Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

SYNTHESIS OF GUANOSINE TETRAPHOSPHATE

(MAGIC SPOT I)

IN SACCHAHOMYCES CEHEVISIAE

Chia

C. Pao,

John Paietta,

and Jonathan

Department

of Genetics

University

of Washington

Seattle, Received

October

A. Gallant

Washington

98195

25,1976

SUMMARY

Guanosine

tetraphosphate

extracts

of Saccharomyces

compound

comigrates

(Magic cerevisiae

with

tests

confirm

diphosphate

found

in bacteria.

cells,

suggesting

its

I)

has been

subjected

authentic

and further

yeast

Spot

ppGpp

identity

to heat

in several

with

it

is

in

formic

shock.

acid

The yeast

chromatographic

systems,

the guanosine-5'-diphosphate-3'-

Oxytetracycline

that

found

inhibits

produced

its

formation

on the mitochondrial

in

ribosomes.

INTRODUCTION Guanosine broad

range

5' diphosphate-3' of processes

Escherichia

coli

(l-3)

in transcription, and very

possible

occurrence

of this

therefore

received

considerable

hunting

expeditions

been recounted sea urchin and cultured absence

mammalian

Hogan,

Gallant,

and Shell,

likely

regulatory

of yeast ll),

and Shell,

nucleotide

in

(7),

(12-13);

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

(Silverman,

human fibroblast,

studies

and CHO cells 314

(Lazzarini,

cells

absence (8),

in

(4-6).

of these

nucleotide of ppGpp has

and Atherly, have

cells

The has

Neurospora

and hamster

mouse ascites

unpublished),

unpublished),

the

Ankystrodesmus

unpublished

as well

eukaryotic

The results negative:

a remarkably

and metabolism

bacteria

mouse embryos

cells

controls

in other

attention.

of ppGpp in mouse 3T3,

(Gallant,

Copyright All rights

(10,

(ppGpp) translation,

have been generally

in studies

embryos

diphosphate

(8,

9),

in press),

confirmed

fibroblast (Martin, unpublished).

the cells

Vol. 74, No. 1, 1977

BIOCHEMICAL

Klein

a compound

(14)

has reported

the slime similar

mold Dictyostelium compound

was adequate This

taken.

ppGpp which

is

could

not

with

tion

by rigorous

shock.

unknown,

but

MATERIALS

evidently

synthetic

complete

concentration

than

separations by Rhaese

same cell

lines

(15)

(13,

of mycoplasma

contamina-

of ppGpp in eukaryotic

on the occurrence

cells.

The nucleotide

governing

involves

its

of ppGpp,

cells

identified

accumulates

formation

mitochondrial

is

Saccharomyces Leland minimum

in yeast

in response cells

is

ribosomes.

The following

Louis,

glyceraldehyde-3-P

enzymes

from yeast

pyrophosphatase were

H. Hartwell

purchased

32 P-phosphoric

polyethyleneimine-cellulose

A364A used in

and was described

medium was of Wickerham

and adenosine

Chemicals: MO.;

cerevisiae

0.5 mM and was supplemented

at 100 ug/ml

Carrier-free

other

under-

AND METHODS

from Dr.

Nucleotides

evidence

we report

The mechanism

obtained

inorganic

in question

reported

the question

cases

to the questionable.

in yeast

and medium:

mixed

of one of the

a

of these

chromatographic

studies

In short,

Strains

lysine

has reported

nucleotides

the compound

culture.

criteria,

since

raises

to ppGpp in

In neither

Moreover,

which

communication,

to heat

lines.

in conventional

in other

from the negative In this

cell

(15)

of the compound(s)

unpublished),

in Rhaese's

ranges

it

(16-18).

be detected

and Lazzarini,

mammalian

of some importance,

comigrate

similar

and Rhaese

characterization

a point

have been described

chromatographically

discoidem,

in certain

chemical

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and uridine

enzymes were

except

histidine,

from

Baker's

yeast

were

thin-layer

obtained sheets

315

The

the phosphate tyrosine

and

respectively),

(647 units/mg Inc.

protein).

and ICN Pharmaceuticals.

from New England were

Co. at St.

phosphokinase

protein

from P-L Biochemicals, acid

(19).

Sigma Chemical

dehydrogenase/3-P-glycerate

from

is

at 20 rig/ml.

purchased

(50 and 150 units/mg

study

previously

(20)

with

this

from

Brinkmann

Nuclear

and

Instruments.

Vol. 74, No. 1, 1977

Labelling wise

BIOCHEMICAL

and measurement

indicated,

contamination

from

samples

the medium

on rich

nutrient

of 32 P-phosphoric

acid

an absorbance to about

for

doubling brium

cells/ml).

acid

pools level,

M KH2P04 in

Pakard

Purification

eluted

were

in a small

then

2 ml 0.01

with

2 ml (each)

In Vitro

elutes

ens ymatic (22)

spots

spots

(yielding

buffer,

with

one cell

complete

equili-

acid.

For

with

5 pl

extracted

were

on

developed

dimension

with

followed

chromatograms

out and counted

corresponding about

column

lo4

cpm per

(Isolab

first

washed

pH 7.4

followed

Inc.

with

by 1.5 were

in a

10 to

preparation)

and

Akron,

Ohio) alcohol

and

by 3 consecutive

elutions

(pH 8.0).

The combined

and resuspended

to be homogenous

to ppGpp from

2 ml methyl

bicarbonate

lyophilized

ppGpp was shown

conversion

cut

of

spectrometer.

steps:

were

the first

were

at

chromatography

The desalted

(18).

1 mCi/ml

exponentially

and removal

were

The sheets

in

absorption

on PEI-cellulose

and Chappell

(21).

of

of 0.2 corresponds

thin-layer

of 2 M triethylammonium

isolated,

development

out

following

M Tris*HCl

TEA-bicarbonate thus

cut

pool,

32 P-phosphoric

Radioactive

QUIK-SEP

to the

diluted

and exogenous

scintillation

of ppGpp:

according

As

dimension

liquid

14 chromatograms

or by plating

to ensure

1.5 M LiCl

and radioactive

Tri-Carb

samples

to the medium

sheets

the second

radioautographed

by plating

out at least

samples

other-

The possibility

growing

carried

loo-p1

unless

aeration.

of experiments

by two-dimensional

containing

grown,

the nucleotide

cultures

were

of isotope

followed

acid

to all

determination

polyethyleneimine-cellulose 2 M formic

plates

To label

Starting

of nucleotide

2 M formic

forced

0.2 at 720 nm. (An absorbance

the nucleotide

determination

were

and eliminated

plates.

was added

addition

between

and under

was tested

agar

nucleotide

after

Cultures

on a Mac Conkey agar

of about

3.4 x lo6

samples

of nucleotides:

at room temperature

of bacterial O.lml

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in distilled

in 2 different

water. systems

of

sheets. of pm:

modifications.

Procedures ppGpp can substitute

316

were

of Glynn

ADP as acceptor

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIGURE 1. Autoradiograph of two-dimensional thin-layer chromatograms of formic acid extracts (20 ~1) of yeast grown at room temperature (left) and after 6 minutes at 38" (right). Nucleotides were extracted in formic acid and resolved on thin-layer of PEI-cellulose together with non-radioactive marker nucleotides as described in MATERIALS AND METHODS and previously (21). The chromatograms were exposed to Kodak non-screen X-ray films for 72 hours. Dotted circles represent the positions where marker nucleotides migrated.

in the

exchange

reaction

glyceraldehyde-3-P

between

dehydrogenase

Glyceraldehyde-3-P

The equilibrium

mmole),

following:

KC1 (0.5

acid

phosphokinase

buffer

at 37'

(0.2

for

Hydrolysis

of ppGpp by Zinc-activated

and 10 units tides

were

of yeast resolved

as described

on PEI-cellulose

far

to the right,

amounts. (0.3

before yeast

thin-layer

MgSO4 (0.05

glyceraldehyde-3-P dehydrogenase Reaction

(21). inorganic

pyrophosphatase

mixture

by two-dimensional

pyrophosphatase:

10 mM Tris*HCl,

317

mmole),

respectively.

and followed

so that

10 pl reaction

glyceraldehyde-3-P

and 0.3 units

in 100 ul containing inorganic

is

NAD (1 umole),

60 minutes

chromatography

+ NADH + ATP(pppGpp)

at pH 7.4

uCi),

at 0.1

thin-layer

ppGpp was hydrolyzed

in catalytic

KH2P04 (1 umole),

32 P-phosphoric

was incubated

reaction

by

phosphokinase:

3-P-glycerate

second

only

and ATP catalyzed

1,3-PP-glycerate

----+

TrisaHCl

mmole),

and 3-P-glycerate mixture

of the

can be present

the

(10 umole),

and 3-p-glycerate

+ ADP(ppGpp)

position

ADP (or ppGpp)

phosphate

+ Pi + NAD --+

1,3-PP-glycerate

contained

inorganic

at 3J0 for sheet.

pH 7.0, 10 hours.

Purified 0.4 mM Zn(OH)2 Nucleo-

BIOCHEMICAL

Vol. 74, No. 1, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

. FIGURE 2. Conversion of yeast ppGpp to pppGpp by glyceraldehyde-3-P dehydrogenase and 3-P-glycerate phosphokinase. ppGpp was purified by preparative thin-layer chromatography from yeast culture which has been shifted from room temperature to 38' for six minutes. Experimental details were described in MATERIALS AND METHODS. Dotted circles represent the positions where marker nucleotides migrated. Radioactivity in pppGpp and ppGpp spots are 806 and 136 cpm respectively. The chromatogram was exposed to Kodak nonscreen X-ray films for six days.

Alkaline

hydrolysis

ppGpp,

of yeast

ppG=:

To 60 ul sample

6 ul each of 0.1 M EDTA and 2 N KOH were

incubation reaction

at 37“, mixture.

approximately Nucleotides

8 ul were

2 M formic

then

containing

radioactive

added.

After

acid

was added

separated

overnight to the

by thin-layer

chromato-

washy . RESULTS FIGURE 1 shows a radioautogram yeast

cells

after

chromatography. evident.

This

heat

shock

A compound compound

of 32 P labelled

and resolved which

comigrates

was quantitatively

318

compounds

by two dimensional exactly adsorbed

with

extracted thin-layer

marker

by activated

ppGpp is charcoal,

from

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lb Time

(mln)

FIGURE 3. Formation of ppGpp by yeast during temperatuss shift-up. A364A was grown in synthetic minimum medium and labeled with P-phosphoric acid at 1.0 mCi/ml at room temperature of about 23" for at least one cell generation before being shifted to 38". 100 ul samples were removed at times indicated. For other information see MATERIALS AND METHODS. In the oxytetracycline experiment, the drug was added 10 minutes before temperature change and to a final concentration of 2 mg/ml. Closed circles represent ppGpp produced without oxytetracycline treatment; open circles, with oxytetracycline.

and was also

labelled

indications,

we isolated

chromatography

compound

of FIGURE 1 both

in our hands

give

Given

32 P-labelled

and subjected

The isolated system

by 14 C-adenine.

it

these

material

preliminary

by preparative

thin-layer

to more stringent

comigrated

with

and Brinckmann

different

separations,

two dimensional

separation

described

chromatographic

properties

distinguish

tests.

authentic

on Merck

rather

favorable

ppGpp in the separation

PEI-cellulose and also

by Randerath the

(21,

compound

plates, in

the double-step

FIGURE 88).

from pppG,

PPPPA, PPGP, PGPP, PPPGPP, PPAPP, and PPPAPP, all

of which

in one or both

The compound

to periodate

of the oxidation,

The isomer is

also

their

seperation

periodate sensitivity

tinguish

them.

at the

same rate

pppGp is

like

systems ppGpp,

tested.

indicating

chromatographically

the two isomers

to alkaline

hydrolysis,

providing

Alkaline

treatment

of the yeast

and to the same degradation

319

nucleotide

product

pppA,

was insensitive

from ppGpp and

differ a simple

ppppG,

2' or 3' position.

indistinguishable

However

These

run differently

an esterified

resistant.

which

drastically

in

way to dishydrolyzed

as alkaline

it

hydrolysis

BIOCHEMICAL

Vol. 74, No. 1, 1977

of authentic (or

ppGpp.

prolonged

yeast

storage)

nucleotide

comigrates

Similarly,

with

pGpp;

authentic

(22)

ppGpp.

reaction

of ppGpp cannot

communication). well.

is

that

the

the same guanosine

FIGURE 3 shows the following

shift

from

ppGpp was about blocked

breakdown

and could a highly

in this

room temperature

passed

this

2'

test

as

after

found

in bacteria.

in yeast

The peak

the

cells

3' -diphosphate

The nucleotide's

of pppG.

but

Sy, personal

by yeast

to 38".

isomer.

enzymatic

reaction,

of ppGpp accumulation

the

product

specific

compound

accumulated

with

be a cyclic

communication;

the yeast

5'-diphosphate

kinetics

10% that

The major

personal

compound

pyrophosphatase

products

to pppGpp

Cashel,

activated

breakdown

provides

FIGURE 2 shows that

We conclude

shock

(23;

RESEARCH COMMUNICATIONS

zinc

one is unknown,

ppGpp can be phosphorylated

isomer

with

two identical

the minor

The Glynn-Chapel1 test;

treatment

yielded

and with

AND BIOPHYSICAL

level

heat

cells attained

accumulation

by

is completely

by oxytetracycline.

DISCUSSION In E. coli,

the

tetracyclines yeast

cells

(24,

ribosomes

which

depends

it

is

likely

which

are

stringent

(25),

synthesis

that

that

respects factor

yeast

is

resonsible

cells

inhibits

to these as well

of E. G,

is

for

endogenous

in some way on activation

by the

in by the

and resembly

In fact,

Richter

enzyme responsible

by mitochondrial contain

by the

produced

antibiotics

(26). the

inhibited

ppGpp formation

the nucleotide

sensitive

can be activated

imply

of ppGpp is

oxytetracycline

in other

the

ppGpp synthesis Our results

Since

ribosomes

has shown that

factor

25).

(FIGURE 3),

mitochondrial prokaryotic

ribosome-linked

ribosomes

an enzyme analagous ppGpp synthesis

for

from yeast. to the stringent

and which

tetracycline-sensitive

(27)

also

mitochondrial

ribosomes. The mechanism It

clearly

by which

does not

to 38" increases

the

involve growth

heat amino rate

shock

induces

acid

ppGpp synthesis

starvation,

of our

320

yeast

since strain.

transfer Moreover,

is

unknown. from

23'

a derivative

Vol. 74, No. 1, 1977

of this

strain

BIOCHEMICAL

with

a temperature

produced

no more ppGpp upon

strain.

Thus,

thing

other

to detect

the

than

signal

the nucleotide

Nonetheless,

in amino

the

signal,

respect,

yeast

acid

is maximal

at 36“

the wild

Kudrna

starved it

above 30",

activating

yeast

is,

is

parental

probably

and Edlin's cells

synthesis

whereas

cytoplasmic

failure

operates

through

is noteworthy,

prctein

some-

(7).

apparently It

enzyme

type

in yeast

ribosomes.

mitochondrial

temperature

than

explains

whatever

of the mitochondrial

increasing

shift

tRNA, which

RESEARCH COMMUNICATIONS

isoleucine

ppGpp accumulation

some activity that

sensitive

temerature

for

uncharged

AND BIOPHYSICAL

falls

in this

sharply

protein

with

synthesis

(28).

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Cashel,

M. and Gallant,

J.

(1969)

2.

Cashel, M. and Gallant, and Lengyel, P. eds.,

J. Cold

3.

Gallant, Advances

J. and Lazzarini, McConkey, E. H.,

R. A. (1975) in Protein ed. Dekker, New York.

Synthesis:

4.

Swanton,

M. and Edlin,

(1972)

Res.

5.

Eccleston, 1370-1376.

6.

Rhaese,

H. J.,

7.

Kudrna,

R. and Edlin,

8.

Buckel,

P. and Bock,

9.

Alberghina, F. A. M., Schiaffonati, Biochem. Biophys. Acta. -312:435-439.

E. D.

Dichtelmuller

Biochem.

(1973)

(1975)

Biochim. L.,

Brandhorst,

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Pirrone, (1976)

A. M., Roccheri, M. C., Bellanca, Developmental Biology @:311-320.

12.

Mamont,

P., Hershko, A., Biochem. Biophys.

13.

Fan,

K.,

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Klein,

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Rhaese,

Fisher, C.

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(1974) in Ribosomes, Nomura, M., Tissieres, A., Spring Harbor Laboratory. New York., pp. 733-745.

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Loewen,

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Gallant,

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McLaughlin, 100:579-584.

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Wickerham,

L. J.

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Randerath,

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Glynn,

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Cashel, M., of Ribosome Copenhagen

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Kaplan, S., Atherly, Sci. US -70:689-692.

25.

Block, R, and Haseltine, A. and Lengyel, P., eds. pp. 747-761.

26.

Clark-Walker, G. D. and Linnane, Commun. E:8-13.

27.

Richter,

D.

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Marmiroli, Biophys.

N., Restivo, F., Zennaro, Res. Commun. -70:589-594.

(1976) Shell,

C. S.,

G.

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P. T. and Hartwell, J.

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