GDP kinase activity associated with salt-washed ribosomes

BIOCHEMICAL

Vol. 78, No. 2, 1977

GDP KINASE ACTIVITY Alice

August

ASSOCIATED WITH SALT-WASHED RIBOSOMES

M. Wertheimer

Department Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and $1. S. Kaulenas

of Zoology, Amherst,

University of Massachusetts Mass. 01003

lo,1977

SUMMARY: In the presence of ATP, lM-salt-washed ribosomes and subunits of both eukaryotes and prokaryotes phosphorylate GDP to 5'GTP. A possible role for the ribosome-associated GDP kinase is presented. INTRODUCTION: protein

synthesis

as well

and elongation, complexed

Hydrolysis

it

with

a specific

factors

Although

a mechanism

bind

The GDP kinase of Escherichia

It

found

coli

for

(I).

that

GDP much more

has been

necessary

elongation

factor.

case of prokaryote

initiation. subunits

as for

has been thought

initiation

GTP in the

of GTP is

initiation

In both

GTP is

brought

initiation to the

has therefore

been puzzling

strongly

they

for

than

exchanging

elongation, with

factor-bound

lM-salt-washed

and mouse liver

ribosome

reported

that

do GTP (2).

one has not been

associated

of

GDP with

found

for

ribosomes here

and

may be part

of

such a mechanism. A scheme for rather

than

initiation

GTP is brought

phosphorylates ATP requirement (b)

eukaryote

recycling

of a small-subunit

the

GDP.

to the

is ribosomes,

The scheme also

for

initiation

of the

stable

ribosomal

of protein eIF2.GDP protein

given

which

and that

incorporates synthesis

complex,

and (c)

postulates the

ribosome

(a)

findings

in eukaryotes

that

of an (3,4),

phosphorylation

(5).

MATERIALS AND METHODS: All ribosome isolations were doreat 4')C. Buffers are given in Table 1. Buffers were adJusted to pH 8.0 at room temperature. Frozen mid-log phase x. __ coli (strain A-19 from General Biochemicals) were suspended in two volumes of Buf. A with DNAase (Sigma) added to a final concentration of 10 ug/ml. After sonication, the suspension was incubated at 4oC for 20 min, then centrifuged for 30 min at 15,OOOg.

Copyright All rights

0 1977 by Academic of reproduction in any

Press. Inc. form reserved.

565

GDP

Vol. 78, No. 2, 1977

Table

1.

Salt

BIOCHEMICAL

Composition

(mM)

of buffers

Tris-HCl

used

NHqCl

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in ribosome

KC1

isolations.

MgAc

Dithiothreitol B-mercaptoethanol

(D) (!4)

Buffer A B C D E F G H I J K

50 50 50 30 10 50 50 50 50 50 50

100 1000 15 45 50 10 10 10 10 1n

25 15 10 25 500 25 60 60 25

15 10 2 1 4 : 3 9 10

5 (M) 5 (M) 0.1 (D) 0.1 (D) O:l

(D)

011 1.0 1.0 0.1

(D) (D) (D) (D)

The supernatant was centrifuged at 105,OOOg for 90 min. The pellet was resuspended in Buf. B and incubated for 8 h at 4OC. In some experiments, Triton X-100 was added to a final concentration of either 0.5% or 1%. The supernatant from clearing at 15,OOOg for 15 min was centrifuged at 105,OOOg for 90 min. The pellets ("l&i-salt-washed ribosomes") were frozen at -7OOC or resuspended in Buf. C to a final concentration of 50-100 A26Gnm units/ml and assayed directly or frozen at -20°C in aliquots. Alternatively, the ribosomes were resuspended in Buf. D and made into subunits by centrifuging into a lo%-30% sucrose gradient made up in Buf. E. Subunit fractions were collected, made 15mM in magnesium, and precipitated by addition of a half volume of cold 100% ethanol. The precipitate was collected by centrifugation at l2,5OOg for 10 min, resuspended in Buf. C, and assayed directly or frozen at -200C in aliquots. Before use, i-ibosomes were incubated at 37OC for at least 10 min. Crude ribosomes were extracted from starved male mice (strain CD, Charles River) essentially as in Ref. 6. The crude pellets were rinsed with distilled water, resuspended in Buf. B containing 0.25M sucrose by two strokes of a hand-held Teflon-glass homogenizer and incubated at O°C for 2 h. Triton X-100 was added to a final concentration of 1% and the solution was cleared at 15,000& for 15 min. The supernatant was pelleted at 105,OOOg for 3 h through a 5-ml cushion of 5OOmM sucrose made up in Buf. B. The pellets ("1M-salt-washed ribosomes") were (a) frozen at -7O'C until use or (b) resuspended in Huf. F to a concentration of 50-100 A26Unm units/ml and assayed directly or frozen at -2OOC in aliquots. Alternatively, the pellets were resuspended in Buf. G, incubated with lmM puromycin for 10 min at hoc, then 15 min at 37OC, and centrifuged into a lo%-30% sucrose gradient made up in Buf. G (modified from Ref. 7). Subunit fractions were collected and treated as g. & subunits, except that resuspension was in Buf. F. Assay conditions are given in the legend to Fig. 1. Assays were run The assay volume was 50 ul. Controls were at 37OC, usually for 120 min. run under the conditions given, but the final resuspension buffer (Buf. C or Buf. F) replaced the ribosomes. Tritium-labelled nucleotides were obtained from New England Nuclear or Amersham/Searle. Unlabelled nucleotides were from Sigma or Calbiochem, and were not purified before use. Stock solutions

566

BIOCHEMICAL

Vol. 78, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of sucrose (Fisher) were treated with diethylpyrocarbonate (Eastman) and filtered before use. Stock salt solutions were filtered through 0.22-vrn Millip re filters. Assay mixtures were precipitated by additiun of formic acid to a final concentration of' ml. After at least 30 min in the cold, the supernatants frc,m a lO-min spiri on a bench centrifuge were spotted onto plastic-backed PEI-cellulose plates (Brinkmann I ns t ruments) that had been washed by The one-dimensional ascending chromatography with distilled water. chromatographs were washed for 1 min in distilled water to remove salts, dried, and developed in 1.5X h312PC4. Twtio ?-dimensional systems were used to identify the GTF. One system was Method 2 of Ref. 8 and the other is given in Ref. 9. Fluorography was done essentialiy as described in Ref. 10. Kodak RP 54 film was used and exposure was for 6 days at -7OOC. Lyniform znnal strips were cut from one-dimensional plates and counted in 5 ml ilf Liquifluor (New England Nuclear). Mcrmalization was done by dividing the counts per minute (cpm) of each strip by the sum of the cpm of all the strips in an assay, givin& the fraction of total tcpm ir; a strip. Peaks could then easily be identified and percent of total cpm in a peak calculated. The specific activity R, in moles of product per min per mole of ribosomes was calculated using the formula

R= where F is a *2hOnm units or GDP in the product peak, A260nm units Error bars in Ref. 11 using was always at RESULTS: g. &

GDP kinase

of subunits

in --E -.coli

of mouse liver

precipitation

step

reticulocyte

subunits

show an activity assays, large

activity (Fig.

activity

small subunits

P,)/(A)

CT),

constant factor that adjusts factors of 10 and converts into pmoles of ribosomes, C is the nmoles of substrate GTP assay mixture, P, is the percent of total cpm in the P, is the percent of total cpm in the control,A is the in the assay,and T is the duration of the assay in minutes. Fig. 1 at the 99% confidence level were calculated as in estimated probable errors. The sum of cpm in an assay least 40,000.

and mouse liver

formation the

(F) (C) (Ps -

or other not

equal

could

1).

given

isolated

be at least

but

with

of our

W-salt-washed is

The formation

factors

had higher

in

The activity

ribosomes,

to that

subunits

is present

not

in the

lM-salt-washed

partly

than

does diminish

Discussion. (a kind

large.

ethanol

Combined

gift

of R. S. Ranu*)

ribosomes.

In all

The activity

due to contamination

of

by small

subunits. GTPase activity * Dept.

of Biology,

is

indistinguishable

from

MIT.

567

zero

of

by the

may be due to the

ethanol

activity

diminished

of subunits

this

ribosomes

in eukaryote

ribosomes

Vol. 78, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.5 (a) GDP Klnose E co11

1.0 :

w

L

s

SL

Cd) GTPase mouse her 0.0

Fig.

1.

The rate

and reduced

of GDP formation

subunits,is

therefore

The magnesium

for

lM-salt-washed

donors.

CTP gives

a lower

than

presence

ribosomes

and monovalent

salt

It

should

SL

However, activity with

ATP).

assays

of any of the

using

in g. coli

and not

that

subunits

and subunits, a turnover

conditionsgiven

be noted

there

in the

(Fig.

1).

and of s. coli rate. caption

to

are two magnesium

optima

ribosomes. 1, CTP, GTP, and UTP can replace

of Fig.

CDP can accept

of mouse liver

by eukaryote

of synthesis

conditions

phosphate

other

zero

mouse liver

the

20% of that

to essentially

the rate

1 are optimal.

Under

than

L

GDP kinase and GTPase activities of lM-salt-washed ribosomes and subunits of E. coli and mouse liver. W: whole ribosomes, about unit per assay; S: small subunits (about 0.33 A260nm unit l *260nm per assay); L: large subunits (about 0.67 A26Gnm unit per assay); SL: small and large subunits combined in one assay at a ratio of 1:2 (about 1 A260nm unit per assay). Assay conditions: (a) Buf. H, 4mM ATP, 0.1mM[3H]GDP (specific activity 200 mCi/mmole (b) left-hand bar: Buf. I, 2mM ATP, O.lmM t3H]GDP; right-hand bar: Buf. J, 2m~ ATP, 0.m 13H]G~p; CC) Buf. K, o.lti 13BlGTP (specific activity 200 mCi/mmole); (d) same as (b) except [3H]GDP Magnesium concentrations have been is replaced by O.lmM [3H]GTP. adjusted for presence of ATP.

and subunits,

Fig.

rI

.I s

w

the

activity

with

and GTP is the Although phosphates,

it

is visual

UTP is half

poorest not yet

donor certain

inspection

ADP, CDP, and UDP as acceptors

corresponding

triphosphates. 568

that

ATP as

with

ATP.

(activity

is

less

if

diphosphates

of chromatographs do not

show the

1;

BIOCHEMICAL

Vol. 78, No. 2, 1977

DISCUSSION: Ref.

12) could

The GDP kinase be caused

(b)

a factor-dependent

the

ribosome,

(c)

ribosome

throughout

activity,

mediated

It

in reverse

isolation

by ribosomal

eukarynte

ribosomes

to approximate

in -____ in vitro

assays

'vlalton could If

and Gill

do not

kinase

associated

contaminants,

that

ribosomes

bound.

is

to

ribosomal

the

to

used. are

and

GTFase

ribosomal

and

the

of

The conditions

similar

those

to

were

initiation

that

used

with

remove most

unlikely

that

of

GDP kinase that

be some of the

ribosome-

subunits

the ITDP

isolation. to eliminate

there

is

no easy way to

in activity

when

may be due to partial ribosomal

ribosomes

of importance

of 0.21 units/mg 100,000 daltons,

'sell

seems possible

during

we

would

would

The decrease

E. coli is

ribosomes

designed

or necessary

by eIF2,

1M salt

eIF2

were

(3,4).

in the

it

with

that

ribosomes

may in fact

into

eIF2

most

with

successful.

with

in eukaryotes

lM-salt-washed

case of eukaryotes,

activity

*We assume a specific activity the molecular weight is about weight for NDP kinase (13).

associated

due to contamination

However,

procedures

factor,

the

were

partitions

occurs If

bound

to the

an authentic

conditions

therefore

are dissociated

no such decrease

is tightly

It

in the

of a contaminant,

tightly

subunits

of

washing

and Gill

isolation

or not they

factors

of the

in vivo,

probable

eIF2.

that

especially

know whether

removal

the

is

do.

by Walton

iue

an NDP kinase

and that

that

GDP kinase

Although

found

twenty-one

It

is due to residual observed

eukaryote

eIF2,

(d)

CTPase,

synthesis.

of every

eIF2.*

from those

activity

is

the

GDP kinase

one out

have bound

eIF2

under

describe

the ribosome-associated that

reaction

GTPase activity

those

(2)

binds

or

in the ATP stimulation

have to have bound

the

the

of protein

be implicated

estimate

the

negligible

are thought

(see also

proteins.

that

is

of residual

that

procedure,

described

of a ribosomal

because

contaminant

RESEARCH COMMUNICATIONS

has been

reaction

observed

a cytoplasmic

because

that

a reverse

activity

seems improbable

acting

activity

by (a)

the

AND BIOPHYSICAL

protein.

shows that in prokaryote

protein a typical

The fact the

activity

initiation,

(2) and that molecular

Vol. 78, No. 2, 1977

BlOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

40s ATP ADP i elF2,GDP

+ 4OS-P

elF2.GTP.40S elF2-GDP

t

40s

60s

i

Fig.

2.

A partial scheme of eukaryotie initiation. The involvement and sequence of events concerning tRNA, mRNA, and factors other than eIF2 are not represented. Reaction (a) involves a stable phosphorylated subunit as an intermediate and requires ATP. The subunit would thus be similar to known NDP kinases(including succinyl thiokinase) which all have stable phosphorylated intermediates (13). The small subunit may be phosphorylated while still attached to the large subunit. Reaction (b) does not involve ribosomal phosphorylation, but does require ATP. The chief feature is that the ribosome phosphorylates and dephosphorylates eIF2-bound GDP and the stable eIF2eGDP complex is thus recycled.

Reaction

2 of Fig.

used in guanosine related

higher

antagonizing If

the

interesting Figure

2 gives

GDP and the Even if

its

than

is

for

understanding

presence

complex described

be taken

into

magnesium (b)

one,

how the

eukaryote

recycled

is only account

570

activity

concentration

presence

it

of GTPase

would

ribosome

initiation

The major are

this

washing.

ribosomal

Introduction.

eIF2-GDP

the activity should

scheme for

may somehow be

to notice

intensive

a true

same substrates

activity

inhibitory),

indeed

in the

the

use of suboptimal 8mM are

As the

of others

or (c) more

a proposed

listed

formation,

(a)

the activity, reaction

route.

The failure

implications

the events bound

reaction.

may be due to

(concentrations

be the likely

tetraphosphate

to that

in E. -- coli

2 would

incorporates

is that

in a simple

when assaying

works. that

feature

a tightly

have

bound

eIF2-

way. contaminant,

nucleotide

cycling

are

BIOCHEMICAL

Vol. 78, No. 2, 1977

in protein

synthesis,

synthesis,

phosphorylation

of the

phosphorylation

binding

of nucleotides of factors

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

control

to components

and ribosomal

of protein

proteins,

and the

role

of initiation.

was supported in part by a grant to ACKNOWLEDGEMENTS: This research AMW would like to MSK from the Research Council, Univ. of Massachusetts. thank G. Brown, R. Duhamel, G.-Fredericks and R. Yenofsky for valuable criticism, andO.Martini and D. Richter for their hospitality and a chance to do an interesting preliminary experiment. REFERENCES 1. 2.

For example, Haselkorn, Annu. Rev. Biochem. Walton, G. M., and Gill, 390,

3. 4.

5.

6. 7.

R., and Rothman-Denes, 42, 397-438. G. N. (1975) Biochim.

L. B. (1?73) Biophys.

Acta

231-245. (1970)

Marcus, A. J. Biol. Chem. 245, 955-961. Schreier, M. H.,and Staehelin, T. (1973) in Regulation of Transcription and Translation in Eukaryotes, edited by E. Bantz, P. Karlson, and H. Kersten, pp. 335-349, Springer-Verlag, New York. Wool, I. G., and StBffler, G. (1974) in Ribosomes, edited by M. Nomura, A. TissiCres, and P. Lengyel, pp. 417-460, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Schreier, IM. H., and Staehelin, T. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 462-465. Sherton, C. C., and Wool, I. G. (197L) J. Biol. Chem. 249, 2258-2267.

a. 9.

10. 11.

12. 13.

Gallant, J., Shell, L., and Bittner, R. (1976) Cell 7, 75-84. Cashel, M., Lazzarini, R. A., and Kalbacher, 3. (1969) J. Chromatogr. 40, 103-109; Cashel, M., and Kalbacher, B. (1970) J. Biol. Chem. 245, 2309-2318. Randerath, K. (1970) Anal. Binchem. 34, 188-205. Margenau, H., and Murphy, G. M. (1956) The Mathematics of Chemistry and Physics, 2nd ed., Ch. 13, D. Van Nostrand Company, Inc., Princeton, N.J. Wertheimer, A. M., (1977) Fed. Proc. 36, 871 (Abstr. No. 3164) (a preliminary report of some of the present material). Parks, Jr., R. E., and Agarwal, R. P. (1973) inThe Enzymes, Vol. VIII, Part A, edited by P. D. Boyer, pp. 307-333, Academic Press, New York.

571