GTP-binding protein(s) in green plant, Lemna paucicostata

Vol. 143, No. 3, 1987 March 30, 1987

RESEARCH COMMUNICATIONS

Pages 908-912

GTP-BINDING

PROTEIN(S)

Kohji

National

AND BIOPHYSICAL

BIOCHEMICAL

GREEN PLANT,

IN

Lemna paucicostata ---

Hasunuma and Kanako Funadera

Institute for Myodaijicho,

Basic Biology, 38 Nishigonaka, Okazaki, 444 Japan

Received February 3, 1987 in GTP-binding protein(s) assayed by [35S]GTP S was detected the extract of Lemna paucicostata containing meXIbrane components. About 15 % of =-- SIGTP S activated GTP-binding protein, separated from unbound [ 5 5SlGTP 1 by gel filtra was retained on Millipore HAWP membrane fi 3 ter. 80 % of'@" S]GTPyS activated protein, mixed with charcoal solution, remained in the supernatant after centrifugation of the mixture. Successive treatments of the assay mixture by charcoal solution established the assay system for the activation of GTP-binding protein. The Km value for the activation 0 1987 Academic Press, Inc. of GTP-binding protein was 8 nM. A growing

lines

in the

transduction

teins the

literature.

In

of

catalyzes

GTP

Transducin-GTP

vated

the

(2).

conversion

proteins

complexes

Ca2+ ions

1,4,5-trisphosphate,

GTP-binding

protein

bisphosphate

to

(4).

inositol

polyphosphoinositide of

a GTP-binding

binding oncogenic

capacity

of

of

and GTPase activity.

ras protein

having

0006-291X/87 $1.50 Copyright 0 I987 by Academic Press, Inc. All rights of reproduction in any form reserved.

light of

reticulum, to

phosphatidyl

and Ni,

Ns

receptor stimulated

be regulated inositol

was activated ras

protein

acti-

GTP to GDP

and diacyl

by a 4,5-

glycerol by the action

has both

Microinjection

a significantly

908

the

by hormone-hormone

1,4,5-trisphosphate

Normal

to transducin-

cyclase,

was suggested

in

photoactivated

hydrolysis

adenylate

endoplasmic

Cleavage

(5).

the

for

pro-

has appeared

transducin-GDP

with

phosphodiesterase protein

signal

an inhibitor

and regulated

by inositol

of GTP-binding

segment,

coupled

from

role

external

of

subunits

are GTP-binding (3).

of

removes

Regulatory

the

rod outer

cGMP phosphodiesterase

and Pi

for

chains

retinal

rhodopsin (1).

evidence

impaired

of

GTP-

mutated

GTPase acti-

by

Vol.

143,

vity

of

proteins green

PC12

these in

culture

on the similar

a stimulation

period

a GTP-binding

in

the

of

paper

we report

and

assay

method

in

in

on the Lemna

not

COMMUNICATIONS

is

reception existence paucicostata

(6).

been

paucicostata

controlled

of

made with

light

of and

also

3',5'-GMP by the

we postulated of

In

GTP-binding

oscillation

by 5 uM cyclic

cycle, the

of

have

Lemna

flowering

light-dark

in

roles

circadian

flowering

Since

protein

present its

a rhythmic

RESEARCH

differentiation

significant

concentrations

(7).

BIOPHYSICAL

observations

We detected

meduim

a dark

AND

a morphological

reports

3',5'-GMP

observed

cells

animal,

plant.

cyclic

BIOCHEMICAL

3, 1987

induced

spite

of

No.

in length

of

an involvement signal.

GTP-binding

In

the

protein(s)

441.

EXPERIMENTAL Strain and medium: Lemna paucicostata 441 has been maintained -__ by A. Takimoto of Kyoto University. The strain is a short day piant. A half strength Hutner's medium with 1 % sucrose was used to grow the plant. Glass distilled-deionized water was used to prepare culture medium. Growth of Lemna: For the maintenance of the strain, a three --frond colonv wastransplantedtothe above medium (50 ml) in a 100 ml Erlemneyer flask at‘every 7 days. twas incubated at 25 OC under white fluorescent light (3.9 W/m z ). For experimental use, the three frond colony wastransplantedto 200 mlofthe above medium in 1,000 ml Roux flasks. After incubation at 25 'C under continuous light for 10 days, about 50 fronds from the culture were transplanted to several petri dishes (6 cm) containing 10 ml each of theabove fresh medium. Afterincubationat 25 OC under white they were transferred to the dark room fluorescent light for 12 hr, 8 hr. at 25 OC for The following procedures were Preparation of crude extract: performed under dim green safelight. After removal of liquid liquid nitrogen was poured into a medium from the above culture, petri dish and the frozen plant was stored at -80 OC in darkness. Whole plants from 12 petri dishes were collected, thoroughly macerated in liquid nitrogen and mixed with 10 ml of extraction buffer containing 25 mM Pipes, pH 6.4, 0.25 mM EDTA, 1 mM MgC12, 0.5 mM diazoacetyl-DL-norleucine methyl ester, 1 mM phenyl methylsulfonyl fluoride and 0.01 mM pepstatin A. The mixture was thoroughly After macerated to frozen powder and then it was warmed to melt. thawing, the extract was centrifuged at15,OOO xg for 20 minat 2 OC. The supernatant (19 ml) was divided into 1 ml aliquots and stored at -80 OC. Assay activity: Assay for3;;phiflgdf3ngCyfm -for GTP-binding [35S]GTP S (Guanosine S'[y-thioltriphosphate, [ mol, NEGyO3OH) to GTP-binding proteins was performed according to Reaction mixture (200 ul) the modified method as described (8). contained 20 mM Pipes, pH 6.4, 0.1 mM EDTA, 0.1 M NaCl, 1.5 mM methylsulfonyl fluoride and crude extract of MC12 r 0.2 mM phenyl Incubation at 24 OC was started by adding the crude extract Lemna. --Reaction was stopped by adding to the preheated reaction mixture. 909

BIOCHEMICAL

Vol. 143, No. 3, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

400 ulofthe ice-coldquenching buffer composed of 20 mM Pipes, pH 6.4, 0.1 % Lubrol PX, 0.1 M NaCl, lO.mM MgCl 1 mM fi-mercaptoethano1 and 0.1 mM GTP. The mixture was filtere 2' with Millipore HAWP membrane filter and washed 8 times with the ice-cold washing buffer containing 20 mM Tris-HCl, pH 7.2, 0.1 M NaCl and 25 mM MgCl . Control experiment were performed by adding the quenching buf 8er The radioactivity on membrane filter before adding crude extract. in control experiments was subtracted from all sample counts. When charcoal was used to remove unbound [35S]GTP S, ice-cold 0.8 ml of charcoal solution containing 5 % of Norit SX'Plus, 1 % bovine serum albumin (Sigma; fraction V), 0.2 % dextran and 20 mM Pipes, pH 6.4 was added in sted of the quenching buffer. After 5 min of chilling in ice, it was centrifuged at 2,000 xg for 10 min at 4 OC. To the 0.7 ml of supernatant, 0.3 ml of charcoal solution was added and centrifuged as above. Resulting supernatant (0.8 ml) was mixed with 5 ml of Aquasol 2 scintillator and the radioactivity was counted. In control experiments, charcoal solution was added before the addition of crude extract, and the radioactivity was subtracted from all experimental counts. Protein was measured by the Bio-Rad protein assay kit with bovine serum albumin as standard.

RESULTS AND DISCUSSION Detection activity

of

of -

transducin

specifically method, from of

GTP-binding

the

trapping

the

activation

of

binding

Using

uCi

0.1

linear

of

for

activation

protein

was performed

in this

of

the

activation

that

in

the

presence

to GTP-binding

but

at

activation

of GTP-binding

protein

incubation

(data

higher

to maintain reduced

retention

of

volume

protein, of the

the

pressures [35 S]GTPyS

To improve binding

shown),

the the reaction

suction for

method

mixture

method

[35S]GTP,S

procedures

was relatively per assay,

the

up to

60 sec.

was about

five-fold

1.0 uCi the at

care

20 set

should

in

be since

reduction

in the

protein. activated

GTP-

were performed.

was increased 910

extract

to be constant,

resulted

this

The kinetics

IO-fold

GTP-binding

to assay

following

for

by

was examined.

and at

pressure

activated

the

work.

protein

this

suction

in

[35S]GTP,

was about

With

Using

activation

[35S]GTPYS,

GTP-binding

(1).

proteins

was linear

of GTP-binding 0.1 uCi

exercised

0.5 uCi

protein

of

not

the

the

membrane filter

filter

protein(s)

of GTP-binding

Rate

using

on the

per assay,

120 set,

for

GTP-binding

[35 S]GTPyS

[35 SIGTPyS

over

Assay

has been performed

Lemna by [35 SIGTPyS

the

protein:

to 2.5 mland

The 2.5 uCi

Vol. 143, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2

60

120 Incubation

100 tie

240

300

(set)

Sephadex G-100 gel filtration of reaction product of [ S)GTPyS and whole plant extract of Lemna. The procedure for the gel filtration of the reaction products through a Sephadex G-100 column (2x32 cm) was described in the text. () Radioactivity in 0.5 ml of each fraction. (---o---) Radioactivity in the supernatant of each fraction treated with charcoal solution. The radioactivity was calculated to the value which was expected when 0.5 mlof fraction was used and all the supernatantwas recovered for counting. ( ***.-...--..a- ) Protein concentration.

Fj$j.L

Fig. & Binding of [35 S]GTPyS as assayed after successive treatments with charcoal solution. ReqctOipn,~:x~~r~3~0S.:GTmd.)Sc~~:a~~~d 2.67 mg of protein and (7~ . . , (---a--) 0.5 nCi (2.5 nM) and (V ) 1.0 uCi (5YnM) Per assay, with the standard error bars shown.

[35S]GTPYS reaction trated

was added to mixture

for

quenching

the

5 min at

buffer

G-100

mM NaCl,

25 mM MgC12 and

ml

into

column

2

of each fraction

fraction

of the

GTP-binding fractions

gel

procedure.

To

0.1

15

of

the

1).

% of

the

for

fuged

radioactivity

in 0.7

the

60 min.

911

pH 7.2,

with

at

of

ml

of

in

the void

0.5

volume of

in the by the

charcoal

20

the same

counting

radioactivities

0.9

ml

on a

membrane localization

fraction,

chilledinice

concen-

was loaded

Radioactivity peak

the

IO-fold

HAWP membrane filter

was mixedand and the

of

PX and eluted

indicating

on Millipore ml

ml

of

20 mM Tris-HCl,

an apparant

filtration Only

with

(Fig.

incubation

The mixture

% Lubrol

0.1

revealed

protein. retained

24 OC, 0.5

equilibrated

fractions

ml

After

was added.

Sephadex

buffer

mixture.

peak

washing

solution

The mixture

was centri-

supernatant

was counted.

BIOCHEMICAL

Vol. 143, No. 3, 1987

About

65 % of radioactivities

in the

remained

in

the

supernatant,

remained

in the

supernatant.

mixtures

of

% of

the

80

the

The result

activated

reaction

free

fraction

with

protein

2 % of

time

of

provided

protein.

charcoal

[35S]GTPyS

ice-chilling

assay

remained method

Successive

solution

will

of

to 5 min,

protein

a rapid

fractions

free

was shortened

GTP-binding

GTP-binding

mixture

only

When the

activated

RESEARCH COMMUNICATIONS

GTP-binding

whereas

and the

[35 S]GTPyS

supernatant.

[35S]GTPyS the

charcoal

AND BIOPHYSICAL

the about in

for

treatments

completely

of

remove

[35S]GTPyS. Kinetics

method

of --

the activation

described

above,

protein

was followed

Results

in Fig.

different

at

of

of

different

of

the Km value

of

the

tion

of

t35~]G~~y~ in

GTP-binding

concentrations

activation

of

[35S]GTPyS from

(I)

the

M.

This

and the

its

of of

activation

for

at

double

GTP-binding

was 8~10~~

transducin

in

least

[35S]GTP,,S.

value

is very

Ni

of

three

5 min.

reciprocal with

the

GTP-binding

at

protein

subunit

Using

p rotein:

the activation

2 show an increase

rate

observed

kinetics

concentrations

Estimation

of -

plot the

of

the

concentra-

close

adenylate

to

those

cyclase

(8). ACKNOWLEDGEMENTS We are grateful to Dr. P.-S. Song for valuable discussion and critical reading of the manuscript and to Dr. A. Takimoto for providing strains of Lemna. We are also grateful to Mrs. Yazawa and Miss T. Imaizumi for excellent technical assistance.

M.

REFERENCES 1.

Bennett,

N. and DuPont,

Y. (1985)

J. Biol.

Chem. 260,

4156-

4168. 2. 3. 4. 5. 6. 7. 8.

Stryer, L., Hurley, J. B. and Fung, B. K-K. (1981) Curr. Top. 15, 93-108. Membr. Transp. Gilman, A. G. (1984) Cells, 577-579. Gill, D. L., Ueda, T., Chueh, S.-H. and Noel, M. W. (1986) Nature 320, 461-464. Cockcroft, S. and Gomperts, B. D. (1985) Nature 314, 534-536. Bar-Sagi, D. and Feramisco, J. R. (1985) Cell%, 841-848. Hasunuma, K., Shinohara, Y., Funadera, K. and Watanabe, M. (1986) Abstr. of XVI Yamada Conference: Phytochrome and Plant Photomorphogenesis, 155. Kurose, H., Katada, T., Haga, T., Haga, K., Ichiyama, A. and Ui, M. (1986) J. Biol. Chem. 261, 6423-6428. 912