Towards Functional Proteomics of Membrane Protein
Complexes in Synechocystis sp. PCC 68031
Mirkka Herranen, Natalia Battchikova, Pengpeng Zhang, Alexander Graf, Sari Sirpiö, Virpi Paakkarinen,
and Eva-Mari Aro*
Plant Physiology and Molecular Biology, Department of Biology, University of Turku, FIN–20014 Turku,
Finland
The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC
6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass
spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown
wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase
(NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the
protein complexes was studied by culturing the wild type and several mutant strains under various growth modes
(photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt.
The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of
CP43⬘ protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the
CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein
complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins
assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth
subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and
are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement
of PSII activity for de novo synthesis of the NDH-1 complexes.
Photosynthetic organisms experience continuous
fluctuation in their growth environment, including
changes in light conditions, temperature, and nutrient
availability. Cyanobacteria, the probable progenitors
of chloroplasts, possess a remarkable capacity to adapt
to a wide range of environmental conditions. Cyanobacteria are primarily photoautotrophic, but some
strains, such as Synechocystis sp. PCC 6803, can also
grow mixotrophically or photoheterotrophically in the
presence of external Glc. This, together with their flexibility to genetic manipulation and simple nutrient
requirements for growth, has rendered cyanobacteria
into a favored model in deciphering the structurefunction relationships and acclimation strategies of
the photosynthetic machinery.
Energy-transducing thylakoid membranes are the
targets for modification by a number of environmental variables. Light, the most important environmental factor for photosynthesis, induces a well-defined
acclimation response, including changes in the
amount and ratio of the two PSs (Fujita, 1997; Hihara,
1999) and in the abundance and composition of the
light-harvesting phycobilisome antenna (Grossman
et al., 1993, 2001). Similar responses in the thylakoid
1
This work was supported by the Academy of Finland.
* Corresponding author; e-mail [email protected]; fax 358 –2–333–
8075.
Article, publication date, and citation information can be found
at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.032326.
470
membrane components also can be observed upon
changes in the nutrient status, such as availability of
iron (Straus, 1994; Sandström et al., 2002). Generally,
iron deficiency induces a down-regulation of the
iron-containing thylakoid components and, when
possible, their replacement with noniron counterparts. A well-known response to iron deficiency is
the derepression of the dicistronic isiAB operon (Vinnemeier et al., 1998), encoding a chlorophyll a (Chl
a)-binding protein CP43⬘ (IsiA) and flavodoxin, respectively (Laudenbach and Straus, 1988; Laudenbach et al., 1988). Recently, CP43⬘ was demonstrated
to form a Chl a-containing auxiliary antenna ring
around PSI trimers in iron-deficient cells (Bibby et al.,
2001; Boekema et al., 2001).
Increase in salinity of the growth medium likewise
was reported to induce the expression of the isiAB
operon (Vinnemeier et al., 1998; Vinnemeier and
Hagemann, 1999) and accumulation of flavodoxin in
salt-stressed cells (Fulda and Hagemann, 1995). This
was suggested to contribute to enhanced cyclic electron transport around PSI upon salt stress (Hagemann et al., 1999). Conversely, salt stress appears to
repress the genes encoding PSI components and subunits of the phycobilisomes (Kanesaki et al., 2002).
Limitation of inorganic carbon (Ci) in aquatic environments also poses a problem for photosynthesis.
As a consequence, cyanobacteria have developed an
extremely efficient carbon-concentrating mechanism,
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Proteomics of Membrane Protein Complexes in Synechocystis
which operates to rise the CO2 concentration up to
1,000-fold around their relatively inefficient Rubisco
enzyme (Badger and Price, 2003). Reverse genetics
experiments have revealed that the uptake of Ci,
either in the form of HCO3⫺ or CO2, is accomplished
by bicarbonate transporters (Omata et al., 1999; Shibata et al., 2002a) and CO2 uptake systems associated
with the function of specialized type I NAD(P)H
dehydrogenase (NDH-1) complexes (Ogawa, 1991a,
1991b; Badger and Price, 2003) in the thylakoid membrane (Ohkawa et al., 2001). Besides CO2 uptake,
NDH-1 complexes play a role in the cyclic electron
transfer around PSI (Mi et al., 1994, 1995) and in the
respiratory electron transfer (Mi et al., 1992; Schmetterer, 1994). Although the analysis of various ndh
gene inactivation mutants has suggested a diversity
of functions for NDH-1 complexes, the structural
basis and protein composition of various hypothetical NDH-1 complexes have remained elusive.
During the past few years, genome-wide microarray studies of Synechocystis sp. PCC 6803 have provided a vast amount of information on global modification of gene expression in response to
environmental variables (Hihara et al., 2001; Suzuki
et al., 2001; Kanesaki et al., 2002; Hihara et al., 2003;
Singh et al., 2003). The challenge of future studies is
to find out how the changes in transcript levels are
realized on the protein level. Here, we have taken a
functional proteomics approach to analyze the dynamic changes occurring in Synechocystis sp. PCC
6803 membrane protein complexes when the cells are
subjected to elevated CO2 concentration, iron depletion, or salt stress or when their growth mode
changes from photoautotrophy to mixotrophy or
photoheterotrophy.
RESULTS
Protein Complexes of Synechocystis sp. PCC 6803
Membranes: Photoautotrophic Growth Conditions
Membrane protein complexes from wild-type (WT)
Synechocystis sp. PCC 6803 cells grown photoautotrophically under air level of CO2 were first separated by blue native (BN)-PAGE. Approximately 20
distinct protein complexes could be observed in silverstained BN gels (Fig. 1). To identify the protein complexes and to resolve their composition, the proteins
from the BN gel lanes were separated in a second
dimension by denaturing SDS-PAGE (Fig. 1), and the
Figure 1. Two-dimensional BN/SDS-PAGE separation of the membrane protein complexes of
Synechocystis sp. PCC 6803. Membranes were
isolated from WT cells grown photoautotrophically under air level of CO2. Upper, Silverstained BN gel lane with electrophoretically separated membrane protein complexes, denoted
above the gel lane. Below the lane, the molecular
size markers (HMW Calibration Kit for Native
Electrophoresis, Amersham Biosciences, Buckinghamshire, UK) are shown in kilodaltons. The
complexes were separated into their respective
subunits by subjecting a BN gel lane to a denaturing SDS-PAGE (lower, silver stained SDSPAGE). The numbered protein spots were excised
from the silver-stained SDS-PAGE gels and identified with MALDI-TOF (see Table I).
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471
Herranen et al.
silver-stained protein spots were identified by mass
spectrometric (matrix-assisted laser-desorption ionization time of flight [MALDI-TOF]) analysis or by
immunoblotting (Fig. 2). Altogether, a total of 53 protein spots were identified as the products of 37 different Synechocystis sp. PCC 6803 genes (Table I).
Complexes Related to Linear Photosynthetic Electron
Flow and ATP Synthesis
PSI became distinguished as three major complexes, apparently representing a monomer, a trimer,
and a high-molecular mass PSI supercomplex (Fig.
1). Moreover, a minor fraction of PSI resolved in BN
gels apparently in a dimeric form. The reaction center
subunits PsaA and PsaB, and PsaD, PsaF, and PsaL
were identified by MALDI-TOF in the PSI complexes
(Table I).
PSII complexes were resolved mainly as monomers
but also as dimers or monomers lacking the CP43
subunit. The reaction center proteins D1 and D2, the
Chl a-binding proteins CP47 and CP43, and the
␣-subunit of cytochrome b559 were identified in this
study. Most small subunits of PSII, however, failed
the identification by MALDI-TOF, whereas subunits
of the oxygen-evolving complex apparently released
from PSII upon membrane solubilization.
The cytochrome b6f (Cyt b6f) complex resolved
mostly as a monomer, although a marginal amount
of dimeric form was also observed in the gels. In both
complexes cytochrome f, cytochrome b6, and subunit
IV were identified.
The ATP synthase complex migrated in the BN gel
slightly faster than the PSII dimer (Fig. 1). Subunits B
and B⬘ of the membrane integral CF0 complex, and
the extrinsic CF1 subunits ␣, ␤, ␦, ␥, and ⑀ were
identified by MALDI-TOF (Table I).
NDH-1 Complexes
Four complexes contained NDH-1 subunits. Based
on their migration (and, correspondingly, the size),
we denoted them as NDH-1L (large), NDH-1M (medium size), NDH-1S1 (small), and NDH-1S2 (Fig. 1).
NDH-1L migrated in close proximity to ATP synthase, NDH-1M practically overlapped with the PSI
monomer, NDH-1S1 migrated slightly faster than the
CP43-less PSII monomer, and NDH-1S2 migrated between the NDH-1S1 and the Cyt b6f monomer. Both
NDH-1L and NDH-1M contained the NdhH, NdhK,
NdhI, and NdhJ subunits, which have been reported
to belong to a NDH-1 subcomplex (Berger et al.,
1993). The NDH-1S1 complex contained another set
of four proteins, identified as NdhF3, NdhD3, CupA,
and sll1735 gene product. High hydrophobicity of the
NdhF3 and NdhD3 subunits resulted in formation of
diffuse protein spots in SDS-PAGE gels (Fig. 1) and
low coverage data in MALDI analysis (Table I). However, antibodies raised against the NdhD3 and
NdhF3 of Synechocystis sp. PCC 6803 explicitly identified these proteins in the NDH-1S1 and NDH-1S2
complexes and moreover, showed the absence of
these subunits in the NDH-1L and NDH-1M complexes (Fig. 2A). The specificity of the NdhD3 and
NdhF3 antibodies is shown in Figure 2B. The NDH1S2 complex is likely to represent either an assembly
or degradation intermediate of NDH-1S1 because it
contained only the NdhF3 and NdhD3 subunits.
Other Membrane Protein Complexes
Figure 2. Identification of the NdhF3 and NdhD3 proteins by immunoblotting. A, After BN/SDS-PAGE, the proteins were silver stained
(left) or electroblotted onto a polyvinylidene difluoride membrane
and immunodecorated first with NdhF3 antibody and subsequently
with NdhD3 antibody (right). Membranes containing 10 ␮g of Chl a
were loaded in the BN-PAGE. A portion of the gels containing all
NDH-1 complexes is shown. NdhH, NdhI, and NdhJ subunits are
marked with arrows. The arrowheads on the right denote the positions of the NdhF3 and NdhD3 subunits in the NDH-1S complexes.
B, Immunoblots demonstrating the specificity of the NdhD3 and
NdhF3 antibodies. Membranes were isolated from WT and ⌬ndhD3
cells grown under air level of CO2. Membrane proteins were separated by SDS-PAGE, electroblotted onto a membrane, and immunodecorated either with NdhD3 or NdhF3 antibody.
472
A small protein complex migrating between the
NDH-1S1 and NDH-1S2 complexes in the BN gel (Fig.
1) contained SbtA protein (Slr1512), which has been
demonstrated as an essential component in a
sodium-dependent bicarbonate transport in Synechocystis sp. PCC 6803 (Shibata et al., 2002a). Interestingly, both proteins (denoted as 26 in Fig. 1) belonging to this complex, albeit having different masses
(30 and 27 kD), were identified as SbtA by MALDITOF (Table I).
In addition, we discovered several minor, previously unknown protein complexes. One of them, containing the slr0151 gene product, was observed in the
close proximity to the PSII monomers (Fig. 1). Two
other protein complexes, containing gene products of
sll1757 and slr0695, respectively, migrated between
the NDH-1S2 and Cyt b6f monomer. The function
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Plant Physiol. Vol. 134, 2004
Proteomics of Membrane Protein Complexes in Synechocystis
Table I. Membrane proteins of Synechocystis sp. PCC 6803 identified by MALDI-TOF
The numbering corresponds to the BN/SDS-PAGE gel in Figure 1, except no. 32, which refers to Figure 6. ORF, Open reading frame in the
Cyanobase. Experimental (Exper.) molecular masses were estimated by comparing the migration of the respective proteins with that of the Mr
markers. Values of theoretical (Theor.) masses for unprocessed proteins are included. Results of searching in the National Center for
Biotechnology Information database using Mascot are presented as: ratios of peptide masses that provided the identity to the total amount of
peptides used in the search, percentages of coverage of full-length protein at 50 ppm, and score values according to Mascot.
No.
ORF
Gene Product
Gene
13
14
15
16
17
18
19
20
21
22
23
24
slr1834
slr1835
slr0737
sll0819
slr1655
slr0906
sll0851
sll0849
slr1311
sll1317
slr0342
slr0343
sll1326
slr1329
sll1327
sll1324
sll1325
slr1330
sll1323
slr0261
slr1280
sll0520
slr1281
sll1732
sll1733
sll1734
psaA
psaB
psaD
psaF
psaL
psbB
psbC
psbD
psbA
petA
petB
petD
atpA
atpB
atpC
atpF
atpD
atpE
atpG
ndhH
ndhK
ndhI
ndhJ
ndhF3
ndhD3
cupA
25
sll1735
26
27
slr1512
sll1577
sll1578
slr2067
slr1128
slr0151
sll1757
slr0695
sll0247
ssr3451
PSI subunit Ia PsaA
PSI subunit Ib PsaB
PSI subunit II PsaD
PSI subunit III PsaF
PSI subunit XI PsaL
PSII CP47 protein
PSII CP43 protein
PSII D2 protein
PSII D1 protein
Cytochrome f
Cytochrome b6
Cytochrome b6f complex subunit IV
ATP synthase ␣ subunit
ATP synthase ␤ subunit
ATP synthase ␥ subunit
ATP synthase B subunit
ATP synthase ␦ subunit
ATP synthase ⑀ subunit
ATP synthase B⬘ subunit
NADH dehydrogenase subunit 7, NdhH
NADH dehydrogenase subunit NdhK
NADH dehydrogenase subunit NdhI
NADH dehydrogenase subunit I, NdhJ
NADH dehydrogenase subunit, NdhF3
NADH dehydrogenase subunit 4, NdhD3
Protein involved in low CO2-inducible, highaffinity CO2 uptake, CupA
Protein homologous to secreted protein
MPB70
Sodium-dependent bicarbonate transporter
Phycocyanin beta subunit
Phycocyanin alpha subunit
Allophycocyanin alpha subunit
Stomatin-like protein
Unknown protein
Unknown protein
Unknown protein
Iron-stress induced Chl-binding protein CP43⬘
Cytochrome b559 ␣ subunit
1
2
3
4
5
6
7
8
9
10
11
12
28
29
30
31
32a
33b
a
CP43⬘ was identified from the gel shown in Figure 6.
b
isiA
psbE
Peptides
%
Cover
Score
Theor.
matched/total
65
65
18
17
16
48
37
31
28
22
18
14
59
59
31
19
18
16
15
43
26
24
23
40
32
45
83.1
81.4
15.7
18.4
16.6
55.9
50.5
39.6
39.9
35.3
25.2
17.5
53.9
51.7
34.6
19.8
20.1
17.9
16.2
45.8
27.5
22.6
20.7
66.6
54.4
50.2
11/21
8/21
9/13
7/11
5/6
9/11
9/12
10/12
9/12
12/15
6/8
3/4
12/31
18/31
15/18
6/9
3/3
6/10
11/13
20/22
14/19
11/16
6/9
3/6
4/6
21/29
13
10
52
33
30
11
17
19
22
39
24
12
27
56
28
31
21
40
42
26
60
52
18
4
6
50
115
90
161
109
98
132
88
135
120
204
80
47
129
250
178
87
78
77
170
159
183
218
106
36
65
253
17
14.2
5/6
53
103
30/27
18
18
18
32
38
28
18
28
10
39.7
18.3
17.7
17.4
35.7
35.0
31.8
19.1
37.3
9.4
13/16
8/22
7/22
5/22
13/20
14/20
6/8
5/7
12/16
3/5
17
60
57
44
42
51
20
38
24
46
145
126
111
105
193
166
156
94
88
64
Low-Mr polypeptide, not seen in Figure 1.
of these novel protein complexes remains to be
investigated.
Effect of Growth Mode on the Synechocystis sp. PCC
6803 Membrane Protein Complexes
Next, we tested the effect of the growth mode
(photoauto-, mixo-, or photoheterotrophy) on the
membrane proteome of Synechocystis sp. PCC 6803.
To induce mixotrophic growth, the WT Synechocystis
sp. PCC 6803 cells were cultured in the light in the
presence of 5 mm Glc. Analysis of the membrane
Plant Physiol. Vol. 134, 2004
sbtA
cpcB
cpcA
apcA
Mass (kD)
Exper.
protein pattern, however, did not reveal any significant changes of whether the WT cells were grown in
the absence (Fig. 1) or presence (data not shown) of
Glc. To induce photoheterotrophic growth conditions, the cell cultures were supplied with the PSII
electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1dimethylurea (DCMU) in addition to Glc and light.
These non-photosynthetic growth conditions induced notable changes in the pattern of membrane
protein complexes (Fig. 3A). In particular, the NDH1S1, NDH-1S2, and SbtA complexes were completely
absent, and only traces of the NDH-1M complex were
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473
Herranen et al.
Figure 3. Membrane proteome of Synechocystis
sp. PCC 6803 under photoheterotrophic growth
mode. Membranes were isolated from WT cells
grown in the presence of 10 ␮M DCMU and 5
mM Glc (A) or from the PSII-less mutant strain LC
grown in the presence of 5 mM Glc (B). Membranes corresponding to 10 ␮g of Chl a were
subjected to BN/SDS-PAGE analysis as described in “Materials and Methods.” The NdhH,
NdhK, NdhI, and NdhJ subunits in the NDH-1L
and NDH-1M complexes are marked with arrows. The arrowheads above the figures denote
the Glc-induced complexes.
present. Conversely, two novel protein complexes in
the range of 210 kD became strongly induced by
these growth conditions and, therefore, were denoted
as “Glc-induced” complexes. Mass spectroscopic
analysis did not reveal the identity of the proteins
belonging to these complexes, possibly because of
strong hydrophobicity interfering the sample
preparation.
To exclude possible DCMU-induced side effects
(Schmitz et al., 1999; Zhang et al., 2000), we analyzed
the membrane protein complexes of the photoheterotrophic mutant strain LC. This non-photosynthetic
mutant is devoid of PSII complexes (Fig. 3B) and
requires external Glc for growth (Mulo et al., 1997).
Similar to the photoheterotrophically grown WT cells
(Fig. 3A), the LC mutant lacked the NDH-1S1, NDH1S2, and SbtA complexes but contained the two “Glcinduced” complexes in its membranes (Fig. 3B). Despite the absence of both NDH-1S complexes, low
quantities of the NDH-1M and NDH-1L complexes
were distinguished in the mutant membranes, with
NDH-1L being the dominant one. Similar results
were obtained with two other photoheterotrophic
PSII-mutant strains PCD and CD (data not shown),
which contain PSII complexes yet unable to support
photoautotrophic growth (Mulo et al., 1997). Thus,
the possibility that DCMU per se caused the changes
in membrane protein complexes can be excluded.
As a control to the mutant LC, we tested the autotrophic mutant strain AR cultured in the presence
or absence of Glc. This strain contains the same four
antibiotic resistance genes as LC but no mutations in
the PSII reaction center protein D1 (Mäenpää et al.,
1993). When grown in the absence of Glc, the membrane protein profile of the strain AR strongly resembled that of the photoautotrophically grown WT
strain (Fig. 4A versus Fig. 1). However, when the AR
cells were grown in the presence of Glc, the membrane protein pattern showed a loss of the NDH-1S1,
NDH-1S2, and SbtA complexes but contained the two
“Glc-induced” complexes (Fig. 4B). In addition, the
amount of the NDH-1M complex drastically decreased so that the NDH-1L complex became dominant. Thus, in regard to the NDH-1, SbtA, and “Glcinduced” complexes, the mixotrophically grown AR
cells differed from the mixotrophically grown WT
cells and resembled the photoheterotrophic PSII mu-
Figure 4. BN/SDS-PAGE separation of membrane protein complexes of the mutant control
strain AR. The AR cells were grown under standard growth conditions either photoautotrophically (A) or mixotrophically (B) in the presence
of 5 mM Glc. Membranes containing 10 ␮g of
Chl a were loaded in the BN-PAGE. The NdhH,
NdhK, NdhI, and NdhJ subunits in the NDH-1L
and NDH-1M complexes are marked with arrows. The Glc-induced complexes are marked
with arrowheads above B.
474
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Plant Physiol. Vol. 134, 2004
Proteomics of Membrane Protein Complexes in Synechocystis
tant strains (Fig. 3B) or WT strain grown in the photoheterotrophic mode (Fig. 3A).
We also analyzed the protein complex profile of
two other photoautotrophic mutant strains, an A2
mutant (Jansson et al., 1987) and a ⌬psbA1 mutant
(for description of the mutants, see “Materials and
Methods”), grown in the presence or absence of Glc.
The membrane protein complexes of these mutant
strains responded similarly to the AR strain to the
availability of Glc as an energy source (data not
shown). It should be noted that all mutant strains used
in this study were grown in the presence of antibiotics.
Membrane Proteome during Growth under
Elevated CO2
To study the effect of CO2 concentration on the
accumulation of membrane protein complexes, we
analyzed the membranes of WT cells grown under
elevated CO2 concentration (1% [v/v] in air). The
most pronounced changes induced by elevated CO2
were strong down-regulation of the NDH-1S1, NDH1S2, and SbtA complexes and a marked reduction in
the quantity of NDH-1M (Fig. 5). The content of the
other membrane protein complexes showed no significant change in response to an elevated CO2 concentration (Fig. 5 versus Fig. 1).
Effect of Iron Deficiency or Salt Stress on the
Membrane Proteome
The effects of iron starvation or increased salinity
on the membrane protein complexes were studied
with WT cells grown for 72 h in the absence of iron or
in the presence of 0.4 m sodium chloride, respectively. Iron deficiency induced a pronounced accumulation of CP43⬘ protein (Fig. 6A). This protein was
found solely in association with the PSI trimers,
forming a big PSI-CP43⬘ supercomplex. This resulted
in an increase in the amount of PSI-CP43⬘ supercomplexes relative to PSI trimers. Otherwise, the membrane proteome strongly resembled that of the WT
cells grown in iron-sufficient conditions (Fig. 1).
The increase in the salinity of the growth medium,
on the other hand, did not induce any noticeable
changes in the membrane protein complexes (Fig.
6B), as compared with the cells cultured in the absence of added NaCl (Fig. 1).
Synthesis of Membrane Protein Complexes
The expression patterns of membrane protein complexes described above represent a steady-state situation under each particular growth condition. To get
insights into the synthesis and turnover of the membrane protein complexes, we performed [35S]Met labeling experiments with WT cells performing photoautotrophic, mixotrophic, or photoheterotrophic
growth. The most distinct feature under all different
growth conditions was the incorporation of radioactive Met into the PSII and its synthesis intermediates
(Fig. 7). Labeling of subunit IV of the Cyt b6f complex
was also observed under all growth conditions, suggesting a higher turnover rate for this protein as
compared with the other Cyt b6f subunits. The
NDH-1 subunits were synthesized in minor amounts.
However, the synthesis of the NDH-1M and particularly the NDH-1S subunits NdhF3 and CupA was
observed only during photoautotrophic and mixotrophic growth. Induction of photoheterotrophic
growth by the addition of PSII inhibitor DCMU (15
min before labeling) completely abolished the incorporation of [35S]Met into these complexes. Labeling
of the SbtA proteins followed closely that of the
NDH-1 subunits under all growth conditions.
DISCUSSION
Figure 5. Effect of elevated CO2 concentration on the membrane
protein complexes. Synechocystis sp. PCC 6803 membranes were
isolated from WT cultures bubbled with 1% (v/v) CO2 in air. Membranes corresponding to 10 ␮g of Chl a were subjected to BN/SDSPAGE analysis as described in “Materials and Methods.” Arrows
mark the NdhH, NdhK, NdhI, and NdhJ subunits in the NDH-1L and
NDH-1M complexes.
Plant Physiol. Vol. 134, 2004
Cyanobacterial membranes are dynamic entities,
changing their protein composition in response to a
wide variety of environmental factors, including
light quality and quantity (Fujita, 1997; Hihara, 1999),
iron availability (Straus, 1994; Sandström et al., 2002),
CO2 concentration (Murakami et al., 1997; Deng et
al., 2003), and salinity (Murakami et al., 1997). These
changes are necessary to balance the energy transduction processes and to minimize photooxidative
damage, thus being crucial for the cyanobacterial
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475
Herranen et al.
Figure 6. Effect of iron deficiency or salt stress
on the membrane protein complexes. WT Synechocystis sp. PCC 6803 cells were grown in the
absence of iron (A) or in the presence of 0.4 M
NaCl (B). Membranes corresponding to 10 ␮g of
Chl a were subjected to BN/SDS-PAGE analysis.
Black arrows mark the NdhH, NdhK, NdhI, and
NdhJ subunits in the NDH-1L and NDH-1M
complexes. White arrow in (A) denotes the
CP43⬘ protein (denoted as 32 in Table I).
growth in the ever-changing environment. Previous
studies on acclimation of the thylakoid protein complexes have addressed mainly the dynamics of the two
PSs. Compared with the vast amount of information
available on the cyanobacterial photosynthetic complexes, far less is known about the composition, structure, and dynamics of other membrane-embedded
protein complexes.
Diversity of the NDH-1 Complexes
The most distinctive novel feature of Synechocystis
sp. PCC 6803 membranes observed in this study was
the multiplicity of complexes containing NDH-1 subunits. The NDH-1 subunits are encoded by at least 10
single copy genes (ndhAIGE, ndhB, ndhCJK, ndhH, and
ndhL) and two multigene families (ndhF family with
three members and ndhD family with six members;
Badger and Price, 2003). Altogether four distinct
NDH-1 complexes, denoted here as NDH-1L, -M, -S1,
and -S2, were resolved in BN-PAGE (Fig. 1).
The multisubunit NDH-1L and NDH-1M complexes showed a similarity in their protein composition. Both complexes contained the NdhH, -K, -I, and
-J subunits (Fig. 1; Table I). In addition, they contained several other, still unidentified proteins. It is
conceivable that these proteins represent more hydrophobic NDH-1 subunits because they were not
effectively digested by trypsin to provide representative sets of peak masses. These subunits must therefore be identified by other methods than MALDI-TOF.
Two smaller complexes, NDH-1S1 and NDH-1S2,
contained a set of proteins different from that of
NDH-1L or NDH-1M. NDH-1S1 consisted of four
proteins, two of which comprised NDH-1S2. The first
hints for the identity of these two common subunits
came from MALDI-TOF analysis. In the spectrum of
the protein with an apparent molecular mass of 32
kD, four peaks were observed that referred to
NdhD3, and in the spectrum of the protein of 40 kD,
three peaks were observed that referred to NdhF3.
476
The reason for obtaining low-coverage data in the
MALDI-TOF analysis is likely to be related to the high
hydrophobicity of the NdhF3 and NdhD3 proteins,
which apparently also induced an anomalous migration of these proteins in the SDS-PAGE. However, the
protein specific antibodies confirmed the identity of
the NdhF3 and NdhD3 proteins in the NDH-1S complexes (Fig. 2). It is noteworthy that the NDH-1L and
NDH-1M complexes lacked the NdhF3 and NdhD3
subunits, whereas the NDH-1S complexes appeared to
lack the subunits belonging to NDH-1L and NDH-1M
(Fig. 2).
Reverse genetics approaches have revealed that
NdhF3, NdhD3, NdhF4, and NdhD4 subunits contribute mainly to the uptake of CO2 (Klughammer et
al., 1999; Ohkawa et al., 2000a, 2000b; Shibata et al.,
2001), whereas NdhF1, NdhD1, and NdhD2 are involved in respiration and cyclic electron transfer
around PSI (Yu et al., 1993; Klughammer et al., 1999;
Ohkawa et al., 2000a). Previous mutant studies by
Ogawa and coworkers also have demonstrated a role
for the NdhB, NdhK, and NdhL subunits in the uptake of CO2 and in the NDH-1-mediated electron
transfer pathways (Ogawa, 1991a, 1991b, 1992; Mi et
al., 1992, 1994; Ohkawa et al., 2000b). Based on these
results, it was hypothesized that there may exist several functionally distinct NDH-1 complexes, all sharing a core encoded by the single-copy genes but
differing in their NdhF and/or NdhD components.
In variance with this hypothesis, our results suggest
that the CO2 uptake complex formed by NdhF3 and
NdhD3 is a separate complex from the other NDH-1
complexes containing single-copy NDH-1 subunits.
It is evident, however, that the NDH-1S complexes are
functionally dependent on NDH-1M (or NDH-1L),
and at the moment, we cannot completely exclude the
possibility that the NDH-1S complexes may represent
subcomplexes of the larger NDH-1 complexes.
In addition to NdhF3 and NdhD3, we identified the
CupA protein (Sll1734, also known as ChpY) in the
NDH-1S1 complex. The genes for NdhF3, NdhD3,
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Plant Physiol. Vol. 134, 2004
Proteomics of Membrane Protein Complexes in Synechocystis
and CupA are clustered in the genomes of several
unicellular cyanobacteria (Klughammer et al., 1999;
Ohkawa et al., 2000b; Maeda et al., 2002) and transcribed together into a tri-cistronic mRNA (Ohkawa
et al., 2000b; McGinn et al., 2003). Similarly to NdhF3
and NdhD3, the CupA protein was suggested to play
a role in high-affinity CO2 uptake because this process was impaired in CupA-deficient mutants
(Klughammer et al., 1999; Shibata et al., 2001; Maeda
et al., 2002). Our data here give the first direct evidence, to our knowledge, that these three proteins
associate with each other to form a membraneembedded protein complex.
Interestingly, the NDH-1S1 complex was found
also to contain a fourth subunit, encoded by sll1735.
The sll1735 gene is located immediately downstream
of the ndhF3/ndhD3/cupA operon (Klughammer et al.,
1999; Ohkawa et al., 2000b; Maeda et al., 2002), but it
is apparently transcribed independently of the other
NDH-1S1 subunits (Ohkawa et al., 2000b). Inactivation of the sll1735 gene had no significant effect on
the growth or uptake of CO2 in cyanobacterial cells
under low CO2 conditions (Klughammer et al., 1999;
Ohkawa et al., 2000b). Our results show that the
Sll1735 protein is an intrinsic component of the
NDH-1S1 complex, although its specific role still remains to be elucidated.
We did not find the constitutively expressed, lowaffinity CO2 uptake complex containing the NdhF4,
NdhD4, and CupB proteins (Shibata et al., 2001;
Maeda et al., 2002). It might be that this complex was
expressed at a relatively low level and, therefore, was
not seen in our gels.
Dynamics of the NDH-1 Complexes
Figure 7. Incorporation of radioactive Met into the membrane proteins in vivo. Synechocystis sp. PCC 6803 WT cells were labeled with
[35S]Met under photoautotrophic (A), mixotrophic (B; light and 5 mM
Glc), or photoheterotrophic (C; light and 15 ␮M DCMU) growth
conditions. Membranes corresponding to 10 ␮g of Chl a were subjected to BN/SDS-PAGE analysis as described. The main labeled
polypeptides, besides the PSII core proteins, are marked with arrows.
The positions of PSII core subunits CP47, CP43, D2, and D1 are
denoted on the right of each figure. SuIV, Subunit IV of Cyt b6f
complex. The position of PSII assembly intermediates is denoted
below C.
Plant Physiol. Vol. 134, 2004
The NDH-1 complexes, particularly NDH-1S1 and
NDH-1S2, were the most dynamic group of membrane protein complexes. The two NDH-1S complexes were strongly expressed in cells cultured photoautotrophically under air levels of CO2, whereas an
increase in CO2 concentration drastically reduced the
expression of these protein complexes (Fig. 1 versus
Fig. 5). This observation is in accordance with the
studies showing that the NdhF3, NdhD3, and CupA
subunits play a role in high-affinity CO2 uptake,
induced during acclimation of cells to low CO2 conditions (Klughammer et al., 1999; Shibata et al., 2001;
Maeda et al., 2002). Accumulation of the NDH-1S
complexes under low CO2 conditions appears to be
regulated at the level of transcription because the
abundance of the ndhF3, ndhD3, and cupA transcripts
was reported previously to become strongly upregulated by Ci limitation (Figge et al., 2001; McGinn
et al., 2003). Iron deficiency or salt stress, on the other
hand, had no distinct effect on any of the NDH-1
complexes (Fig. 6).
Intriguingly, accumulation of the NDH-1S complexes was also affected by the growth mode of the
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Copyright © 2004 American Society of Plant Biologists. All rights reserved.
477
Herranen et al.
cells. Pulse labeling experiments of photoautotrophically grown WT cells revealed ongoing synthesis of
NDH-1S complexes (Fig. 7A), which resulted in high
steady-state amounts of these complexes in the membrane (Fig. 1). Similar results were obtained using
mixotrophically cultured WT cells (Fig. 7B), indicating
that the cells relied on active CO2 uptake even in the
presence of external organic carbon source. Analysis
of metabolic fluxes using the carbon isotope labeling
technique has demonstrated that during mixotrophic
growth of WT Synechocystis sp. PCC 6803, carbon is
assimilated mainly via the Calvin cycle, whereas the
catabolism of Glc has only a minor role in carbon
metabolism (Yang et al., 2002). In contrast, no NDH-1S
complexes were present in membranes of WT cells
performing photoheterotrophic growth in the presence of DCMU (Fig. 3A). The [35S]Met labeling experiments revealed no synthesis of the NDH-1 complexes
under these non-photosynthetic conditions (Fig. 7C).
This suggests that photosynthetic activity, and the
activity of PSII in particular, is required for NDH-1S
accumulation. This is consistent with the inhibition of
light-induced transcription of the ndhF3/ndhD3/cupA
operon by DCMU (Figge et al., 2001). Moreover, at the
functional level, DCMU was shown recently to specifically inhibit the uptake of CO2 by the high-affinity
uptake system (Maeda et al., 2002; Shibata et al.,
2002b), which is represented here by the NDH-1S1
complex.
Similar to photoheterotrophic WT cells, the nonphotosynthetic PSII mutant strains were devoid of
NDH-1S complexes (Fig. 3B; data not shown). This
confirms the importance of PSII function for NDH-1S
accumulation and excludes the possibility that the
lack of NDH-1S in photoheterotrophic WT cells was
a nonspecific side effect caused by DCMU. Somewhat surprisingly, the down-regulation of NDH-1S
content was also observed when the photosynthetic
mutant cells AR, A2, and ⌬psbA1 were grown mixotrophically in the presence of Glc (Fig. 5; data not
shown). Although the physiological process behind
this phenomenon remains to be solved, it is highly
likely that the mutant cells grown under energetically demanding antibiotic stress become more amenable to exploit external Glc as their energy source
than the unstressed WT cells. This would reduce the
need for CO2 fixation via the Calvin cycle and result
in down-regulation of the CO2 uptake complex
NDH-1S.
It is noteworthy that the accumulation of the
NDH-1S and the NDH-1M complexes was closely
paralleled under all growth conditions. This further
supports the idea obtained by mutant analysis that
these two complexes probably function in cooperation. The observed down-regulation of the NDH-1M
content under elevated CO2 (Fig. 5) is consistent with
the recent results of Deng et al. (2003) showing reduced amounts of the NdhH, NdhI, and NdhK subunits in cells grown under high CO2. The NDH-1L
478
complexes, in contrast, appeared relatively unaffected by the different growth conditions. However,
the mutant cells grown under antibiotic stress generally contained a higher amount of NDH-1L complexes than the WT cells (Figs. 3B and 4). Based on
the differences in the accumulation patterns, it is
tempting to speculate that the NDH-1M and NDH-1L
complexes, despite having at least partly similar subunit composition, may play functionally different
roles. The NDH-1M might operate together with
NDH-1S to facilitate the uptake of CO2, whereas the
NDH-1L might be specifically involved in respiratory electron transfer chain and become up-regulated
under energetically demanding stress conditions.
This notion is further supported by a nearly complete
lack of NDH-1M and by a strong up-regulation of the
NDH-1L complex in a PSI-less mutant strain of Synechocystis sp. PCC 6803 (N.V. Battchikova, P. Zhang,
and E.-M. Aro, unpublished data).
Dynamics of PSI and PSII Complexes
Previous studies of thylakoid adaptation mainly
have addressed the dynamics of the two PSs and
their light-harvesting antenna (Fujita, 1997; Hihara,
1999; Grossman et al., 2001). Although our experimental system was not quantitative enough to reveal
modulations in the relative steady-state amounts of
PSI and PSII under the different growth conditions, a
very distinct accumulation of CP43⬘ protein (IsiA)
associated with PSI supercomplexes was evident
when Synechocystis sp. PCC 6803 cells were grown in
iron-depleted growth medium (Fig. 6A). Recently,
the CP43⬘ protein was shown to form an additional
Chl a-containing antenna around PSI trimers (Bibby
et al., 2001; Boekema et al., 2001), increasing the
absorption cross section of PSI up to 100% (Andrizhiyevskaya et al., 2002). This was suggested to
compensate the decrease in the PSI and phycobilisome content because of limited iron supply for their
synthesis (Bibby et al., 2001). However, we often
observed trace amounts of CP43⬘ and PSI supercomplexes even in cells grown in the presence of iron.
Accumulation of the CP43⬘ protein may represent a
more general acclimation response because the transcription of the isiAB operon (encoding CP43⬘ and
flavodoxin, respectively) was inferred recently to be
induced also under oxidative stress conditions (Jeanjean et al., 2003).
Higher expression levels of the isiAB operon also
have been observed in cells grown under salt stress
(Vinnemeier et al., 1998; Vinnemeier and Hagemann,
1999). However, under our experimental conditions,
the salt stress did not induce up-regulation in the
amount of the CP43⬘ protein (Fig. 6B). In fact, the lack
of any distinctive changes in the steady-state levels of
photosynthetic proteins or protein complexes under
salt stress conditions agrees with earlier studies demonstrating that plasma membrane, the outer mem-
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Plant Physiol. Vol. 134, 2004
Proteomics of Membrane Protein Complexes in Synechocystis
brane, and the cytosol are the main targets of salt
stress via the accumulation of osmolytes and systems
for active extrusion of sodium ions (for review, see
Joset et al., 1996).
Despite the relatively static image of PS complexes
obtained from the steady-state data, the pulse labeling experiments revealed active synthesis of these
complexes in cells grown photoauto-, mixo-, or photoheterotrophically (Fig. 7). The most notable feature
was the active synthesis of PSII, and particularly of the
PSII reaction center protein D1, under all conditions
tested. The D1 protein is prone to continuous light
damage, degradation, and de novo synthesis to maintain the photosynthetic activity (Aro et al., 1993). This
so-called PSII repair cycle was characterized by labeling of PSII core monomer intermediate complexes
and, thus, was shown to be strongly operational independently of the growth mode of the cells.
Other Membrane Protein Complexes
A protein complex containing the SbtA protein(s)
was among the most dynamic complexes in the cyanobacterial membranes. Inactivation of the sbtA gene
severely depresses the Na⫹-dependent HCO3⫺ uptake in Synechocystis sp. PCC 6803 (Shibata et al.,
2002a). Thus, the SbtA protein complex seen in our
gels most likely represents the low CO2-induced,
Na⫹-dependent HCO3⫺ transporter suggested by
Shibata et al. (2002a). Expression of the SbtA protein
complex closely followed the alterations in the expression of NDH-1S under all growth conditions,
suggesting that these two Ci uptake mechanisms
share a common regulatory pathway.
Expression of two novel protein complexes, denoted as the “Glc-induced” complexes, was strongly
induced under photoheterotrophic growth conditions (Fig. 3) and in mixotrophic mutant cultures
(Fig. 4B). Although the exact role of these protein
complexes awaits the identification of their constituent subunits, their induction under conditions where
the cells were likely to rely on external Glc for their
growth strongly suggests that they are involved either in the uptake of Glc or in the oxidation of
NADPH formed by Glc catabolism. The scarcity of
these complexes in the mixotrophic WT cultures is in
line with the observation that the Glc catabolism
plays only a minor role in the mixotrophically grown
WT cell cultures (Yang et al., 2002).
The rest of the membrane protein complexes
showed no drastic modulation under the various
growth conditions. However, we observed three
novel protein complexes, including sll1757, slr0151,
and slr0695 gene products, respectively. Although
the physiological role of these proteins remains to be
solved, we show here that these proteins are components of membrane-embedded complexes.
We conclude that the BN/SDS-PAGE is a powerful
method for analysis of the composition and dynamPlant Physiol. Vol. 134, 2004
ics of membrane protein complexes of cyanobacterial
cells. Of particular interest was the observation of
four distinct NDH-1 complexes, whose accumulation
was regulated by environmental cues. Furthermore,
we demonstrate that the gene products of the ndhF3/
ndhD3/cupA operon associate together as a protein
complex and introduce a new subunit for this CO2
uptake complex. In addition to the already known
cyanobacterial membrane components, several new
protein complexes, formed by proteins with unknown functions, are reported. The function of these
novel protein complexes remains to be elucidated.
MATERIALS AND METHODS
Strains and Growth Conditions
A Glc-tolerant strain of Synechocystis sp. PCC 6803 (Williams, 1988) was
used as the WT. In addition, seven mutant strains were studied. In the
mutant ⌬ndhD3, a kanamycin resistance cassette interrupts the ndhD3 gene
(Ohkawa et al., 2000b). The other six mutant strains have mutations in the
psbA genes, encoding PSII reaction center protein D1. The mutant strain
⌬psbA1 has a chloramphenicol resistance cassette replacing the silent psbA1
gene. In the mutant A2, the psbA1 and psbA3 genes are insertionally inactivated with kanamycin and chloramphenicol resistance genes, respectively,
leaving the psbA2 as the only functional psbA gene (Jansson et al., 1987).
Strain AR, a derivative of A2, has an additional ⍀-fragment (streptomycin
and spectinomycin resistance cassette) after the psbA2 gene (Mäenpää et al.,
1993). The strains LC (⌬T227-Y246), CD (⌬G240-V249), and PCD (⌬R225V249) are derivatives of AR, containing additional deletions in the psbA2
gene in a region corresponding to the D-E loop of the D1 protein (Mulo et
al., 1997). The strains LC, CD, and PCD are obligate photoheterotrophs,
whereas all the other strains are capable in photoautotrophic growth.
All strains were grown in 200-mL batch cultures of BG-11 medium
(Stanier et al., 1971) buffered with 20 mm HEPES-NaOH (pH 7.5) at 32°C.
The cultures were maintained under continuous light of 50 ␮mol photons
m⫺2 s⫺1 under air level of CO2 unless otherwise indicated. The growth
media for the mutant strains were supplemented with appropriate antibiotics
(Jansson et al., 1987; Mäenpää et al., 1993; Mulo et al., 1997) at concentrations
of 2.5 ␮g mL⫺1 for chloramphenicol, 5 ␮g mL⫺1 for kanamycin and streptomycin, and 10 ␮g mL⫺1 for spectinomycin. The growth media for the photoheterotrophic mutants were supplied additionally with 5 mm Glc.
To induce mixotrophic or photoheterotrophic growth of WT or photoautotrophic mutant cultures (AR, A2, and ⌬psbA1), the cells were grown in the
presence of 5 mm Glc or Glc together with 10 ␮m DCMU (Sigma, St. Louis),
respectively. Measurements of oxygen evolution by a Clark-type oxygen
electrode (Hansatech, Kings Lynn, UK) confirmed that the 10 ␮m concentration of DCMU prevented oxygen evolution in vivo. Iron stress was
induced by replacing ferric ammonium citrate with ammonium citrate in the
growth media. Salt stress was induced by growing the cells in the presence
of 0.4 m NaCl. During all treatments, the cells were let to adapt to prevailing
conditions for several generations before harvesting at the logarithmic
growth phase (A750 0.6–0.8, measured with Spectronic Genesys 2 spectrophotometer, Spectronic Instruments, Inc., Rochester, NY).
Isolation of the Membrane Fraction
Membranes were isolated essentially as described by Gombos et al.
(1994). Cells were harvested by centrifugation (6,000g for 8 min at 4°C) and
washed twice with washing buffer (50 mm HEPES-NaOH [pH 7.5] and 30
mm CaCl2). The cells were then broken in isolation buffer (50 mm HEPESNaOH [pH 7.5], 30 mm CaCl2, 800 mm sorbitol, and 1 mm ⑀-amino-n-caproic
acid) by vortexing them in the presence of glass beads (150–212 microns;
Sigma) for 8 min at 4°C. Unbroken cells and glass beads were removed by
centrifugation at 550g for 5 min. The membranes were pelleted from the
supernatant by centrifugation at 17,000g for 20 min, resuspended in storage
buffer (50 mm Tricine-NaOH [pH 7.5], 600 mm Suc, 30 mm CaCl2, and 1 m
glycinebetaine), and stored at ⫺70°C. All steps were performed in dim light
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Copyright © 2004 American Society of Plant Biologists. All rights reserved.
479
Herranen et al.
at 4°C. Isolated membranes were highly enriched with thylakoids but
apparently also contained fragments originating from plasma membrane.
Determination of Chl Concentration of
Isolated Membranes
trypsin autodigestion products (842.5094 and 2,211.1046 m/z). Proteins were
identified as the highest ranking result by searching in the National Center
for Biotechnology Information database using Mascot (http://www.matrixscience.com). The search parameters allowed for carbamidomethylation of
cystein, one miscleavage of trypsin, and 50 ppm mass accuracy. For positive
identification, the score of the result of [ ⫺ 10 ⫻ Log(P)] had to be over the
significance threshold level (P ⬍ 0.05).
The Chl a content of the isolated membranes was determined according
to Arnon (1949).
ACKNOWLEDGMENTS
BN-PAGE/SDS-PAGE
BN-PAGE was performed according to Kügler et al. (1997), with the
modifications of Cline and Mori (2001). Cyanobacterial membranes were
washed with washing buffer (330 mm sorbitol, 50 mm BisTris [pH 7.0], and
250 ␮g mL⫺1 Pefabloc [Roche Diagnostics, Indianapolis]), collected by centrifugation (18,000g for 10 min), and resuspended in resuspension buffer
(20% [w/v] glycerol, 25 mm BisTris [pH 7.0], 10 mm MgCl2, 0.1 units ␮L⫺1
RQ1 RNase-Free DNase [Promega, Southampton, UK], and 250 ␮g mL⫺1
Pefabloc) at Chl a concentration of 1 mg mL⫺1. An equal volume of resuspension buffer containing 4% (w/v) n-dodecyl ␤-d-maltoside (Sigma) was
added under continuous mixing, and the solubilization of membrane protein complexes was allowed to occur for 50 min on ice, followed by 10 min
at room temperature. Insoluble material was removed by centrifugation at
18,000g for 20 min. The supernatant was mixed with 0.1 volumes of Coomassie Blue solution (5% [w/v] Serva blue G, 100 mm BisTris [pH 7.0], 30%
[w/v] Suc, 500 mm ⑀-amino-n-caproic acid, and 10 mm EDTA) and loaded
onto 0.75-mm-thick 5% to 12.5% (w/v) acrylamide gradient gel (Hoefer
Mighty Small mini-vertical unit, Amersham Pharmacia Biotech, Uppsala),
containing 500 mm ⑀-amino-n-caproic acid. Electrophoresis was performed
at 4°C by increasing voltage gradually from 50 up to 200 V during the 4.5-h
run. For separation of proteins in the second dimension, the lanes of the BN
gel were excised and incubated in SDS sample buffer containing 10% (v/v)
␤-mercaptoethanol and 6 m urea for 45 min at room temperature, followed
by 15 min at 40°C. The lanes were then layered onto 1-mm-thick SDS-PAGE
gels (Laemmli, 1970), with 14% (w/v) acrylamide and 6 m urea in the
separating gel. The proteins were visualized by silver staining (Blum et al.,
1987, except that the stop buffer was replaced by 0.4 m Tris and 2.5% [v/v]
acetic acid), by autoradiography ([35S]Met labeled proteins), or by electroblotting the proteins onto a polyvinylidene difluoride membrane (Immobilon
P, Millipore, Bedford, MA) and immunodetection by protein-specific antibodies using the CDP-Star Chemiluminescent Detection Kit (New England
Biolabs Ltd., Hertfordshire, UK). The NdhD3 antibody was raised against
the amino acids 185 to 196 and 346 to 359, and NdhF3 antibody was raised
against the amino acids 28 to 41 and 439 to 453 of the respective proteins of
Synechocystis sp. PCC 6803 (Eurogentec, Seraing, Belgium).
Labeling of Membrane Proteins in Vivo
Synechocystis sp. PCC 6803 WT cells were grown photoautotrophically or
mixotrophically under standard growth conditions until the logarithmic
growth phase and adjusted to Chl a concentration of 10 ␮g mL⫺1. Thereafter, the cell suspensions were pulse labeled with 3 ␮Ci mL⫺1 [35S]Met
(⬎1,000 Ci mmol⫺1, Amersham Biosciences UK Ltd.) for 30 min under
conditions supporting photoautotrophic, mixotrophic, or photoheterotrophic growth. The photoheterotrophic growth conditions were induced by
supplying the photoautotrophically grown cell culture with 15 ␮m DCMU
15 min before labeling. Labeling was terminated by adding 1 mm nonradioactive Met and 0.5 mg mL⫺1 chloramphenicol, and the cells were harvested
by centrifugation at 4°C. Membranes were isolated and subjected to BN/
SDS-PAGE as described. After BN/SDS-PAGE, the gels were dried, and the
proteins were visualized by autoradiography.
Identification of Proteins by MALDI-TOF
In-gel trypsin digestion and sample preparation for MALDI-TOF analysis
was done manually according to Shevchenko et al. (1996). Samples were
loaded onto the target plate by the dried droplet method using ␣-cyano-4hydroxycinnamic acid as a matrix. MALDI-TOF analysis was performed in
reflector mode on a Voyager-DE PRO mass spectrometer (Applied Biosystems, Foster City, CA). Internal mass calibration of spectra was based on
480
We thank Dr. Teruo Ogawa for the ⌬ndhD3 mutant, Dr. Bruce Diner for
the ⌬psbA1 mutant, and Dr. Wim Vermaas for the PSI-less mutant. The mass
spectrometer group in the Turku Centre for Biotechnology is thanked for
advice in protein analysis.
Received August 26, 2003; returned for revision September 17, 2003; accepted October 14, 2003.
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