Abstract‘In vitro’ phosphorylated thylakoid polypeptides were studied by means of different electrophoretic techniques. A highly resolving two-dimensional electrophoresis method, recently developed in the laboratory using CHAPS and SDS as detergent for electrofocusing, allows the separation of each of the LHCP apoproteins into several molecular species. Those having more acidic isoelectric points correspond to the phosphorylated forms

Ambard-Bretteville, F.

Dubertret, G.

Rémy, R.

Elsevier - Publisher Connector 

Separation of phosphorylated from non-phosphorylated LHCP polypeptides by two-dimensional electrophoresis 

Volume 188, number 1 FEBS 2777 August 1985 
Separation of phosphorylated from non-phosphorylated 
LHCP polypeptides by two-dimensional electrophoresis 
R. R&my, F. Ambard-Bretteville and G. Dubertret* 
Laboratoire & Photosynth&e and *Laboratoire de Cytophysiologie de la Photosynth&e, CNRS, 91190 Gg-sur- yvette, 
France 
Received 21 May 1985 
‘In vitro’ phosphorylated thylakoid polypeptides were studied by means of different electrophoretic tech- 
niques. A highly resolving two-dimensional electrophoresis method, recently developed in the laboratory 
using CHAPS and SDS as detergent for electrofocusing, allows the separation of each of the LHCP apopro- 
teins into several molecular species. Those having more acidic isoelectric points correspond to the phosphor- 
ylated forms. 
Two-dimensional electrophoresis Electrofocusing Light-harvesting complex 
Pea 
Phosphorylation Polypeptide 
1. INTRODUCTION 
Higher plants and algae are able to regulate light 
energy distribution between their two photo- 
systems. This regulation is thought to involve the 
reversible phosphorylation of LHCP polypeptides. 
According to the actual model of regulation, a 
mobile part of LHCP, when phosphorylated, 
migrates from appressed thylakoid regions (rich in 
PS II) to non-appressed regions (rich in PS I). The 
reverse movement occurs when LHCP is dephos- 
phorylated. The abundant literature published on 
this topic in the last decade can be found in recent 
reviews [l-4]. At present, among the many ques- 
tions raised by LHCP phosphorylation, a major 
problem is knowledge of the exact amount of 
Abbreviations: CFl , chloroplast coupling factor; 
CHAPS, 3-[(3-cholamidopropyI)dimethyhtmmonio]-l- 
propanesulfonate; chl, chlorophyll; DCMU, 3-(3’ ,4’- 
dichlorodiphenyl)-1 , 1 -dimethylurea; IEF, isoelectrofo- 
cusing; LHCP, light-harvesting chlorophyll a/b-protein 
complex; Ll, L2, L3, LHCPl, LHCPZ, LHCP3; Lx, 
LHCPx - the light-harvesting complex of photosystem 
I; PS, photosystem; SDS-PAGE, SDS-polyacrylamide 
gel electrophoresis 
LHCP which can be phosphorylated and, to this 
aim, to achieve the biochemical separation of 
phosphorylated and non-phosphorylated LHCP 
forms. Conventional methods such as SDS-PAGE 
do not allow this separation. Several years ago, we 
developed 2-D electrophoresis to characterize dif- 
ferent lines of higher plants, especially Brassica, by 
their mitochondrial or chloroplast polypeptides 
[5]. Unfortunately, as mentioned in [5-71, LHCP 
polypeptides generally run as streaks in 2-D 
separations due to their hydrophobicity. Recently, 
using CHAPS as detergent, we greatly improved 
the resolution of our 2-D analyses. From total 
thylakoid polypeptides or purified LHCP, we suc- 
ceeded in the separation of LHCP polypeptides in- 
to several molecular species differing in isoelectric 
point [7]. Here, we demonstrate that phosphory- 
lated LHCP polypeptides can be separated from 
non-phosphorylated ones. 
2. MATERIALS AND METHODS 
2.1. Plant material and chloroplast isolation 
The investigations were carried out with pea 
(Pisum sativum L. cv. Kelvedon Wonder) grown in 
a greenhouse at 22°C and 15 h day length. 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
00145793/85/%3.30 0 1985 Federation of European Biochemical Societies 43 
Volume 188, number 1 FEBS LETTERS August 1985 
Chloroplast isolation was performed according 
to [8]. 
2.2. Thylakoid phosphorylation 
The method in [8] was followed except that 
chlorophyll concentration in the incubation 
medium was 150pg/ml and ATP was 1OOpM. Il- 
lumination was provided by a white light 
(500pE/m2 per s). When required, 10 &i 
[Y-~~P]ATP (Amersham, spec. act. 3000 Ci/mM) 
were added to the reaction mixture. Phosphory- 
lated samples were defined as those illuminated for 
15 min followed by 15 min in the dark in the 
presence of 20 mM NaF. Dephosphorylated 
samples differed in that NaF was added only after 
the dark period. Non-phosphorylated samples 
were defined as having 30 min dark period with 
ATP before adding NaF. 
2.3. Fluorescence measurements 
Room-temperature fluorescence induction tran- 
sients were performed on a laboratory-built device 
[9] after dilution of the samples at 5 pug chl/ml and 
in the presence of 2 x lo-’ M DCMU. 
Fluorescence emission spectra were recorded at 
77 K as in [lo] with samples containing about 
10 pg chl/ml. 
2.4. Two-dimensional electrophoresis 
Two kinds of 2-D electrophoresis were per- 
formed differing in the nature of the 1-D. We used 
either SDS-PAGE of undenatured chlorophyll- 
protein complexes as in [lo, 1 l] or IEF performed 
with acetone-depigmented thylakoids. This IEF 
technique using CHAPS and SDS as detergent has 
been detailed in [7]. The second dimensions were 
performed in 12Vo acrylamide gels according to 
[121. 
3. RESULTS 
3.1. Fluorescence controls of the phosphorylation 
state of thylakoids 
Fig.1 presents the fluorescence induction tran- 
sients. As demonstrated in [8], the magnitude of 
the variable component is reduced in the 
phosphorylated sample compared to the non- 
phosphorylated one. Moreover, the induction 
curve is rather exponential instead of sigmoidal. 
The dephosphorylated sample shows an in- 
44 
NP 
DP 
P 
Fig. 1. Room-temperature induction transients in 
presence of 2pM DCMU of phosphorylated (P), 
dephosphorylated (DP) and non-phosphorylated (NP) 
thylakoids. 
OD 
.E 
3 
‘0 
t 
W 
g 
H 
W 
: 
A 
LL 
0.4 
0.3 
0; 
0.1 
c 
I-
I-
I- 
I- 
+ ,‘, , , 1 I I I 
700 750 
WAVELENGTH (nm) 
Fig.2. Low-temperature (77 K) fluorescence emission 
spectra of phosphorylated (- - -) and non- 
phosphorylated (-) thylakoids. Spectra are 
normalized to 735 nm. Excitation, 470 nm. 
Volume 188, number 1 FEBS LETTERS August 1985 
termediate curve where the maximum level of 
fluorescence observed with the non-phosphory- 
lated sample is not reached. This is probably due 
to insufficient dephosphorylation, the occurrence 
of photodestruction being unlikely, since pigment 
photobleaching is not observed, Under the 3 condi- 
tions investigated, the initial level of fluorescence 
is constant. Fig.2 compares the fluorescence mis- 
sion of phosphorylated and non-phosphorylated 
samples. The short-wavelength PS II fluorescence 
emissions decrease relative to the PS I 735 nm 
emission indicating that, as in [13], an increased 
proportion of the absorbed excitation energy is 
partitioned to PS I in the phosphorylated sample. 
3.2. Tw~-dirnen~~u~~~ analysis of p~~~p~ory~ated 
chlorophyll-protein complexes 
Comparison of fig.3A and B confirms that the 
major [y-32P]ATP-labelled polypeptides are those 
of LHCP, either in its oligomeric forms (LHCPI 
lSt Dimension 
w 
and LHCP2) or monomeric form (LHCP3). The 
1-D separations presented on the right-hand side of 
fig.3A and B and in fig.4 show that the 2 con- 
stitutive LHCP polypeptides of 29 and 27 kDa are 
labelled. Other polypeptides about 43, 33, 20 and 
11 kDa show faint labelling (figs 3B and 4). It is 
noteworthy that a 24 kDa polypeptide (arrow in 
fig.3A) that we mentioned earlier as constitutive of 
a light-harvesting complex corresponding to the 
peripheral antenna of PS I 1101 is non- 
phosphorylated (fig.3A,B). 
3.3. Two-dimensional analysis of phosphorylated 
t~ylako~d poiypeptides 
FigSA shows a typical 2-D electrophoresis of 
phosphorylated thylakoid polypeptides. Up to 90 
polypeptides are detected. As reported in 171, the 
location of LHCP spots was performed from the 
2-D analysis of purified LHCP. Here, LHCP is 
resolved into at least 5 polypeptides. Two major 
Fig.3. Two-dimensional electrophoresis of phosphorylated pea thyiakoids. First dimension, chlorophyll-protein 
complexes resolved by mild electrophoresis. Second dimension, polypeptides resolved by SDS-PAGE. (A) Coomassie 
blue-stained gel. (B) Autoradiogram of the gel in A. Large arrowheads to the right of A and B show, as control, a 1-D 
eiectrophoresis of thylakoid polypeptides. The smaI1 arrowhead in A indicates the 24 kDa polypeptide constitutive of 
the light-harvesting complex of the peripheral antenna of PS I. 
Volume 188, number 1 FEBS LETTERS August 1985 
m 
; 
r 
. 
: 
: i 
A B 
67 
43 
30 
20 
14 
Fig.4. SDS-PAGE of phosphorylated thylakoid 
polypeptides. (A) Protein markers in order of decreasing 
Mr: phosphorylase b, bovine serum albumin, 
ovalbumin, carbonic anhydrase, soybean trypsin 
inhibitor, a-lactalbumin. (B) [32P]ATP light-labelled 
thylakoid polypeptides tained with Coomassie blue. (C) 
Autoradiogram of the gel in B. (D) Autoradiogram of 
[32P]ATP dark-labelled polypeptides. 
spots correspond to the 29 kDa apoproteins and 
two minor spots belong to those of 27 kDa. One 
additional spot is present at 26 kDa as is more ob- 
vious from the details in fig.SC taken from a better 
2-D separation of this region. From the 
autoradiogram of gel A shown in fig.SB as well as 
in the details in fig.SD, it is clear that labelled 
LHCP polypeptides are those which possess more 
acidic isoelectric points. These phosphorylated 
polypeptides are indicated by the open arrows in 
figSA. By comparing in figSA and B the intensity 
of staining and radiolabelling, it appears that the 
29 and 27 kDa polypeptides seem equally labelled 
relative to their protein content. 
Apart from LHCP polypeptides, the other 
phosphorylated polypeptides previously observed 
are no longer detected. We explain their disap- 
pearance by their low labelling and by the more 
drastic solubilization process performed for 2-D 
analysis. 
IEF 
7 6 5 t3H 
IA --- 
-* --- - _ 
6 . .6:6 * . P” 
Fig.5. Two-dimensional gel electrophoresis of 
[3ZP]ATP-labelled thylakoid polypeptides. IEF in the 
first dimension, SDS-PAGE in the second dimension. 
(A) Coomassie blue-stained gel. Filled arrows, non- 
phosphorylated LHCP polypeptides; open arrows, 
phosphorylated ones. To the right is a 1-D control with 
the same sample. (B) Autoradiogram of the gel in A. (C) 
Details of LHCP spots resolved by 2-D electrophoresis 
from a gel similar to that in A. Polypeptides stained with 
Coomassie blue. (D) Details of the autoradiogram 
corresponding to C. 
46 
Volume 188, number 1 FEBS LETTERS August 1985 
4. DISCUSSION AND CONCLUSION 
Here, we have studied the phosphorylation of 
thylakoid chlorophyll-protein complexes and 
polypeptides. The fluorescence data were given as 
control of the phosphorylation state and will thus 
not be discussed. Among the chlorophyll-protein 
complexes, only monomeric and oligomeric forms 
of LHCP were highly phosphorylated. The 
chlorophyll-protein complex representing the 
peripherical light-harvesting antenna of PS I first 
demonstrated and named LHCPx by us in non- 
denaturing gels [10,14] and then LHCl by other 
groups [15-191 is non-phosphorylated. In addi- 
tion, in agreement with other data, minor polypep- 
tides of 43, 35, 33, 20 and 11 kDa were also found 
to be phosphorylated. The original result which is 
presented here comes from the application of a 
highly resolving 2-D electrophoretic separation we 
recently developed [7] which, for the first time, 
allows discrimination between phosphorylated and 
non-phosphorylated LHCP apoproteins. It was 
recently reported that in barley, the high-Mr 
apoprotein of LHCP consists of at least 3 peptides 
with the same Mr but different isoelectric points 
[20]. We confirm this finding for the larger 
apoprotein of LHCP (29 kDa) with more resolving 
analyses and show that forms having more acidic 
isoelectric points are phosphorylated. The isoelec- 
tric point difference between phosphorylated and 
non-phosphorylated 29 kDa forms is estimated at 
less than 0.1 pH unit. These observations can also 
be applied to the 27 kDa polypeptide. 
It is noteworthy that we are at present unable to 
prove that phosphorylation induces an acidic shift 
of the 29 and 27 kDa polypeptides. All attempts 
we have made to obtain full labelling of LHCP 
apoproteins have failed. Conversely, under 
dephosphorylating conditions, each of the 29 and 
27 kDa polypeptides always remains composed of 
at least 2 different electrofocusing spots detected 
by Coomassie blue or silver staining even if all 32P 
labelling disappears from acidic LHCP forms (un- 
published). If we keep in mind that quantitative 
variations in the relative amounts of stained 
polypeptides are difficult to estimate, the possibili- 
ty of a molecular difference between phosphory- 
lated and non-phosphorylated polypeptides cannot 
be excluded. To support this suggestion, LHCP is 
now known to be encoded in the nucleus by multi- 
ple genes [21]. To clarify this, we believe that 2-D 
analysis and purification of these LHCP polypep- 
tides could, in the future, bring forth new informa- 
tion about this subject. 
ACKNOWLEDGEMENTS 
The authors are grateful to A. de Courcel for 
improvement of the English paper and thank Mrs 
Montaggioni for typing the manuscript and R. 
Boyer for the photographic work. 
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47 


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Separation of phosphorylated from non-phosphorylated LHCP polypeptides by two-dimensional electrophoresis

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