(Received for publication, February 16, 1995; and in revised form, May 2, 1995)
From the
A nuclear-encoded polypeptide of 6.1 kDa was identified in
isolated photosystem II (PSII) reaction center from Spinacia
oleracea. The hydrophobic membrane protein easily escapes staining
procedures such as Coomassie R-250 or silver staining, but it is
clearly detected by immunodecoration with peptide-directed IgG. This
additional subunit was found to be present in PSII reaction centers
previously known to contain only the D1/D2/cytb
Light-induced photosynthetic water oxidation and plastoquinone
reduction takes place in the thylakoids of cyanobacteria, algae, and
plants. These redox mediated reactions are catalyzed by a multisubunit
membrane complex designated as photosystem
II(1, 2, 3, 4) . This membrane
protein complex has been shown to consist of more than 25 different
polypeptides with relative molecular masses ranging from 47 down to 3
kDa. The minimum subcomplex that can evolve oxygen and release protons
is referred to as the PSII
However, based on sequence
homology between the L and M subunits of the reaction center complex of
purple bacteria and the D1 and D2 protein, the concept of a D1/D2
reaction center heterodimer was proposed also for higher
plants(5, 6, 7) . Indeed, such a PSII
reaction center complex consisting of the D1, D2 proteins, cytochrome b
Previously several low molecular mass
polypeptides of 10-3 kDa have been identified in various types of
photosystem II preparations (12, 13, 14) using polyacrylamide gels of
high resolution. Most of them have one hydrophobic stretch predicted to
be a transmembrane
In this paper
we report on the identification and isolation of a nuclear-encoded
6.1-kDa polypeptide. It is shown that the 6.1-kDa protein is an
additional, previously undetected subunit of the PSII reaction center
of higher plants. The molecular mass of the polypeptide was calculated
on the basis of the number of amino acids of the homologous protein
deduced from the Arabidopsis thaliana gene ((37) ).
Although the purified spinach polypeptide has a molecular mass of 4.6
kDa as estimated from SDS-urea polyacrylamide gels, we prefer to
designate it 6.1-kDa polypeptide throughout the manuscript.
To remove excess detergent,
the PSII membrane fragments were washed with medium: A, 10 mM MES-NaOH, pH 6.5, 5 mM MgCl
Immunoblotting was performed onto
PVDF membranes (0.2 µm) according to Towbin et al.(34) using a semidry blotting system (Millipore).
Immunodecorations were visualized either using the ECL (enhanced
chemoluminescence) technique (Amersham) or the alkaline
phosphatase system (Bio-Rad).
Purified IgG was used in dilutions
from 1/500 up to 1/10
Additionally,
O
Figure 1:
Denaturing, silver stained SDS-PAGE of
the purified low molecular mass protein (lanes 1 and 2), and PSII membrane fragments (lane 3). Lane 1 shows an immunoblot using the site-specific antiserum raised
against the 6.1-kDa protein.
At room temperature the absorption spectrum of the purified
low molecular mass protein had a maximum at
N-terminal amino acid
sequencing of the isolated protein was performed after SDS-PAGE
followed by electroblotting onto PVDF membranes. Nineteen amino acids
could be determined (Table 1) supporting and extending
preliminary data(13, 14) , but also confirming that
the site-directed antibody indeed identified the 6.1-kDa protein. A
comparison of the obtained N-terminal sequence with those from Triticum aestivum(14) , Chlamydomonas reinhardtii(36) , and that deduced from randomly obtained cDNA from A. thaliana(37) revealed that the first 10 amino
acids were almost identical. With respect to C. reinhardtii(36) only three deviations were found in: position 7
(S/N), 8 (T/G) and 9 (E/D). The comparison with A. thaliana revealed only one deviation in position 18 (M/S). Interestingly,
in position 6 of the purified 6.1-kDa protein a double, equally sized,
signal was obtained, suggesting 50% of methionine and glutamine in this
position. The significance and reason for this are not clear at the
moment.
Figure 2:
A, immunoblots of purified 6.1-kDa protein (1), PSII reaction center (2), O
Intact
normal thylakoids and right-side-out thylakoids reacted only very
weakly with the 6.1-kDa directed antibodies, while inside-out
thylakoids showed a very strong reaction (roughly 10 times stronger
compared to thylakoids). The much stronger IgG immune response with the
inside-out thylakoids suggests that the N terminus of the 6.1-kDa
protein is on the lumen side of the membrane. This finding leads us to
further investigate the gene recently obtained from A.
thaliana(37) , corresponding to the isolated 6.1-kDa
protein. An analysis of the presequences of the precursor of the
6.1-kDa protein deduced from A. thaliana turned out to have a
predicted length of 79 amino acids. This should be compared to the
mature protein that is predicted to consist of only 54 amino acids. The
presequence reveals some typical features common with other transit
sequences found for lumen polypeptides such as the extrinsic 10-, 16-,
23-, and 33-kDa subunits of photosystem II. Some of the typical
features are: it starts with the dipeptide composed of methionine-
alanine and has a central part enriched in the hydroxylated amino acids
serine and threonine as well as in positively charged amino acids
arginine and lysine(38, 39) . It ends with the
consensus sequence tripeptide alanine-X-alanine, where X represents a variable amino acid residue.
The mature 6.1-kDa
protein deduced from A. thaliana gene (37) contains a
hydrophobic region in the middle, which is predicted to be a
transmembrane
Figure 4:
Schematic drawing of the 6.1-kDa protein
with respect to its special features concerning localization,
orientation, and nearest neighbors.
Figure 3:
Immunoblots of PSII membrane fragments
cross-linked with EDC/Chl ratio (w/w). Control (lane 1), 1:1, lane 2; 2:1, lane 3; 4:1, lane 4; 10:1, lane 5; and 20:1, lanes 6-9, using the
antiserum against the 6.1-kDa protein (lanes 1-6), D1 (lane 7), D2 (lane 8), and cytb
In this work it is shown that PSII reaction center complexes
contain one additional, previously not detected low molecular mass
subunit. This finding was affirmed by analyzing several types of PSII
reaction center complex preparations and using two different
immunoassays (immunoblotting and ELISA). Using both methods unambiguous
immunodecorations of the 6.1-kDa polypeptide were obtained in
stoicheiometric amounts for the PSII reaction center complex
preparations thus far analyzed. The purity of the used PSII reaction
center complexes was assured by silver-stained SDS-urea polyacrylamide
gels, their absorption spectra (Fig. 2), and by determining the
number of chlorophyll a molecules/P680 (4-6
chlorophyll/reaction center, not shown). Furthermore cross-linking
experiments in combination with specific immunodetections using
anti-D1, anti-D2 and anti-psbE gene product showed a close
association of the 6.1-kDa protein with the heterodimeric D1/D2
proteins of the PSII reaction center complex and the subunits of the
cytb
The 6.1-kDa
protein is likely to be an integral membrane protein, as various salt
wash treatments, including Tris, were not able to remove the 6.1-kDa
protein from PSII membrane fragments. The high hydrophobicity of the
protein was further established by its low polarity index (32%)
determined from an amino acid analysis of the isolated protein. The
corresponding protein deduced from the A. thaliana gene (37) was predicted to be a membrane spanning protein with one
At the
inner thylakoid surface the N-terminal tail of the 6.1-kDa subunit
appears to be shielded by the extrinsic 33-kDa protein which in turn is
in close association with PSII reaction center complex. This would
suggest that the N terminus of the 6.1-kDa protein is in close vicinity
to the extrinsic 33-kDa protein. However, in isolated
O
At the moment no specific function can be ascribed to the
6.1-kDa protein. From its absorption spectrum with a maximum in the UV,
no prosthetic group could be inferred to be associated with the
isolated protein, but it cannot be totally excluded that it could have
been lost during the isolation. However, a data base sequence homology
search for functional motifs did not give any suggestions or
indications for the function of the protein. In summary, these findings
seem to exclude the possibility that the 6.1-kDa protein is directly
involved in redox processes of PSII. This polypeptide could provide
ligands for binding manganese as the site of the water oxidase is
considered to be located near to the D1 and D2 proteins and to CP47.
Recently Tang et al.(46) described the
purification of a photochemically active reaction center complex
deprived of the psbI gene product and the
heterooligomeric cytb
Considering the interesting result
that the 6.1-kDa protein is the only nuclear encoded subunit in the
PSII reaction center complex, one tends to speculate that it could play
a regulatory role in the assembly of PSII. In this case the four
negatively charged residues of the protein, located on the outer
surface of the thylakoid membrane, may have a crucial function. Other
interesting functional roles for the 6.1-kDa protein could be to
provide docking domains for other proteins, like the extrinsic subunits
regulating the water oxidizing enzyme, or CAB proteins of the inner
antennae. Alternatively, the 6.1-kDa protein could be involved in the
high turnover of the D1 protein within the PSII reaction center under
high light. Further experiments are in progress to elucidate the
function of this newly discovered component of the PSII reaction center
complex.
We thank Dr. H. Salter for his help in data base
searching, Prof. W. Cramer and Dr. A. Szczepaniak for providing a
peptide-directed antiserum elicited against the psbE gene
product, and Prof. B. Andersson and Prof. G. Renger for their interest
and support. We also thank Dr. P.-I. Ohlsson (University of Umeå)
for performing the sequence and amino acid analyses.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
proteins and the psbI gene product. Furthermore,
cross-linking experiments using
1-(3-dimethylaminopropyl-)3-ethylcarbodiimide showed that the nearest
neighbors were the D1 and D2 proteins and the
cytb
. The 6.1-kDa protein was purified by immune
affinity chromatography. N-terminal sequence analysis of the isolated
protein confirmed the identity of the 6.1-kDa protein and enabled
finding of strong similarities with a randomly obtained cDNA from Arabidopsis thaliana. Using enzyme-linked immunosorbent assay
in combination with thylakoid membrane preparations of different
orientation, the N terminus of the protein, predicted to span the
membrane once, is suggested to be exposed at the lumen side of the
membrane. Consequently the 6.1-kDa protein seems to be the only subunit
in the PSII reaction center that is nuclear encoded and has its N
terminus on the lumen side of the membrane. These findings open for new
interesting suggestions concerning the properties of photosystem II
reaction center with respect to the photosynthetic activity, regulation
and assembly in higher plants.
(
)core complex. This
detergent-solubilized complex contains 10-13 different
polypeptides, four manganese ions, at least one calcium ion, and about
50 chlorophyll a molecules.
has been isolated from higher plants (8, 9, 10) . This so-called D1/D2 reaction
center heterodimer has now been shown to bind all of the redox
components needed for the primary photochemistry of PSII(11) .
Notably all the protein subunits in this complex have been found to be
chloroplast encoded.
-helix. They have been found to be encoded in
the chloroplast genome and referred to as psbH-psbN
gene products (for a summary see (2) ). One, the psbI
gene product, has been found to be present in the D1/D2 heterodimer of
higher plants(15, 16) . The function of these small
subunits, as well as the organization within the PSII complex are,
however, hitherto unknown. Using mutants from Synechocystis PCC
6803 where the psbH, psbK, or psbJ
genes have been deleted, it was recently shown that these subunits were
not essential for water oxidation and/or electron transport. Therefore,
it was suggested that they could fulfill a regulatory or structural
function within PSII (17, 18, 19) . The psbL gene product (with an approximately molecular mass of 5
kDa) has been suggested to be involved in stabilizing the Q
binding niche(20) . Besides these, at least three further
nuclear-encoded low molecular mass polypeptides have been suggested to
be present or associated with various types of PSII
preparations(12, 13, 14) .
(
)
Isolation of PSII Preparations
Thylakoid
membranes and PSII membrane fragments were isolated from spinach
chloroplasts(21) . PSII membrane fragments contained 220
Chl/reaction center with a Chl a/b (w/w) ratio of
2.0. Oxygen evolving PSII core complexes were either purified after
-N-octyl glucoside solubilization using the procedure
described by Ghanotakis and Yocum (22) or
-dodecylmaltoside as reported by Haag et al.(23) . PSII reaction center complexes have been purified
from PSII membrane fragments using three different types of protocols:
1) following the method originally described by Nanba et al.(8) , 2) a modified procedure reported by Seibert et
al.(10) , and 3) finally following a technique recently
developed by Bóza et al. (24). Light-harvesting
complexes of PSII were purified following the method described by Burke et al. (25) . Inside-out and right-side-out thylakoid
membranes were isolated using the aqueous polymer two-phase system
(Dextran T-500/polyethylene glycol, Carbowax 3350) as described
previously(26, 27) .
, 15 mM
NaCl, 2 mM sucrose. To release extrinsic polypeptides or
partly integrated proteins from the thylakoids the following media were
used: 1 M NaCl, 10 mM MES-NaOH, pH 6.5; 1 M
CaCl
, 10 mM MES-NaOH, pH 6.5; 0.8 M Tris,
pH 8.4. The samples were centrifuged and finally resuspended in storage
medium (medium A containing 400 mM sucrose). The Chl contents
as well as the Chl a/b ratios were determined using the method
described by Porra et al.(28) .
Protein Analysis
Polypeptide analyses were either
carried out by SDS-urea-PAGE using the buffer system described by
Laemmli (29) using a resolving gel of 17.5% acrylamide
containing 0.1% (w/v) SDS and 4 M urea or using the system
developed by Schägger and von Jagow(30) . Polyacrylamide
gels were stained with silver according to Oakley et
al.(31) . The relative molecular mass of the polypeptide
was determined by plotting the log of relative molecular masses as a
function of the relative mobilities. Molecular markers were purchased
from Pharmacia (low range marker, 16.9-2.5 kDa) or Amersham
(Rainbow marker ). Protein concentrations were measured by
Markwell et al.(32) .
N-terminal Sequence Analysis
N-terminal sequencing
of the 6.1-kDa polypeptide was either directly performed from the PVDF
membranes using essentially the method described by
Matsudeira(33) . The sequence was obtained by Edman degradation
and pulse liquid phase sequencing using an Applied Biosystems
sequenator (ABS 477A).
Immunological Experiments
A polyclonal antiserum
was raised in a rabbit against the N-terminal 15 meric oligopeptide
(LVDERMSTEGTGLPF) derived from a partial sequence obtained for the
mature polypeptide(13, 14) . The purity of the
oligopeptide was tested by reversed phase high performance liquid
chromatography using an isocratic gradient of acetonitrile and 0.1%
trichloroacetic acid and ion spray mass spectrometry (calculated and
measured M = 1754). The oligopeptide was
coupled to keyhole limpet hemocyanin by chemical cross-linking using m-maleimidobenzoic acid N-hydroxysuccinimide ester.
The immunization was carried out following standard procedures. IgG was
purified from the antiserum using protein A-Sepharose chromatography
according to standard techniques.
. ELISA multititer plates were first
coated with a variety of different PSII samples (usually 0.5-1
µg Chl for PSII membrane fragments, salt-washed PSII membrane
fragments, and O
-evolving PSII core complexes). After that
the plates were washed three times with PBS containing 0.05% (v/v)
Tween 20 (PBS
). The cavities were blocked with 5%
(w/v) skimmed milk in PBS
for 1 h at 37 °C and
washed again as described above. The first antibodies were then added
and incubated at 4 °C overnight. The cavities have been washed as
described, horseradish peroxidase coupled to goat-anti-rabbit IgG was
added in 5% (w/v) skimmed milk in PBS
(dilution
1/20,000), and incubated for 1 h at room temperature. The
immunodecorations were detected in situ using the ECL method.
Chromatographic Procedures
Approximately 1 mg
ml purified IgG has been coupled onto CNBr-activated
Sepharose using standard techniques. When using PSII membrane fragments
(2 mg/ml Chl) as starting material, they were solubilized in 1% (w/v)
-dodecylmaltoside and 1% (v/v) Triton X-100 for 30 min.
Unsolubilized material was sedimented by low speed centrifugation and
an aliquot of the supernatant (approximately 1-2 mg protein)
loaded onto the immunoaffinity column that was equilibrated in a buffer
containing 20 mM MES-NaOH, pH 6.5, 4 mM sucrose, 10
mM NaCl, 10 mM CaCl
, 0.1% (w/v)
-dodecylmaltoside. Subsequently PSII polypeptides have been
isocratically eluted by 0.25 M NaCl, 0.5 M NaCl, 0.5 M NaCl, and 0.5% Triton X-100 in the same buffer. The bound
6.1-kDa polypeptide was finally detached from the affinity column by
0.2 M glycine-HCl, pH 2.5, 0.1% (w/v)
-dodecylmaltoside.
After elution the fractions were immediately titrated to pH 7.5 with an
aliquot of 2 M Tris, pH 9.8.
-evolving PSII core complexes were used as starting
material. In this case the PSII core complexes were applied directly on
to the column without any presolubilization. The 6.1-kDa protein was
separated from the other subunits and detached from the column
following the same procedure as described above for PSII membrane
fragments. The protein was concentrated by vacuum centrifugation,
usually followed by precipitation in precooled acetone at -20
°C to remove surplus detergent and salt.
Absorption Spectroscopy
The absorption spectrum of
the purified polypeptide was recorded at room temperature using a
Shimadzu UV 3000 spectrophotometer from 200 to 800 nm (slit width, 1
nm). Cytb was determined by difference
spectroscopy from the reduced (hydroquinone-reduced for the high
potential and Na
S
O
-reduced for
total cytb
) minus
K
[Fe(CN)
]-oxidized form using an
extinction coefficient of 17,500 M
cm
(35) . The absorption spectra of the
PSII reaction center complexes (solubilized in 25 mM MES-NaOH,
pH 6.5, 10 mM CaCl
, 0.025% (w/v)
-dodecylmaltoside) were recorded at room temperature with a Chl
concentration of 5 µM using a Beckman DU 64
spectrophotometer. The optical path length was 1 cm and the scan speed
500 nm/min.
Cross-linking Experiments
PSII membrane fragments
were cross-linked at constant Chl concentrations of 250 µg/ml in
assay medium (50 mM MES-NaOH, pH 6.5, 15 mM NaCl, 5
mM MgCl, 400 mM sucrose) on ice for 30
min in the dark using EDC/Chl ratios (w/w) from 1:1 up to 20:1. The
samples were centrifuged (12,000
g, 10 min, 4 °C),
the supernatants saved for control, and the pellets resuspended in 1 ml
of assay medium. The washing procedure was repeated twice under the
same conditions, and the pellets were finally resuspended in 250 µl
of assay medium. O
-evolving PSII core complexes were
cross-linked at a constant Chl concentration of 25 µg/ml using the
same EDC/Chl ratios as described above. Cross-linking was performed on
ice for 30 min in the dark. The polypeptides were precipitated in
precooled acetone at -20 °C for 30 min (total volume 1.25
ml). The polypeptides were sedimented for 20 min (12,000
g), the supernatants discarded, and the pellets prepared for
electrophoretic analysis.
Quantification of the 6.1-kDa Polypeptide
The
6.1-kDa protein was immunodecorated with known amounts of peptide
specific IgG and the immuneresponse was quantified using a densitometer
(Molecular Dynamics Densitometer and Image Quant software). The amount
of 6.1-kDa protein was calculated at the saturation level of the immuno
decoration. The protein was then referred to the number of chlorophyll
molecules/PSII reaction center. The following mean values were measured
and used for the various PSII preparations; PSII membrane fragments 220
Chl and PSII core complexes 50 Chl. For comparison the same procedure
was applied for cytb. In the case of
cytb
, the calculations based on immunotitration
experiments were confirmed by difference spectroscopic determination
Identification and Purification of the 6.1-kDa
Polypeptide
A N-terminal site-specific IgG was used to produce
an immunoaffinity column. To purify the protein, PSII core complexes
were loaded onto the affinity column. The PSII core complexes were
found to bind tightly to the column, indicating that the epitope was
surface-exposed in these preparations. The low molecular mass
polypeptide was purified by first stepwise isocratically detaching
other PSII polypeptides from the column by increasing the NaCl
concentration from 10 to 500 mM and adding Triton X-100 to a
final concentration of 0.5% (v/v). Finally, the low molecular mass
protein was eluted in glycine-containing buffer at low pH. The
purification method was highly efficient since almost no protein was
lost while detaching other PSII polypeptides from the affinity column.
The low molecular mass component was exclusively detected in the
glycine-eluted fraction (Fig. 1, lanes 1 and 2). Fractions obtained after each step were routinely probed
by SDS-urea-PAGE followed by immunoblotting.
Notably, the low
molecular mass polypeptide did not stain with Coomassie R-250 and only
very weakly with silver stain after prolonged development (Fig. 1, lane 2). In addition, the isolated protein
tends to bind high concentrations of lipids and/or detergent that leads
to a distorted band after electrophoresis. A focused band, however, was
obtained after acetone precipitation of the polypeptide and
resolubilizing it in SDS-containing buffer (Fig. 1, lane
2).
= 276 ±
1 nm with a shoulder at 280 ± 1 nm (not shown) indicating that
no chromophores were associated with the isolated form of the protein.
Using immunotitration, the number of 6.1-kDa proteins/PSII reaction
center was estimated to be 1-2.
Localization of the 6.1-kDa Polypeptide
In an
attempt to localize the low molecular mass protein of 6.1 kDa within
PSII, and to gain information on its nearest neighbors, different
thylakoid membrane preparations and PSII complexes were investigated by
means of immunoscreening. Under denaturing conditions, i.e. after SDS-PAGE followed by Western blotting, the 6.1-kDa
polypeptide was unambiguously identified in all types of PSII
complexes, but not in LHCII preparations. Of particular interest is
that the 6.1-kDa protein was found in stoicheiometric amounts in PSII
reaction center complexes (see Fig. 2A, lanes
1-7 and Table 2). The purity of the used PSII
preparations is shown by a silver-stained SDS-polyacrylamide gel in Fig. 2B and additionally for the PSII reaction center
complex by its absorption spectrum (Fig. 2C). The
presence of the 6.1-kDa protein in the PSII reaction center complex was
also further established by analyzing three different types of
preparations (see ``Materials and Methods''). All three gave
a strong positive immunoreaction with the 6.1-kDa site-directed IgG
(only one of these is shown in Fig. 2). The 6.1-kDa protein
could neither be removed from the PSII membrane fragments by Tris
washing at high pH nor by high salt concentrations indicating that the
6.1-kDa protein is an integral membrane protein component.
-evolving
PSII core complex (3), LHCII (4), PSII membrane
fragments (5), Tris-washed PSII membrane fragments (6), and thylakoids, using the antiserum raised against the
6.1-kDa protein. B, silver-stained SDS-PAGE of PSII membrane
fragments (7), PSII reaction center (8). The upper arrow indicates aggregated PSII reaction centers at the
interface between the spacer gel and the separation gel. C,
absorption spectrum of the PSII reaction center complex used in A and B.
The
Western blot analysis clearly shows that the 6.1-kDa protein is present
in all PSII samples; however, to obtain information on the topology of
the protein it is necessary to preserve it from denaturation.
Therefore, we used a more appropriated ELISA technique (Table 2).
Again the strongest immunodecoration was observed in PSII reaction
center and in the PSII core complexes, meaning that the N-terminal tail
of the 6.1-kDa protein was highly surface-exposed in these two
preparations (see Table 2). Interestingly, the PSII membrane
fragments revealed a rather weak antibody reaction in ELISA. After
washing the PSII membrane fragments with NaCl to remove the two
extrinsic 23- and 16-kDa proteins, the immunoresponse was also very
weak. However, in PSII membrane fragments completely deprived of all
three extrinsic proteins (33, 23, and 16 kDa) either by Tris or
CaCl washing, the epitope was clearly accessible and a
strong and distinct immunoresponse was detected. Obviously a removal of
the extrinsic 23- and 16-kDa proteins did not expose the N terminus of
the 6.1-kDa protein, while an elimination of the extrinsic 33 kDa (psbO) polypeptide did. This indicates that the N terminus of
the 6.1-kDa protein would be located somewhere in the vicinity of the
33-kDa protein. However, the O
-evolving PSII core complexes
contained the psbO gene product(23) , and the N
terminus of the 6.1-kDa protein was still recognized by the
peptide-directed IgG. This could be due to an induced conformational
change in the 33-kDa protein binding region or that the 33-kDa protein
is partly lost during the preparation or the ELISA procedure.
-helix. Interestingly, just at the outer surface of
the thylakoid membrane, close to the hydrophobic region, four
negatively charged amino acids are located (Fig. 4). A sequence
homology search against the Swissprot and Prosit database did not
reveal any significant homology to other proteins or functional motifs,
except for the corresponding 6.1-kDa protein in C.
reinhardtii, T. aestivum, and spinach. The calculated
molecular mass of the deduced mature protein from A. thaliana gene (37) is 6.043 kDa. The relative molecular mass of the
purified polypeptide was estimated to be 4.6 kDa on the applied
SDS-PAGE system. The reason for the discrepancy in relative molecular
masses between the purified polypeptide and that previously described
is not clear at the moment, however, different gel systems have been
used(13, 14) .
Nearest Neighbor Analysis by Chemical Cross-linking with
EDC
To further establish that the 6.1-kDa protein is in close
contact with polypeptides of the PSII reaction center, chemical
cross-linking experiments were performed to investigate the nearest
neighbors of the small subunit in PSII. Using a zero cross-linker, EDC,
that contains no spacer between the two reactive groups and mainly
conjugates proteins via -,
-amino groups, and in addition via
carboxyl groups, it was possible to identify the proteins next to the
6.1-kDa subunit. PSII membrane fragments were cross-linked with various
EDC/Chl (w/w) or EDC/protein (w/w) ratios. The conjugates were analyzed
by SDS-urea-PAGE and screened with peptide-specific IgG directed
against the 6.1 protein after electroblotting and visualized by ECL.
The result of such an experiment using PSII membrane fragments is
demonstrated in Fig. 3(lanes 1-6). It was found
that with increasing ratios of EDC/Chl the intensity of two bands in
the 36-38 kDa and 14-16 kDa region enhanced in parallel.
The main part of the 6.1-kDa protein, however, remained not
cross-linked, showing that the cross-linking reaction was not
quantitative. Using a set of different polyclonal antisera, we tested
various PSII polypeptides for being cross-linked by EDC with the
6.1-kDa protein. The D1 and D2 proteins were identified in the
cross-linked complex with a relative molecular mass of 36-38 kDa
and Cytb
was found in the smaller cross-linked
complexes of 14-16 kDa (see Fig. 3, lanes
7-9). Using PSII core complexes instead of PSII membrane
fragments in the cross-linking experiment, the same cross-linked
products were detected with the 6.1-kDa protein IgG (not shown), which
further supports the conclusion deduced from the experiments using PSII
membrane fragments.
(lane 9).
It is interesting that chemical cross-linking of
PSII membrane fragments with EDC did neither influence the redox
properties of the heme group of Cytb nor did it
shift its maximum (
= 560 ± 1 nm, data
not shown). As the heme group of cytb559 is considered to be
located toward the outer surface of the thylakoid
membrane(40) , this suggests that the EDC-mediated
cross-linking between the 6.1-kDa protein and Cytb
takes place preferentially on the lumen side via the N- and
C-terminal regions of the two polypeptides, respectively.
. The possibility that the 6.1-kDa protein
is loosely or accidentally associated with PSII during preparation can
be excluded by the fact that the whole PSII complex binds to the
affinity column specifically interacting with the N terminus of the
6.1-kDa protein. Only after extensively washing the column (with 0.5 M NaCl and 0.5% Triton X-100) could the other reaction center
complex proteins be detached from the 6.1-kDa protein. So far the PSII
reaction center complex has been thought to be composed of the D1-,
D2-proteins, the
- and
-subunits of
cytb
, and the psbI gene
product, 4-6 chlorophyll a molecules, two pheophytins
and one to two
-carotenes(9, 10, 14, 41) .
However, due to its unusual amino acid composition the polypeptide
stains poorly with Coomassie R-250 and only very weakly with silver.
This probably provides an explanation that the 6.1-kDa protein has
previously escaped detection. On the basis of the finding presented
here, it is suggested that the 6.1-kDa protein is an additional
integral component of the PSII reaction center complex.
-helix. The N terminus of the 6.1-kDa protein was found to be
oriented into the lumen of the thylakoids (Fig. 4) since it is
not recognized by the N-terminal site-directed IgG in right-side-out
thylakoids, but clearly in inside-out thylakoids. No immunodecorations
were observed with PSII membrane fragments. The reason for this
discrepancy is not yet clarified (see Table 2). One possibility
for the detectability of the N terminus in inside-out thylakoids could
be that either the 33-kDa protein was partly lost during the
preparation of the inverted vesicles or while washing the samples in
the cavities of the microtiter plates and therefore the epitope was
exposed to the surface. This would be in line with our observation that
extrinsic polypeptides can easily be removed from inside-out thylakoids
by salt concentrations of 100-200 mM NaCl (data not
shown). The orientation of the 6.1-kDa protein is supported by an
analysis of its presequence that was found to have high homology with
those of other known lumen-directed polypeptides of PSII, like the
three extrinsic subunits of 33, 23, and 16 kDa. Interestingly, this
means that the orientation of the 6.1-kDa protein is opposite to that
of the other proteins found in the PSII reaction center complex. The N
termini of both the D1 and the D2 polypeptides are on the stroma side
of the membrane(43, 44) . Also the psbI gene
product, another monotopic membrane protein, has its N terminus on the
outside of the thylakoid membrane(45) . The topology of
cytb
has recently been determined by means of a
peptide-directed antiserum specifically reacting with the C terminus of
the
-subunit (42) and using hybrid
-subunits of
cytb
in combination with site-specific
antisera(40) . Both the N termini are located on the stroma
side of the thylakoid membrane(40, 42) .
-evolving PSII core complexes the epitope is recognized by
the IgG despite the 33-kDa protein being present. This is probably a
reflection of the fact that the N terminus is more susceptible to the
environment in isolated PSII core complexes than in PSII membrane
fragments.
. The implication of this
finding is that at least these small subunits cannot be involved in
pigment binding (46) .
, cytochrome b 559; EDC,
1-(3-dimethylaminopropyl-)3-ethylcarbodiimide); MES,
2-N-morpholinoethanesulfonic acid; PBS, phosphate-buffered
saline; psb, photosystem b or II; PVDF, polyvinylidene
difluoride; PAGE, polyacrylamide gel electrophoresis; ELISA,
enzyme-linked immunosorbent assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.