Thylakoid membrane protein phosphorylation in correlation with photosynthetic membrane activation

BIOCHEMICAL

Vol. 91, No. 4, 1979 December

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

28,1979

Pages 1377-l

382

THYLAKOID MEMBRANE PROTEIN PHOSPHORYLATION IN CORRELATION WITH PHOTOSYNTHETIC MEMBRANE ACTIVATION by Richard

Bgliveau

and Guy Bellemare

Dgpartement de biochimie Facult6 des sciences et de g&ie Universitg Lava1 Qdbec, Qu6. GlK 7P4 Received

November

5,1979

SUMMARY: The phosphorylation of five g. gracilis thylakoid membrane polypeptides was studied, in isolated chloroplasts. Using C3*Pl labelling, in the light, we found that phosphorylation was inhibited by ethanol and DCMU. Inhibition curves were characteristic of photosynthetic inhibition. Cy-3*Pl ATP labelling was used to distinguish between two groups of phosphoproteins: the first one, includes protein I, II, V which require only ATP for phosphorylation while the second one includes protein III and IV whose phosphorylation is light-requiring. Phosphorylation of protein III and IV was inhibited by CCCP, NH4C1 and DCMU, and was reversible in the dark.

We have roplasts

shown previously

can use light

different

thylakoid

be light-driven

labelling

requiring

for

its

that

not

phosphorylation:

Euglena

of energy This

by CCCP.

could

isolated

source

proteins.

and inhibited

for

was coming

as the bnly membrane

phosphate

(1)

show if

five

was found

the use of c3*P!

any of the

as the ATP used

from photophosphorylation,

chlo-

to phosphorylate

phosphorylation

However,

pracilis

proteins

to

ortho-

was light-

in phosphorylation

the phenomenon

has to be light-

driven. Protein reported degree (1).

phosphorylation

by Bennet

(21,

of similarity The present

phosphorylated MATERIALS Chloroplast trophically, and washed

and the

with study

proteins

those

of thylakoid results

obtained

obtained

with

shows that is

osphorylation

membrane with pea,

has already Euglena

been

show some

as we already

outlined

of two of the

five

light-requiring.

AND METHODS isolation: Euglena gracilis (strain Z) was grown photoheteroin the medium described by Price (3). Cells were harvested twice with distilled water, followed by centrifugation at 1000 0006-291X/79/241377-06$01.00/0 1377

Copyright @ I979 by Academic Press, Inc. All rights of reproduction in anyform reserved.

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

X g for 2 minutes, then washed with grinding buffer Tb (Cl,15 M Sucrose, 0,15 M Sorbitol, 1% Ficoll, 15 mM KCl, 5 mM HEPES-KOH pH 6.8, 5 mM mercaptoethanol). The cells were resuspended in this buffer at 0,5 g/ml, broken in a French pressure cell at 1500 psi, and collected in 4 volumes of Tb. The broken cells were centrifuged at 100 x g for 1 minute, the supernatant centrifuged at 1000 x g for 3 minutes and the chloroplast pellet was resuspended in a small volume of Tr (40 mM Tricine-KOH pH 8.4, 0,33 M Sorbitol). This suspension was centrifuged at 100 x g for 3 minutes and the pellet containing remaining cells was discarded. The chloroplasts were used without further purification. Thylakoid membranes were isolated according to Vasconcelos (4). Incorporation assays: Incorporation was done as described (l),but the reaction was stopped with 33 1.11 100% TCA. After a 2 hour precipitation, the chloroplast proteins were centrifuged at 12000 x g for 1 minute, the pellet washed with 1 ml ethanol-ether (1:l) and then with 1 ml ether, air dried and resuspended in denaturating buffer. Electrophoresis was done as described before (5). Ethanol and DCMU inhibition plots were obtained by incubating 100 ~1 reaction mixture with either DCMU an ethanol in varying amounts.50 1-1 aliquots were plated on paper discs and treated to estimate the 13*PJ orthophosphate incorporation into proteins (1). CY-3*Pl ATP was synthesized according to Reeve and Huang (6).

1

A

2:o

110 CuCrul

Fig.

1: Effect

of DCMU on total

protein

3:o JAN

phosphorylation

Incubation was at 20°C. Isolated chloroplasts containing 20 ug of chlorophyll in 100 ul of Tr were incubated with carrier-free CS*P?-orthophosphate (20 ucijfrom New England Nuclear, Boston. Each reaction mixture contained a specific amount of DCMU in methanol. 50 ul sliquots were withdrawn after a 10 minutes incubation, and plated on paper discs to be treated.

1378

BIOCHEMICAL

Vol. 91, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2

4

6

8

% ETHANOL

Fig.

2: Effect of ethanol on total protein phosphorylation Conditions as in fig. 1, but with varying amounts of ethanol.

RESULTS: Molecular Ferguson

IO

(v/v)

plot,

weight

estimation

gave 28000,

of the

24500,

14700,

phosphorylated

13600,

proteins,

WOO,

for

bands

using I to V

respectively. The inhibitory [32P1

orthophosphate'is

up to 0.75 falls

to 50% then ethanol

the

percentage

ethanol

dark,

in fig. (V/V)

causing

a 50% inhibition

Fig.

3 shows the results

[32 PI the

phosphorylation proteins dark,

down to less

of ethanol

either

I, but

that

than

phosphorylation detected

III

using

is no inhibitory

effect

10% at 5 @I.

2.

incorporation

The inhibition

The inhibition

curve

is proportional

added to the reaction

mixture,

to 7.5% of

of the phosphorylation. of incorporation

in the

or [v-32,]

ATP.

of the

five

proteins

is

in the

dark

and that

with

and V are phosphorylated protein

There

phosphorylation,

[:32 P1 orthophosphate

r3* Pl orthophosphate

being II

1.

at 1 PM, the

is presented

using

of DCMU on total

shown in fig.

uM DCMU, but

for

with

action

either

and IV are

still

1379

in the light-requiring.

light

and in the

We can see that light-driven,

light

r'(- 32PJ ATP, or in the We can

no

Vol. 91, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Ill-

lllIV -ii=

1 2.

Fig.

3: Radioautography

of

Incubation

Fig.

time

04

the

phosphoprotein

was 10 min.

The

gel

was

treated

as

described

A.

Incorporation

with

r32P1

orthophosphate,

in

the

light.

B.

Incorporation

with

c32P:!

orthophosphate,

in

the

dark.

C.

Incorporation

with

Cy-32P!

ATP,

in

the

light.

D.

Incorporation

with

cy-ZPl

ATP,

in

the

dark.

4: Effect of a dark phosphoproteins Chloroplasts

transition,

were

NHaCl,

incubated in

the

in

the

A.

Incorporation

B.

Incorporation 10 minutes

C.

Incorporation

with

5 mM NHkCl.

D.

Incorporation

with

10 uM DCMU.

E. Incorporation

with

1 1.11 methanol.

Upper

part,

15 hours

2.

Lower

part,

4 hours

light,

with'[y-32P1

on the

five

ATP.

light.

after a 5 minutes dark period.

1.

DCMU and methanol

(1).

exposure exposure

1380

light-incubation

followed

by

a

Vol. 91, No. 4, 1979

also

observe

BIOCHEMICAL

that

five

new phosphorylated

(Mw = 123000,

85100,

TCA treatment

and disappear

These

four

branes

do not

not

shown).

In fig. IV is for

59200,

bands

(data

sample

the

is kept

in the dark

DISCUSSION:

for

orthophosphate

into

phorylation,

the

because the

effect

of ethanol

trical

gradient,

five

labelled

is treated

is

explained

also

causing

in

III

and IV,

between

proteinase

K.

thylakoid

mem-

the

the

a 5 minutes

inhibition

to 90°C

shown).

the

appear

of band

if

proteins

by its

III

and

solvent

used

We can also reaction

light

mixture

incubation.

incorporation

of r 3*P!

was made via

photophos-

of DCMU and ethanol. PSI1

and PSI, falls

interaction

an inhibition

us to see if

isolated

(not

phosphorylation

ATP, we bypass

with

not by methanol,

chloroplast

protein

7, 8, 9, 10)

are resistant

1, 2) show that

transfer

total

enabling

they

after

of the drastic

electron

and the

of bands

(fig.

(6,

effect

10 minutes,

Our results

RESEARCH COMMUNICATIONS

the phosphorylation

same inhibitory

a de-phosphorylation

rylation,

if

by NH4CL and DCMU, but

observe

rv-32P1

30000);

show up significantly

DCMU: CCCP has the

inhibited

bands

42000,

4, we can see that

inhibited

DCMU blocks

AND BIOPHYSICAL

ATP synthesis down;

with

the

step

any of the five

caused

is

inhibitory

the membrane

of ATP synthesis

the limiting

As

elec-

(7).

Using

by photophospho-

phosphoproteins

is really

light-requiring. We found in the

dark,

confirming

orthophosphate and V are

no incorporation

is

that

driven

proteins

reaction

is

III

either

reversible

bition

activation

of the

in the

light

and IV are phosphorylated in the dark

The possible membrane

phosphorylation

by photophosphorylation

phosphorylated

ATP, but

of C32P! orthophosphate

in the

(fig.

correlation light

strongly

of DCMU, NH4Cl and CCCP on the

(fig.

proteins 3).

or in the dark only

protein with

Proteins

[ 32P I,

II

using

c~-~~Pl

light

and this

in the

4). of this

is

five

into

phenomenon confirmed

phosphorylation

1381

with

the thylakoid

by the

drastic

of bands

III

inhiand IV

Vol. 91, No. 4, 1979

(fig.

by the

gradients that

The involvment

4).

proven

BIOCHEMICAL

dissipate

and membrane

effect

inhibitory

gradients

seems to be definitely membrane

thus

RESEARCH COMMUNICATIONS

in this

phosphorylation

implication

gradients.

necessary

exogenous

for

the

cy- 32P! ATP.

a new phenomenon to a high-energy

Electron phosphorylation

Protein

implied

is

of membrane

of NH,,Cl and CCCP, uncoupling

and pH membrane are

even with

transfer

of DCMU and the

effect

electrical

two proteins,

thylakoid

of electron

inhibitory

by the

AND BIOPHYSICAL

agents transfer of those

phosphorylation

in the

transition

of the

state.

ACKNOWLEDGEMENTS: We w%sh to thank Johanne Saucier and Colette Tremblay for skillful1 technical assistance. This work was supported by grants from National Research Council No A6923 and FCAC (Province de Qu6bec). REFERENCES: 1.

BGliveau,

R.,

2.

Bennet,

3.

Price,

4.

Vasconcelos, J.L. (1976)

5.

Chua,

6.

Reeve,

7.

Witt,

J. C.A.,

N.H. A.E., H.F.

Bellemare,

(1977) Vallee, A.C., Plant

G. (1979)

Nature R.L.

(1971)

R.C. Quat.

(1962)

P. (1975) (1979) Rev.

iv-803.

344-346. Plant

Physiol.

Mendiola-Morgenthaler, Physiol. -58: 87-90.

and Bennoun, Huang,

269:

BBRC &3 (3):

L.R.,

P.N.A.S. Nucl.

of Biophys.

1382

(U.S.)

-37 : 428-433. Floyd,

-72: 2175-

AC. Res. 6 (1): I(4):

G.L.,

365-477.

81-90.

Salisbury, 2179.