppGpp may be synthesized in order to maintain

Cell 252

some ribosomes in an inactive state. It has been shown that ppGpp will block the binding of amino acyl tRNA to ribosomes (Manzocchi, Tarrago, and Allende, 1973), thereby rendering the ribosomes inactive. (b) There is also the intriguing possibility that more than one category of ribosome may exist during the early stages of organogenesis. Ribosomes functional during specialized protein synthesis may be repressed in early development by ppGpp; other ribosomes involved in cell proliferation would be unaffected by this nucleotide. This speculation would correlate with observations indicating that the differentiative step is sensitive to inhibitors which suppress the formation of new ribosomes @utter, Pictet, and Morris, 1973). The possible regulatory role for ppGpp during embryonic development is of interest, and other regulator molecules of this type have been suggestalthough the latter ed (Mamont et al., 1972), “pleiotypic mediator” does not appear to correspond with ppGpp, since it was postulated to be formed at the cell membrane and to accumulate under conditions of growth restriction. Experimental

Procedures

Ribosome Extraction E. coli ribosomes were extracted from a 6 I L-broth culture in late stationary phase. The cells were harvested by centrifugation at 10,000 x g for 20 min. The cell pellets were frozen until extracted. About 30 gm wet weight of cells were suspended in sufficient Buffer C (see below) to yield 100 ml total volume. The suspension was sonicated for 3 min in the presence of DNAase at a concentration of IO pg/ml. The disrupted cell preparation was centrifuged at 30,000 x g for 30 min to remove intact cells and debris. The clear supernate was centrifuged for 2 hr at 35,000 RPM in a Type 50 Beckman rotor to pellet the ribosomes. Pellets were resuspended in 40 ml of Buffer C, divided into two equal portions, layered onto 19 ml cushions of 40% sucrose (w/v) made in Buffer C, and centrifuged at 27,000 RPM for 20 hr in a Type 27 Beckman rotor. The supernate was siphoned off, and the clear pellet of ribosomes was washed with 1 ml of Buffer C and resuspended into 5 ml Buffer C. Following another centrifugation at 27,000 RPM for 5 hr in the same tubes, the pellets were resuspended in 5 ml of Buffer C and pooled, Small aliquots were frozen until used for assays. In one experiment, 234 mg of ribosomes were obtained from 32.4 gm of cells. This procedure is a modification of the method described by Revel et al. (1968). Mammalian ribosomes were prepared from embryonic or adult tissues. The tissue was suspended in Buffer A (5 vol for adult tissue; 20 vol for embryonic) and homogenized in a Ten-Broeck all-glass hand homogenizer. The homogenates were centrifuged at 12,000 x g for 10 min in a Sorvall centrifuge at 4°C. The supernates were made 1% with Triton X-100 and centrifuged at 50,000 RPM for 90 min in a Type 65 Beckman rotor. The pellets were suspended in Buffer B by hand homogenization, and cleared by centrifugation at 12,000 x g for 10 min in the Sorvall centrifuge. The cleared solution was centrifuged at 50,000 RPM for 75 min, and the pellets were resuspended in Buffer B and again cleared by low speed centrifugation. The ribosomes were loaded over a 6 ml cushion of Buffer B containing 17% sucrose, and centrifuged at 50,000 RPM for 3 hr. The pellets were resuspended in Buffer B containing 50 mM KCI and ethanol-magnesium precipitated (Kaulenas, 1971). The precipitated ribosomes were either stored

frozen at -20°C assays.

or resuspended

directly

in Buffer

C and used

for

Thin Layer Chromatography Chromatography was performed on glass plates covered with 1% PEI Cellulose. Unless stated otherwise, complete separation of ppGpp and pppGpp from GTP and other known nucleotides was achieved by using 1.5 M KH2P04 as the solven’t in a one dimensional system. Samples of 5 pl were spotted without carrier at a position about 2 cm above the edge of the plate, and the solvent was allowed to ascend to at least 16 cm above the origin. Better resolution was achieved when the sample spots were still damp at fhe time the solvent front passed the origin (Cashel, Lazzarini, and Kalbather, 1969). Samples of the in vitro reaction mixtures contained sufficient quantities of GTP and ATP to allow detection by viewing the chromatograms under a UV light. This step, along with the exposure of x-ray films to plates containing 3*P-labeled compounds, greatly facilitated the localization of radioactive nucleotides. Those areas of the chromatograms containing radioactivity were scraped into scintillation vials and counted in toluene-based Liquidfluor (New England Nuclear). Tritium-labeled samples were corrected for quenching by the channels ratio method. Double-labeled samples were corrected for quenching of tritium and for the spillover of 3*P into the 3H window (Irr, 1972). Buffer A Buffer A contains 0.05 M magnesium acetate, sucrose.

M Tris-HCI (pH 7.6), 0.1 M NH4CI, 0.005 0.005 M 2-mercaptoethanol, and 0.25 M

Buffer I3 Buffer B contains 0.05 M Tris-HCI (pH 7.6), 0.24 M magnesium acetate, 0.005 M 2-mercaptoethanol, sucrose.

M KCI, 0.01 and 0.25 M

Buffer C Buffer C was used in all assays and for the preparation of E. coli ribosomes. Mouse ribosomes were ethanol precipitated and resuspended in the same buffer prior to use in the assays. Buffer C contains 0.05 M Tris-acetate (pH 8.0); 0.015 M Mg acetate, 0.06 M potassium acetate; 0.027 M ammonium acetate: 1 mM EDTA, and 0.1 mM dithiothreitol. Tissue Preparation Embryos and adults were obtained from a randomly bred colony of CF strain mice maintained in our laboratory. Timed pregnant females were isolated on the day that a vaginal plug was discovered, and this was designated as the zero day of pregnancy. Adults were killed by cervical dislocation, and the organs and/or the uteri were transferred to a petri dish containing cold physiological saline. The embryos were freed from the uterus, and fetal membranes by gross dissection. The embryonic organ rudiments were pooled following dissection with irridectomy knives. The pooled organs were rapidly frozen and stored at -70°C until the ribosomes were extracted. Acknowledgments This work was supported, in part, by a grant Institutes of Health to B. R. U. and by a grant Stanka Foundation to J. I. Received

July

12, 1974;

revised

August

from from

the National the Peter M.

16, 1974

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