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MAHARASllTRA.
INDIA
Photosynthesis III. Structure and Molecular Organisation of the Photosynthetic Apparatus
Edited by George Akoyunoglou
~.
© 1981 Balaban International Science Services, Philadelphia, Pa.
\
3
,
89 4
LATERAL ORGANIZATION OF THE CHLOROPHYLL - PROTEIN COMPLEXES
OF SPINACH THYLAKOIDS
Jan M. Anderson and B. Andersson *
Division of Plant Industry CSIRO, Canberra, ACT 2601, Australia
Abstract The distribution of chlorophyll-p r otein complexes between
appressed and non- appressed membrane regions was studied by a comparison
of the amounts of chlorophyl l-protein complexes resolved by SDS PAGE
from thylakoids and various subchloroplast membrane fractions. There is
a substantial depletion of photosystem 1 complex, and an enrichment of
photosystem 2 complex and light - harvesting complex in the appressed
grana partitions. The higher enrichment in the grana partition fraction
compared to grana stack fract i ons (derived from ' French press or digitonin
methods) suggests that grana photo system 1 complex is restricted to nonappre ssed grana end membranes and margins , and grana part i tions possess
only photosystem 2 complex and light - harves tin g complex. In contrast ,
stroma thylakoids are highly enriched in pho t osystem 1 complex . They
have also some 10- 20% of photosystem 2 comp lex and light harvesting
complex . The ratio of light - harvesting complex to photosystem 2 complex
is rather constant for appressed and non-appressed membrane regions,
suggesting a close structural organization be tween these two complexes.
The spatial separation of most of photosys tem 2 located mainly i n
grana partitions, from that of photosystem 1 located in non- appressed
regions has important consequences for both the distribution of excitation
energy and photosynthetic electron transport between the photosystems.
INTRODUCTION
As well as an asymmetric distribution of components across membrane
bilayers, there is also increasing evidence for a non-random distribution
l
of components in the lateral plane of some membranes ,2. The structural
compartmentation of most higher plant thylakoids into 'stacked and
unstacked regions with different freeze - fracture appearance is well
3
known • In addition, a functiona l heterogeneity is suggested by several
S
4
Studies. The chloroplast coupling factor and ferredoxin - NADP+ reductase
are located only on the outer surfaces of non- appressed thylakoids.
In
Contrast, photosystem 2 seems to be located mainly in the appressed
*
Present address:
Department of Biochemistry, University of Lund, Sweden
23
'-.A
Anderson and Andersson
24
grana
..
part~t~ons
5,6
I
Membrane fractionation studies after detergent or
7
mechanical fragmentation also suggest a compartmentation of function ,
Heavy fractions containing grana stacks have both photosystems, with
more photosystem 2 than photosystem I, while light fractions originating
from stroma thylakoids have mainly photosystem 1 properties.
A recent
fractionation procedure, using aqueous polymer two-phase partition of
Yeda press-disrupted thylakoids, gives a photosystem 2-enriched fraction
which consists mainly of partition thylakoids devoid of grana end membranes
and
.
marg~ns
8-10
This study compares subchloroplast fractions derived from appressed
(grana partitions) and non-appressed thylakoids (stroma thylakoids, grana
end membranes and grana margins) with respect to their relative content
of the three main chlorophyll-protein complexes
11
. These are the photo-
system 1 P700-chlorophyll a-protein complex, the photosystem 2 P68012 13
chlorophyll ~-protein complex and the light-harvesting complex'
The
chlorophyll-proteins were resolved by a sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE) method which allows most of
the chlorophyll to remain attached to protein, thereby permitting the
relative amounts of chlorophyll associated 1-lith the photosystems to be .
determinei 2 ,13.
METHODS
Spinach chloroplasts isolated in 50 roM phosphate buffer (pH 7.2), 100
roM KCl, 300 roM sucrose were disrupted by passage through the French
. 15 as d escr1'b e d prev10us
.
1y. Su b c hI orop 1 ast
press 14 or b y d"~g1ton1n
membrane fractions were isolated by differential centrifugation (Fig. 1).
Digitonin or
French press
!
Grana
stacks
Veda press and
phase partition
l!
B
F-lO, D-lO
Y-l00
F-144, D-144
PS2 enriched
PSl mainly
Fig. 1.
Fractionation scheme.
1
Grana
partition
83
> PS2
enriched
. ~
:
.,
25
Anderson and Andersson
The French press fractions (F-lO and F-lOO) represent the 10,000 x
100,000 x
& pellet
& and
fractions, and the digitonin fractions (D-lO anp D-144),
represent the 10,000 x
~
and 144,000 x £ fractions, respectively.
Chloroplasts were fragmented also by Yeda-press treatment as described
previously8
The supernatant after centrifugation at 40,000 x
B for 30
min was recentrifuged at 100,000 x £ for 1 h yielding a Y-lOO fraction.
The pellet (Y-40) was passed twice more through the Press in a low-salt
9
buffer before aqueous polymer two-phase partition
Two main fractions
8 9
(T2 and B3) were collected; B3 is highly enriched in photosystem 2 '
9
and originates mainly from the grana partitions ,10
Chloroplasts and subchloroplast fractions were solubilized prior to
use at 4°C with 0.3 M Tris-HCl (pH 8.8), 10% glycerol, 0.375% SDS
(SDS/Chl of 7.5).
Discontinuous polyacrylamide gel electrophoresis was
13
carried out as described before • The relative distribution of chlorophyll
in the chlorophyll-protein complexes in the gels was determined by
scanning at 675 and 670 nm. Antibodies against purified light-harvesting
16
complex
were obtained by standard immunization procedures.
RESULTS
The relative distribution of chlorophyll in the chlorophyll-protein
complexes of spinach thylakoids and various subchloroplast fractions was
13
determined by SDS PAGE at 4°C
(Fig. 2). The P700-chlorophyll~­
protein-complex of PS 1 (termed here PS 1 complex) is resolved as two
P700-chlorophyll
~~_CPla
(CPla and CPl)
which together have photoreactive P700 and 120
~CPl
chlorophyll a molecules with fluorescence
-
LHCp1
tzz~~zI~ LHCP
~-proteins
2
" " CPa
LH CP 3
properties of photosystem 1 only
lY
•
The third
chlorophyll a-protein (PS 2 complex) (CPa) is
-
considered from indirect evidence to be as sociated with photosystem 2 (cf. 17). The three
1-3
~/~-protein bands resolved (LHCP
)
chlorophyll
are all associated with the light-harvesting
complex since they have similar spectral pro13
perties and polypeptide compositions
Some
chlorophyll has been dissociated from proteins'
and moves at the gel front as detergentchlorophyll micelles (FC).
Fig. 2.
Chlorophyll-protein complexes resolved by SDS PAGE
13
,
26
Anderson and Andersson
~PS1complex
~PS2 -
DLHCP
complex
P:8 FC
Ud
69
65
52
27
83
Chloroplasts
60
65
Y-100
67
60
20
F-lO
0-10
F-144
0-144
Fig . 3. Relative chlorophyll distrib~tion (% of total chlorophyll) of
chlorophyll - protein complexes of spinach thyla~ '.oids, French press (F-IO.
F-144), digitonin (D-lO, D-144) and'Yeda press (Y-lOO) fractions and the
grana partition fraction B3 .
The relative amounts of chlorophyll associated with the three main
chlorophyll - protein complexes are shown in the histograms of Fig. 3.
For
unfractionated thylakoids some 27% of the total chlorophyll is
associated with PS 1 complex (CPla and CPl). 52% with the light-harvestin
complex ( LHCpl-3) and 13% with the PS 2 complex (CPa).
The amounts of
chlorophyll -protein complexes associated with the subchl.oroplast fraction
vary considerably from those found in unfractionated thylakoids.
Thylakc
fragments derived from stroma thylakoids (F-144, D-144, Y-lOO) all had mt
more PS 1 complex, and much less PS 2 complex and light-harvesting
complex compared to thylakoids.
The relative amount of chlorophyll
27
Anderson and Andersson
Table 1. Ratios of the amounts of chlorophyll-protein complexes in
thylakoids and sub chloroplast fractions.
CP1 /C Pa
LHCP/ CPl
LHCP/C Pa
Unfractionated thylakoids
2.2
1.9
4. 1
Grana stacks (F·1 0)
1.5
3.0
4.6
Partition region (83)
0.72
6.5
4.7
Stroma thylakoids (Y-100)
15
0.24
3.6
associated with the PS 1 complex is about three times greater than is
f ound in unfractionated thylakoids, and there is a corresponding decrease
in PS 2 complex and light-harvesting complex.
The heavy grana stack
fractions isol ated from the French press (F-lO) and digitonin (D-lO)
procedures are both depleted in CPI and enriched in CPa and LHCP comp lexes.
The depletion of PS 1 complex (about three times less chlorophyll
compared to that of unfractionated thylakoids) is even more pronounced
with the B3 fraction isolated by phase- partition.
Fraction B3 consists
9
of inside-out vesicles derived mainly from the grana partitions ,10
Most of the chlorophyll of fraction B3 is found in LHCP (65%) and CPa
(14%) whereas CPla and CPl together only account for 10% of the total
chlorophyll.
The greater depletion of PS 1 complex in fraction B3 compared
to those of the grana stack fractions (F-lO, D-lO) suggests that grana
photosystem I is located mainly in the non-appressed membrane regions.
Table 1 summarizes the ratios between the relative amounts of chlorophyll
associated with the three main chlorophyll -protein complexes in thylakoids
and subchloroplast membrane fragments.
A high enrichment of PS I
complex in non-appressed thylakoids (CPla + CPl/CPa
=
15) is matched by
a high enrichment of PS 2 complex in appressed thylakoids (CPl/CPa
0.72).
=
Significantly, the LHCP/CPa ratios are rather constant in all
subchloroplast fractions, in contrast to the LHCP/CPl ratios which show
considerable variations.
This suggests that the light-harvesting complex
in the native membrane is associated mainly with photosystem 2 reaction
centre complex rather than with photosystem I reaction centre complex.
An antibody against the Triton X-IOO purified light - harvesting
16
complex
was tested against thylakoids, Y-40 fraction (slightly enriched
in LHCP) and stroma thy1akoids (Y-IOO).
d
Very weak agglutination was
seen with Y-IOO vesicles, in contrast to strong agglutination with the
Y-40 vesicl es.
A very weak agglutination was observed with normal
stacked chloroplast thylakoids, but if the thylakoids were des tacked by
suspension in low salt buffer prior to antibody incubation, a strong
28
Anderson and Andersson
agglutination was seen.
This supports our evidence for depletion of
LHCP not only in stroma thylakoids, but also in exposed regions of grana
end membranes and margins.
DISCUSSION
These results point to a pronounced lateral heterogeneity in the
distribution of chlorophyll-protein complexes between appressed and nonappressed regions of spinach thylakoids.
PS 2 complex and light-harvestin1
complex are located mainly in appressed regions, while PS I complex is
located in non-appressed regions.
These results agree with previous
I
.
6-8 ' IS
measurements of photochemical activities in subchloroplast fract10ns
The present method, however, allows for the first time direct measurement
of the amount of
chlorophyll associated with each photosystem.
Might the depletion of Ps I complex be even greater in grana partitions
in vivo than indicated by the experimental values obtained for the B3
fraction?
Fraction B3 contains residual amounts of coupling factor,
demonstrating some contamination with non-appressed membranes.
the amounts of chlorophyll-protein
were calculated by (a)
comple~es
Therefore,
in the partition regions
assuming different amounts of total chlorophyll
in non-appressed and appressed membrane regions, and
(b)
subtracting
the experimental amounts of chlorophyll-proteins found in the "best"
non-appressed membrane fraction (Y-IOO) from that found in unfractionated
thylakoids (Table 2).
ASSUming 30% of the total chlorophyll is located
in the non-appressed membrane regions, the calculated proportions of
chlorophyll-protein complexes (Table 2) correspond very well to those
obtained experimentally with the BJ fraction (Fig. 3), which is contaminatedl
with some non-appressed membranes.
These calculations point to an extrRmely
low content of PS 1 complex in the partition region.
It seems also that
the amount of total chlorophyll associated with non-appressed membrane
regions of spinach thylakoids is at least 30%.
Thus, our experimental
Table 2. Calculated amounts (% of total chlorophyll) of chlorophyllprotein complexes in grana partitions.
Assumed proportions of
total chlorophyll in
non-appressed thylakoids
20%
25%
30%
35%
40%
Calculated amounts of chlorophyllprotein complexes in grana partitions
CP1 + CP1a
CPa' LHCp 1-3
17
14
9
4
0
15
16
16
17
17
61
64
67
71
75
29
Anderson and Andersson
EJ
[J
PSl
PS2 (reaction centre complex + LHCP)
9 Coupling
factor
Fig. 4. Schematic model for the lateral distribution of photosystem 1 and
photosystem 2 between appressed and non-appressed thylakoid membranes of
spinach chloroplasts 11 •
results, together with the calculations (Table 2) point to an extreme
lateral heterogeneity in the distribution of the chlorophyll-protein
complexes (Fig. 4).
We suggest that PS 1 complex is found only in the
5
non-appressed membrane regions, together with ferredoxin-NADP+ reductase
4
and chloroplast coupling factor . While PS 2 complex and light-harvesting
complex are located mainly in grana partitions, a certain amount {1020%) of the light-harvesting complex and PS 2 complex is located also
in non-appressed membranes.
The demonstrated localization of the chlorophyll-protein complexes in
different membrane regions has important consequences for both the transfer
of light energy from the antennae pigments to the reaction centres of
photosystems 1 and 2, and electron transport between the photosystems.
A
mobile electron transport carrier is required to carry reducing equivalents
from photosystem 2 in the grana partitions, to photosystem 1 located out
in non-appressed membranes.
The lipid-soluble molecules of the plastoquinone
pool would seem to be the most likely electron transport carriers, because
of their greater concentration compared to other electron transport
components.
Such amphipathic molecules have fast diffusion rates in the
18
. Moreover, the rate limiting step in photo-
lateral plane of membranes
synthesis is the re-oxidation of reduced plastoquinone by P700+ 19
Two extreme models have been proposed for the sharing of light
energy between photosystem 2 and photosystem 1.
In the continuous array
model (favoured by most studies) the pigment bed is shared between the
photosystems, while in the separate package model, the photosystems
exist as discrete entities with no shared pigments (cf. 20,21).
Our
studies suggest that both situations are found in grana-containing
30
Anderson and Ande1'8lOll
chloroplasts.
Host of the photosystem 2 complex and its associated
light-harvesting complex, located in the appressed grana partitions,
cannot be in direct contact with PS 1.
Any sharing of light excitation
energy between photosystem 2 and photosystem 1 must occur in the nonappressed membrane regions, rather than as hitherto assumed in grana
partitions.
ACKNOWLEDGEHENTS
We are indebted 'to Dr. 1.J. Ryrie, A.N.U. for the agglutination
experiment.
A European Molecular Biology Organization Fellowship for B.A.
is greatly appreciated.
REFERENCES
1
DePierre, J.W. and Ernster, L.
201-262
2
Israe1achvi1i, J. (1978) in Light Transducing Hembranes (Deamer
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3
Staehelin, L.A.
4
Miller, K.R. and Staehelin, L.A.
5
Jennings, R.C., Garlaschi, F.,H. Gero1a, P.D. and Forti, G.
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Anderson, J.M.
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Cherry, R.J.
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20
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21
Thornber, J.P. and Barber, J. (1979)
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31
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