Download A New Subunit of Cytochrome b6f Complex Undergoes Reversible

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 22, Issue of June 2, pp. 17072–17079, 2000
Printed in U.S.A.
A New Subunit of Cytochrome b6f Complex Undergoes Reversible
Phosphorylation upon State Transition*
Received for publication, February 22, 2000, and in revised form, March 15, 2000
Published, JBC Papers in Press, March 21, 2000, DOI 10.1074/jbc.M001468200
Patrice Hamel‡, Jacqueline Olive§, Yves Pierre¶, Francis-André Wollman储, and
Catherine de Vitry储**
From the ‡Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569, the §Institut Jacques
Monod, Université de Paris VII, 75005 Paris, France, the ¶Laboratoire de Physico-Chimie Moléculaire des membranes
Biologiques, CNRS UPR 9052, Institut de Biologie Physico-Chimique, 75005 Paris, France, and the 储Physiologie
Membranaire et Moléculaire du Chloroplaste, CNRS UPR1261, Institut de Biologie Physico-Chimique, 75005
Paris, France
A 15.2-kDa polypeptide, encoded by the nuclear gene
PETO, was identified as a novel cytochrome b6f subunit
in Chlamydomonas reinhardtii. The PETO gene product
is a bona fide subunit, subunit V, of the cytochrome b6f
complex, because (i) it copurifies with the other cytochrome b6f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b6f mutants accumulating little of the complex, and (iii) it
colocalizes with cytochrome f, which migrates between
stacked and unstacked membrane regions upon state
transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane ␣-helix topology with two large hydrophilic
domains extending on the stromal and lumenal side of
the thylakoid membranes, with a lumenal location of the
N terminus. Subunit V is reversibly phosphorylated
upon state transition, a unique feature that, together
with its topological organization, points to the possible
role of subunit V in signal transduction during redoxcontrolled short term and long term adaptation of the
photosynthetic apparatus in eukaryotes.
The cytochrome bc complexes such as bc1 and b6f are ubiquitous in energy-transducing membrane systems; their major
metabolic function is to couple the oxidation of quinols to the
translocation of protons across the membrane, resulting in the
establishment of a transmembrane electrochemical proton gradient required to drive the synthesis of ATP (1). Beside its role
in photosynthetic electron transport, the cytb6f1 complex is also
recruited for redox sensing and signal transduction in chloroplasts (2, 3). The cytb6f complex plays a key role in the supramolecular reorganization of the photosynthetic apparatus
* This work was supported by the CNRS UPR 1261 and by NIGMS,
National Institutes of Health Grant GM48350 (to S. Merchant for
P. H.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s) AF222893.
** To whom correspondence should be addressed: Physiologie Membranaire et Moléculaire du Chloroplaste, CNRS UPR 1261, Institut de
Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris,
France. Tel.: 33-1-58-41-5000; Fax: 33-1-58-41-5020; E-mail: catherine.
[email protected].
1
The abbreviations used are: cyt, cytochrome; LHC, light-harvesting
complex; LHCP, light-harvesting complex proteins; PQ, plastoquinone;
PCR, polymerase chain reaction; PSI, photosystem I; PSII, photosystem
II; su, subunit; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; Hecameg, 6-O-(N-heptylcarbamoyl)methyl-␣-D-glucopyranoside.
upon state transitions: changes in the redox state of plastoquinones (PQs) that bind to cytb6f complexes allow a photosynthetic cell to adapt to changes in light quality as well as to
changes in intracellular ATP levels (4). At the molecular level,
plastoquinol binding at the Qo site of cytb6f complexes on the
lumen side of the thylakoid membranes activates a kinase that
phosphorylates the light-harvesting antenna on the stromal
side of the membranes (5–9). How the plastoquinol binding
signal is transduced across the membrane for kinase activation
remains unknown. This reversible protein phosphorylation
process controls the migration of the antenna and the cytb6f
complex between the stacked membrane regions containing
photosystem II (PSII) and the unstacked membrane regions
containing PSI (10). Studies with Chlamydomonas reinhardtii
in vivo have shown that state I corresponds to a low phosphorylation of the peripheral antenna proteins that are mainly
associated with PSII, with the photosynthetic apparatus being
set for a linear electron flow from PSII to PSI aimed at carbon
fixation; in state II, the peripheral antenna proteins become
heavily phosphorylated, are mainly associated with PSI and
the photosynthetic apparatus is set for cyclic electron flow and
supplies high ATP levels (4, 11).
C. reinhardtii displays the same photosynthetic apparatus
as that of vascular plants but shows dispensable photosynthesis. Its cytb6f complex has been purified and comprises at least
seven subunits (12). cytf, cytb6, and subunit (su)IV are encoded,
respectively, by the chloroplast genes petA, petB, and petD (13),
whereas the Fe2S2 Rieske protein is encoded by the nuclear
gene PETC (14). In addition, there are three small 4-kDa
hydrophobic subunits, each forming a single transmembrane
␣-helix, products of the chloroplast genes petG (15) and petL
(16), and the nuclear gene product PETM (17). Recently, a
putative fourth small subunit has been identified as a product
of a chloroplast gene petN in higher plants (18). This gene is
located in the nucleus in Volvox (19) and in C. reinhardtii as
suggested by homologies to expressed sequence tags (20).
Last, a nucleus-encoded 19-kDa polypeptide, termed suV,
had been proposed to associate with the rest of the cytb6f
subunits in C. reinhardtii, based on its presence in cytb6fenriched fractions and its absence or low representation in
cytb6f-deficient mutants (21).
We report here the biochemical, molecular, and topological
characterization of suV, encoded by the gene termed PETO and
identified just after PETN, and provide evidence that it is
conserved in other photosynthetic eukaryotes. The potential
roles played by this cytb6f phosphoprotein in photosynthetic
electron transport, redox sensing, and signal transduction are
discussed.
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This paper is available on line at http://www.jbc.org
Cytochrome b6f-associated Phosphoprotein
EXPERIMENTAL PROCEDURES
C. reinhardtii Strains—Wild-type and mutant strains ⌬petB and
⌬petD (22), petC-⌬1 (6), and ATP synthase mutant F54 (23) were grown
on Tris acetate-phosphate medium, pH 7.2, at 25 °C under dim light
(5– 6 ␮mol of photons m⫺2 s⫺1).
suV Protein Microsequencing—Thylakoid membranes (1.5 mg of
chlorophyll/ml) from the F54 mutant, deficient in ATP synthase, were
solubilized 15 min at 4 °C in the presence of 2.2% Mega-8 (w/v), 20 mM
Tricine, pH 8.0, 3 mM KCl, and 3 mM MgCl2. The solubilized supernatant, which is enriched in cytb6f complex was separated on 12–18%
SDS-polyacrylamide gels in the presence of 8 M urea, and electrotransferred onto polyvinylidene difluoride membranes in a semidry system
as described previously (24). Sequencing of the band containing suV
and of a tryptic fragment was performed according to Edman degradation by J. d’Alayer (Laboratoire de Microséquençage des Protéines,
Institut Pasteur, Paris).
DNA and RNA Analyses—C. reinhardtii cDNA library in phage
␭gt10 (25) was generously provided by L.-G. Franzén (Department of
Plant Physiology, Botanical Institute, Göteborg University, Sweden).
Phage DNA of the cDNA library was prepared out of 1013 phage particles from liquid Escherichia coli culture as described previously (26).
Desalted oligonucleotides were purchased from Oligo Express (Paris,
France). Polymerase chain reaction (PCR) procedure followed was as in
Ref. 6, and the annealing temperature was 55 °C. Two phage-specific
primers (5⬘-TGAGCAAGTTCAGCCTGGTTAAGTC-3⬘; 5⬘-GCTTATGAGTATTTCTTCCAGGGTA-3⬘) and a degenerate sense primer deduced from the N-terminal sequence of suV 5⬘-CTGCAGCAGCC(G/T/
C)GT(A/G/T/C)CTGAAGAAGGC(G/T/C)TTCCAGGA-3⬘ were designed
to amplify the 3⬘ part of the PETO cDNA. Either of the two phage
primers in combination with the sense degenerate primer led to a
1.1-kilobase (kb) PCR amplification product when 0.5–1 ␮g of library
DNA was used as a template. An antisense primer, 5⬘-CCCATCACGCCCCAGCTCCC-3⬘, chosen from the sequence of the 1.1-kb PCR product
was used to amplify, with either phage-specific primer, a 0.7-kb PCR
product corresponding to the 5⬘ part of the PETO cDNA. 1.3-kb full
length cDNA was reconstituted using megaprime PCR by mixing the
1.1- and 0.7-kb products (obtained with different phage primers) and
both phage primers. Sequencing was performed on both strands by
Genome Express (Paris, France). Total RNA analysis was performed
according to a previous study (27).
Protein Isolation, Separation, and Analysis—Biochemical analyses
were carried out on cells grown to a density of about 2 ⫻ 106 cells per
milliliter. For polypeptide analysis, samples were resuspended in 100
mM 1,4-dithiothreitol and 100 mM Na2CO3 and solubilized in the presence of 2% SDS at 100 °C for 50 s. Polypeptides were then separated on
12–18% SDS-polyacrylamide gels in the presence of 8 M urea, cells, and
thylakoid membranes were loaded at chlorophyll constant. Heme staining was detected by peroxidase activity of heme-binding subunits using
3,3⬘,5,5⬘-tetramethylbenzidine as described previously (21). Immunodetection using antisera raised against cytf, suIV, Rieske protein in combination with 125I-labeled protein A, or an enhanced chemiluminescence
method was carried out as in Ref. 6. Thylakoid membrane proteins were
purified as done previously (24). Cytb6f complexes were purified by
6-O-(N-heptylcarbamoyl)-methyl-␣-D-glucopyranoside (Hecameg; Vegatec, Villejuif, France) solubilization of thylakoid membranes (12).
Polypeptide extraction from thylakoids were performed by two cycles of
10-min incubation with various dissociating agents at room temperature followed by freeze/thaw as described previously (28). After
Hecameg solubilization, the supernatant was subjected to sucrose gradient centrifugation and gradient fractions were then assayed for kinase activity using [␥-32P]ATP and casein as substrate according to a
previous study (5).
Antibodies Preparation—Anti-PSII PsbB, anti-Rieske, and anti-cytf
antibodies were raised against the entire polypeptides. Anti-suV was
raised against a synthetic peptide containing the sequence of the N
terminus of mature suV (LQQPVLKKAFQDDTP) coupled to ovalbumin
through an extra COOH-tyrosine and prepared by Neosystem (Strasbourg, France). Anti-cytf monoclonal antibody was kindly provided by
O. Vallon (Institut de Biologie Physico-Chimique, Paris). Anti-PSII
OEE2 preparation was described previously (29).
Proteolysis of Thylakoid Membranes—Thylakoid membranes were
washed twice with 20 mM ammonium phosphate, pH 7.4, without protease inhibitors. Various concentrations of protease V8 or of trypsin
were added to thylakoid membranes at a final chlorophyll concentration
of 1 mg/ml. Half of each sample was sonicated for 10 s on ice. Trypsin
and V8 treatments were performed at room temperature for 20 min,
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and the reaction was arrested by adding 2 mM phenylmethylsulfonyl
fluoride as a protease inhibitor. The samples were analyzed by urea/
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting.
Electron Microscopy—Immunocytochemistry on thin sections was
performed according to a previous study (29). Immunogold labeling of
thylakoid membrane vesicles was performed according to earlier work
(30). Anti-suV antibody raised against a synthetic peptide designed
from the sequence of the N terminus of mature suV and anti-cytf
monoclonal antibody recognizing the lumenal domain cytf were used at
dilutions of 200 and 10, respectively, for single or double immunogold
labeling of the vesicles.
Protein Phosphorylation in Vivo—Cells grown at 3 ⫻ 106 cells/ml
were incubated for 120 min, under dim light (5 ␮mol of photons m⫺2
s⫺1), in a phosphate-depleted medium containing 1 ␮Ci/ml of
[32P]orthophosphate or 2 ␮Ci/ml of [33P]orthophosphate. After resuspension in 20 mM Hepes, pH 7.5, 5 mM MgCl2, 10 mM NaCl, the
prelabeled cells were placed for 30 min in either state I or state II
conditions. For state I, cells were illuminated at 50 ␮mol of photons m⫺2
s⫺1 in the presence of 10⫺5 M 3,4-dichlorophenyl-1,1-dimethylurea to get
oxidation of the PQ pool. For state II, the PQ pool was reduced by
placing dark-adapted cells in anaerobic conditions using 20 mM glucose
and 2 mg/ml glucose-oxidase as described previously (31). State I and II
cells were rapidly broken at 4 °C in a French pressure cell at 4000 psi
after adding 10 mM NaF, 10 mM EDTA, 0.1 M sucrose, and protease
inhibitors to the suspension medium. Thylakoid membrane proteins
were prepared as in Ref. 25 with 10 mM NaF and 10 mM EDTA added
in all buffers.
suV Immunoprecipitation—In vivo 33P-labeled thylakoid membrane
proteins were solubilized in the presence of 2% SDS at 100 °C for 50 s
and diluted 10-fold in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM
EDTA, 2% Triton X-100, 10 mM NaF, 200 ␮M phenylmethylsulfonyl
fluoride (buffer A). Protein A-Sepharose CL4B (Amersham Pharmacia
Biotech) was incubated 30 min in 1 ml of distilled water, and washed
three times in 1 ml of buffer A. To 1 ml of protein A-Sepharose CL4B in
buffer A 30 ␮l of suV antiserum was added or none as a control and
samples were incubated 1 h at 4 °C and washed once in 1 ml of buffer
A. The diluted solubilized thylakoid membrane proteins were mixed
with the pellets of protein A-Sepharose, which had or not been in
presence of suV antibodies; samples were incubated 1 h at 4 °C, and
washed three times in 1 ml of buffer A. Samples were resuspended in
100 mM 1,4-dithiothreitol and 100 mM Na2CO3 and solubilized in the
presence of 2% SDS at 100 °C for 50 s for polypeptide analysis.
RESULTS
Sequence and Topology of the PETO Gene Product—We have
previously identified a nucleus-encoded polypeptide of 19-kDa
apparent molecular mass that was absent from the thylakoid
membranes of cytb6f mutants (21). To avoid its copurification
with some ATP synthase subunits that display a similar apparent molecular mass, we recovered suV (PetO) from the ATP
synthase-deficient mutant F54, using a thylakoid membrane
solubilization with Mega-8, which selectively extracts cytb6f
complex. After gel electrophoresis and transfer onto nitrocellulose membranes, we were able to microsequence 31 residues of
the N terminus and one internal tryptic fragment of purified
suV (Fig. 1).
The cDNA for suV was cloned by PCR amplification of a C.
reinhardtii cDNA library with a degenerate primer derived
from the N-terminal microsequence and phage-specific primers. The nucleotide and amino acid sequences (GenBank™
accession number AF222893) are shown in Fig. 1 and are
aligned with the sequence obtained from N terminus sequencing. These sequences are homologous to ones found within a
library of recently released expressed sequence tags (cDNA
sequences) of C. reinhardtii (20).
The methionine codon, CACCAUGGCC, has features of an
initiation codon with a consensus sequence similar to that of
higher plants, i.e. AACAAUGGCC (14). The bipartite transit
peptide is 51 residues long and shows features typical of a
lumenal-targeting sequence. The first domain is analogous to
stroma-targeting peptides: it comprises an alanine in the second position, a short uncharged N-terminal region, a central
region rich in residues with basic (R, K) or small (A, S) side
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Cytochrome b6f-associated Phosphoprotein
FIG. 2. Putative transmembrane topology of suV.
FIG. 1. Nucleotide and amino acid sequence of suV. The deduced
amino acid sequence is in boldface, and the amino acid sequence determined by Edman degradation is in italics. X corresponds to unidentified
amino acid residues. The internal fragment sequence is GLAPXAL. The
open inverted triangle indicates the putative intermediate processing
site. The black inverted triangle indicates the processing site of the
mature protein. The hydrophobic regions likely to correspond to transmembrane ␣-helices are underlined. The potentially phosphorylated
threonine residues in the predicted phosphorylation motifs (italics) are
underlined. The GenBank™ accession number is AF222893.
chains, a consensus sequence VXA just before the putative
intermediate processing site. This latter feature is observed in
many transit peptides for nuclear-encoded chloroplast proteins
of C. reinhardtii such as the Rieske protein, PSI subunits (P28,
P30, and P37), ATPase subunit ␥, heat shock protein HSP70B,
phosphoribulokinase and ADP-glucose pyrophosphorylase
large subunit. The second domain is homologous to a thylakoid
transfer domain, with a central hydrophobic region, and a
consensus cleavage site AXA for the lumenal-processing peptidase. C. reinhardtii suV transit peptide was not recognized as
a chloroplast transit peptide by most prediction programs, because C. reinhardtii chloroplast peptides share more features
with mitochondrial than do higher plant chloroplast presequences (32).
The mature protein is 147 residues long and has a calculated
molecular mass of 15.2 kDa. A central hydrophobic region is
long enough to span the membrane once in an ␣-helical conformation (Fig. 2). The lateral amphipathy of this putative ␣-helix
is low.
Northern blots probed with the entire open reading frame of
suV revealed a 1.3-kb transcript that corresponds to the size of
the insert amplified from the cDNA library (data not shown).
We also detected a few longer transcripts in low amounts,
which might correspond to splicing intermediates of the primary PETO transcript.
The PETO Gene Product, suV, Behaves as a Genuine Subunit
of cytb6f Complex—The antiserum raised against the N terminus of suV recognized only suV in the thylakoid membrane of
C. reinhardtii (Fig. 3A). The steady-state level of suV was
greatly diminished in whole cell protein extracts and in purified thylakoid membrane proteins from cytb6f-deficient mutants that do not accumulate significant levels of any of the
cytb6f complex subunits (see ⌬petB or ⌬petD mutants, Fig. 3, A
and B, which lack, respectively, cytb6 and suIV). In contrast,
suV still accumulated together with the other transmembrane
subunits of cytb6f in mutants lacking specifically the Rieske
protein, such as the petC-⌬1 mutant (Fig. 3C).
suV was recovered together with the other subunits of the
cytb6f complex in the supernatant after solubilization with
Hecameg of the thylakoid membranes (Fig. 4). Upon centrifugation of the supernatant on a sucrose gradient, part of suV
remained associated with the cytb6f complex. However, some
suV spread over fractions of higher density, most likely due to
aggregated forms of the isolated subunit. suV fully dissociated
from the other cytb6f subunits during the next purification
steps that consisted of a chromatography on a hydroxyapatite
column followed by a desorption step in the presence of high
concentrations of ammonium phosphate.
suV Is a Transmembrane Protein with Its N Terminus Facing the
Lumen of the Thylakoid Membranes—We investigated the binding
of suV to the thylakoid membranes using various dissociating
treatments (Table I). Membranes were treated with chaotropic
agents or incubated at high ionic strength or alkaline pH. Neither
cytf, cytb6, suIV, nor PetG were extracted by these treatments, as
expected from their transmembrane anchoring. In contrast, typical
peripheral membrane polypeptides (OEE1) were released in the
supernatant. As reported earlier, the Rieske protein showed an
intermediate susceptibility to dissociating conditions (28), although
it has been clearly identified as a transmembrane protein in the
three-dimensional structure of bc1 complexes (33, 34). suV behaved
similarly to the Rieske protein: Although it was not released from
the membranes at high ionic strength, it readily dissociated with
chaotropic agents such as KSCN (see also Fig. 8) or at alkaline pH.
Cytochrome b6f-associated Phosphoprotein
FIG. 3. Immunodetection of suV of cytb6f in wild-type and
cytb6f mutants. A, immunoblot labeled with an antiserum raised
against the N terminus of the mature suV of thylakoid membranes from
wild-type C. reinhardtii (WT) or from a mutant lacking cytb6 and
accumulating little of the rest of the cytb6f complex (⌬petB). B, immunodetection of cells and thylakoid membranes from wild-type C. reinhardtii and from a mutant lacking suIV and accumulating little of the
rest of the cytb6f complex (⌬petD). C, immunodetection of cells and
thylakoid membranes from wild-type C. reinhardtii and from a mutant
lacking the Rieske protein and accumulating most of the rest of the
complex (petD-⌬1). Levels of three subunits of the b6f complex, cytf,
Rieske protein, and suV were revealed by immunodetection. A subunit
of PSII was immunodetected as a loading control (PsbB). Proteins were
separated by urea SDS-PAGE. Blots were revealed using 125I-protein A.
Biochemical evidence for a transmembrane orientation of
suV, with the N terminus facing the lumen, was provided by
analysis of proteolysis experiments. Thylakoid membrane vesicles were incubated in the presence of protease V8 (endoproteinase Glu-C) and then subjected or not to sonication. Before
sonication, most of the thylakoid vesicles are in a right-side-out
position and exogenous proteases have no access to the lumen
side of the membranes. In contrast, upon sonication, the vesicles burst and exogenous proteases have access to both sides of
the membranes. suV was degraded by proteases in the absence
of sonication, in contrast to the OEE2 protein used here as a
control for a lumen resident protein (Fig. 5). Thus, suV exposes
some protein motifs to the stromal side of the thylakoid membranes. However, the N-terminal-directed antibody detected a
partly protease-protected suV fragment in unsonicated vesicles. This fragment was no longer detected after sonication.
Similar observations were made using trypsin as an exogenous
protease (experiments not shown). The selective detection of
this fragment in unsonicated thylakoid vesicles shows that the
N-terminal domain of suV extends in the lumen. The V8-produced fragment of 11-kDa apparent molecular mass should
correspond to a truncated product of suV at its stromal C
terminus, at E96, E109, or E115, leaving together the transmembrane and N terminus domains that have a predicted size
of 10 –12 kDa. However, its limited accumulation argues for the
high susceptibility of the truncated product to endogenous proteases, whether they are membrane-associated or lumen-located. This high protease susceptibility of suV is also substantiated by in vivo experiments, which show a drastic decrease in
suV content in strains that do not accumulate cytb6f complexes
(Fig. 3). This behavior is in marked contrast with that of OEE2,
which is stable in the thylakoid lumen in the absence of PSII
assembly (28). Therefore, we conclude from these assays with
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exogenous proteases that, in agreement with the sequence
data, suV behaves as a transmembrane protein with the N
terminus facing the thylakoid lumen and the C terminus extending into the stroma.
We then compared the immunogold labeling of thylakoid
membrane vesicles that were exposed either to the anti-suV
antibody, which recognizes the N-terminal sequence of the
mature protein, or to a monoclonal antibody that recognizes a
lumen-located motif of cytf. Evidence for the lumen location of
this cytf epitope came from its immunodetection in mutant
cells expressing only a soluble form of cytf that lacks both the
C-terminal transmembrane ␣-helix and the stromal stretch of
the polypeptide chain (data not shown). Immunogold labeling
showed an unambiguous colocalization of these two antibodies
on the same inside-out vesicle (Fig. 6), giving further support to
the localization of the N terminus of suV on the membrane
lumenal side.
A Protein Phosphorylated in State II—When the intersystem
electron carriers of the photosynthetic electron transport chain
switch from an oxidized (state I) to a reduced (state II) steady
state, the photosynthetic apparatus undergoes a change in its
supramolecular organization such that most of light-harvesting complex II (LHCII) and some cytb6f complex gather next to
PSI in the stromal lamellae membrane regions (10). The supramolecular reorganization of the thylakoid membranes can
be detected by immunocytochemistry (Table II). In this experiment, thin sections of broken cells of C. reinhardtii, pretreated
in either of the two states, were incubated with colloidal goldcoupled antibodies specific for PSII, LHCII, or cytb6f antigens.
The movement of LHCII or cytb6f complexes toward PSI-enriched unstacked membrane domains in state II is demonstrated in Table II by the increase in the ratio of immunogold
labeling of unstacked versus stacked membrane region in state
II as compared with state I. In contrast PSII remained in the
stacked membrane regions in the two states. suV displayed the
same behavior as cytf, being enriched in the unstacked regions
in state II as compared with state I. This change in lateral
distribution between the two states further supports the association of suV with the rest of the cytb6f complex, which shows
lateral displacement upon state transitions.
State transitions are accompanied by reversible changes in
the phosphorylation of several thylakoid membrane polypeptides. The major phosphoproteins that were previously identified in C. reinhardtii (31) belong either to the light-harvesting
complex proteins (LHCP) family or to PSII (Fig. 7A). In particular, phosphorylation of the LHC polypeptides increases in
state II conditions, this process being dependent upon the
presence of the cytb6f complex (5). Therefore, this increased
phosphorylation is not observed in mutants lacking cytb6f complexes (Fig. 7A). The pattern of phosphoproteins detected in
state II showed the presence of an additional component that
has the same electrophoretic mobility as suV. This phosphorylated protein is absent in cytb6f mutants that show very little
accumulation of the cytb6f complex subunits and do not undergo state transition (Fig. 7A). Fig. 7B shows that this change
in phosphorylation was not accompanied by a change in the
steady-state level of membrane-bound suV between states I
and II. That this phosphoprotein is indeed a phosphorylated
form of suV was further confirmed by its behavior upon extraction with chaotropic agents or detergents (Fig. 8A and data not
shown). Furthermore, the phosphorylated protein can be immunoprecipitated by the anti-suV antiserum (Fig. 8B). Thus
suV is a cytb6f subunit that can be reversibly phosphorylated
upon state transitions.
Conservation of suV in Other Photosynthetic Eukaryotes—We
found no homologues of suV within the fully sequenced cya-
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Cytochrome b6f-associated Phosphoprotein
FIG. 4. suV through purification steps of cytb6f complex. Thylakoid membranes were solubilized with Hecameg and centrifuged; S,
supernatant; P, pellet. The supernatant was centrifuged on a 10 –30% sucrose gradient, and fractions were collected. The peak of cytb6f complex
is indicated by b6f max. The complex purified after hydroxyapatite column chromatography is designated b6f HA. All fractions from sucrose
gradient and hydroxyapatite purification were analyzed by urea/SDS-PAGE and levels cytf, Rieske protein, and suV were revealed by immunodetection. Sucrose fractions were assayed for kinase activity using [␥-32P]ATP and casein as substrate.
TABLE I
Extraction of thylakoid proteins by various dissociating treatments
(⫺) less than 15% extraction, (⫹) 15–70% extraction, (⫹⫹) more than 70%. cytf, cytb6, suIV and PetG are four intrinsic subunits of cytb0f
complex. OEE1 is PSII extrinsic subunit.
cytf
cytb6
suIV
PetG
Rieske
suV
OEE1
Tricine
20 mM
NaCl
2M
Nal
1.5 M
KSCN
2M
DTT-Na2C03
85 mM
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹
⫺
⫺
⫺
⫺
⫹/⫹⫹
⫹
⫹⫹
⫺
⫺
⫺
⫺
⫹⫹
⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫹/⫹⫹
⫹/⫹⫹
⫹⫹
FIG. 5. Analysis of the transmembrane topology of suV by proteolysis of thylakoid membrane vesicles. Thylakoid membrane vesicles were incubated with increasing protease V8 concentrations. Vesicles in the presence of the protease were sonicated (⫹) or not (⫺).
Samples were analyzed by urea/SDS-PAGE and immunodecorated using antisera against the lumenal protein OEE2 and against a peptide of
the N terminus of suV. Nonstromal suV fragments protected in nonsonicated vesicles and degraded in sonicated vesicles are indicated by
the arrow.
nobacterial genomes available in data banks. However, a homologue to suV (GenBank™ accession number AF110791) was
identified among cDNAs from Volvox (19), which is a another
green alga closely related to C. reinhardtii. A BLASTP 2.0
alignment (35) showed 147 identities out of the 198 residues of
suV from C. reinhardtii. In particular, the consensus processing sites, hydrophobic segments, and putative phosphorylation
motifs are conserved.
Because the N-terminal peptide sequence that we have used
to prepare antibodies to suV is well conserved between C.
reinhardtii and Volvox, with 12 identities out of 15 residues, we
attempted to see whether the antipeptide would crossreact
with suV candidates from other photosynthetic eukaryotes. A
protein of similar apparent molecular weight as suV was indeed recognized in thylakoid membranes from spinach (Fig. 9).
This cross-reaction argues for the presence of suV in higher
plant cytb6f complexes.
FIG. 6. Lumenal localization of the N terminus of suV by immunogold labeling of thylakoid membrane vesicles. Inside-out
membrane vesicles were colabeled with an antibody against the N
terminus peptide of suV (large gold beads) and with an antiserum
against a lumenal segment of cytf (small gold beads) indicating that
suV has a transmembrane orientation.
DISCUSSION
A Transmembrane Protein Associated with the cytb6f Complex—We first identified suV as a nucleus-encoded polypeptide
of apparent molecular mass of 19 kDa that was present in
cytb6f-enriched fractions and deficient in thylakoid membranes
of C. reinhardtii mutants lacking the cytb6f complex (21). We
therefore proposed that suV was a genuine subunit of the cytb6f
complex. This conclusion was subsequently challenged on the
basis that suV was not recovered in highly purified and active
cytb6f preparations (12, 36). Indeed, we show here that suV
dissociates from cytb6f complex during the purification steps.
Thus suV behaves as a loosely bound partner of the protein
complex and is not required for plastoquinol/plastocyanin oxidoreductase activity. However, it should be considered as a
Cytochrome b6f-associated Phosphoprotein
17077
TABLE II
Labeling relative densities in unstacked relative to stacked membrane
regions in immunocytomicroscopy
Thin sections of thylakoid membranes were immunolabeled using
protein A labeled with colloidal gold particles. Gold particles were
counted on unstacked and stacked membranes, and membrane lengths
were measured. Ratios of immunogold labeling densities in the unstacked over the stacked membrane regions (du/ds) are indicated.
Antigen
State I
State II
LHCII
PSII-CP47
cytf
suV
0.62
0.34
0.52
0.53
1.29
0.28
0.96
0.92
FIG. 8. Extraction of thylakoid membrane phosphoproteins in
state II and suV immunoprecipitation. A, autoradiogram (left) and
immunodetection of suV (right) of thylakoid membrane proteins from in
vivo 33P-labeled wild-type cells in state II (mb) and after 2 M KSCN
treatment, (P) pellet, (S) supernatant. B, autoradiogram (left) of thylakoid membrane proteins from in vivo 33P-labeled wild-type cells in state
II (mb) and after immunoprecipitation (immunopre.) using protein ASepharose without suV antiserum (⫺) as a control or with suV antiserum (⫹); immunodetection of suV (right).
FIG. 7. Reversible phosphorylation of suV upon state transitions. A, autoradiogram of thylakoid membrane proteins from in
vivo32P-labeled cells of wild-type (⫹b6f) and of a mutant lacking cytb6f
complex (⫺b6f). State I, oxidizing conditions; state II, reducing conditions. suV and labeled antennae (CP26, CP29, LHCP13, LHCP11,
LHCP17) and PSII subunits (PsbC, PsbD, PsbH) are indicated. B,
comparative immunodetection of suV and cytf in thylakoid membranes
in state I and in state II.
subunit of cytb6f complexes in situ, because (i) it is found in
association with the cytb6f complex during the first purification
steps, (ii) it follows the same lateral redistribution along the
stacked and unstacked thylakoid regions as cytf upon state
transition, and (iii) it cannot accumulate in a protease-resistant form in the membranes in the absence of the other cytb6f
subunits.
Analysis of the sequence of the PETO cDNA allowed us to
predict the topology of the polypeptide chain in thylakoid membranes. Because it displayed a typical bipartite transit sequence with an ANA motif before the N terminus of the mature
protein, we expected suV would have its N terminus domain
located on the lumen side of the membrane. Indeed, the N
terminus location of suV in the lumen was supported by proteolysis experiments and immunogold labeling. The presence of
a hydrophobic stretch of 17–19 residues suggested that suV
formed one transmembrane ␣-helix connecting two hydrophilic
domains of about 65 residues each. The hydrophobicity of the
transmembrane ␣-helix, 1.9 kcal/residue on the Goldman-Engelman-Steitz scale, is in the range of the other predicted
transmembrane ␣-helices of cytb6f subunits (17). The release of
suV from the membranes with chaotropic agents or in alkaline
pH, when the Rieske protein but not the rest of the cytb6f
complex is extracted, suggests a peripheral location of its transmembrane span with respect to the helix bundle of suIV and
FIG. 9. Enhanced chemiluminescence immunodetection (ECL)
of suV in spinach chloroplast (spi.) compared with that in C.
reinhardtii thylakoid membranes (chl.). Heme proteins, as the
secondary peroxidase antibody, can oxidize luminol and are also revealed by ECL.
cytb6. This distal position of the ␣-helix of suV within the cytb6f
complex is consistent with its progressive release during cytb6f
purification.
What is the Function of suV?—suV is required neither for
electron transfer nor for the dimerization of the isolated cytb6f
complex, because a Hecameg-based purification procedure
yields suV-depleted protein complexes that are fully active and
dimeric (12). However, we cannot exclude a role of suV in the
electron transfer in vivo or in the supramolecular organization
of the photosynthetic apparatus. For example, PufX has only
an indirect role in electron transfer in Rhodobacter sphaeroides: this small transmembrane polypeptide absence retards
quinone exchange between the reaction center and the bc1
complex probably due to the PufX role in the supramolecular
organization of the photosynthetic apparatus (37).
Searches of the GenBank showed no sequence homologies
between suV and proteins of known function, and no specific
motif speak for any obvious biochemical activity. Therefore, the
function of suV can only be speculated upon. There are neither
histidines nor cysteines in the sequence of the mature protein.
17078
Cytochrome b6f-associated Phosphoprotein
Thus suV has no heme-binding motif and cannot correspond to
cytG, a protein that interacts with cytb6 in green algae (38, 39).
The most conspicuous feature of suV is its phosphorylation in
state II. suV probably corresponds to a phosphoprotein of similar apparent molecular weight that has been detected in
higher plant thylakoids but not in cytb6f mutants (40 – 42).
Indeed, suV has a homologue in higher plants, as shown here
by the specific immunological cross-reaction we observed with
spinach thylakoids. Because the putative higher plant homologue is phosphorylated on threonine residues (43), we dismissed two putative serine phosphorylation sites in the stromal
domain of suV (indicated in Fig. 1 and conserved in Volvox):
S82KID, which matches the consensus site for the casein kinase
II phosphorylation site ((S/T)XX(D/E)), and S135KK, which
matches the consensus site for the protein kinase C phosphorylation site ((S/T)X(R/K)). The most likely residues for suV
phosphorylation are then two threonine phosphorylation sites
T109LK, T126KK, which both match consensus sites for protein
kinase C. These putative phosphorylation sites are located in
the C-terminal, stromal-exposed, domain in a sequence context
that differs from the other sites of protein phosphorylation
previously identified in the thylakoid membranes (44).
suV is the only known cytb6f-associated subunit that is reversibly phosphorylated upon state transitions in C. reinhardtii. It suggests that suV is a possible key partner of the
phosphorylation-mediated state transition process in photosynthetic eukaryotes. That the PETO gene is absent from cyanobacterial chromosomes is consistent with such a function
because cyanobacteria use a different, although presently
poorly understood, mechanism to perform state transitions
(45). State transitions in photosynthetic eukaryotes encompass
the redox-controlled activation of a kinase and the subsequent
redistribution of LHCII and cytb6f next to PSI, once LHCII
proteins are phosphorylated. We found no evidence that suV
could act as a kinase by itself, because there are no known
kinase motifs in its amino acid sequence. Moreover, the peaks
of kinase activity that are detected along the sucrose gradient
loaded with the cytb6f-enriched supernatant (5) do not match
suV distribution (Fig. 4). The available data rather suggest
that suV could play a role in the activation of the LHCII kinase.
There is a need for signal transduction upon kinase activation
by reduced plastoquinol: the redox sensor for kinase activation
is the Qo site of the cytb6f complex, which is located on the
lumen side of the membranes (6 –9), whereas the catalytic site
of the kinase is on the stromal face of the thylakoid membranes
where reside all of the target sites for protein phosphorylation.
The redox activation of the kinase via changes at the Qo site of
cytb6f complexes is most likely accompanied by significant conformational changes of the Rieske protein in the thylakoid
lumen (7–9). suV transmembrane topology with two large hydrophilic domains extending on both sides of the membrane has
the features of a typical signal-transducing protein. The extended N-terminal domain of suV in the lumen is well suited to
sense the structural changes of the Rieske protein. The single
transmembrane helix of suV, whose flexibility is supported by
the suV sensitivity to chaotropic agents, could transduce conformational changes to the phosphorylatable stromal C-terminal domain that interacts with the LHCII kinase. It would then
remain phosphorylated as long as the kinase is activated. The
need for some signal-transducing protein in thylakoid membranes also stems from the recent finding that an immunophilin-like lumenal membrane protein (46) regulates the stromal
activity of a membrane-bound phosphatase (47). The transducer protein has not yet been identified.
Redox sensing at the plastoquinone pool level has also been
advocated for long term adaptation of the photosynthesis ap-
paratus involving changes in the stoichiometry of the reaction
centers and/or antenna proteins (48). In particular, the ratio of
PSII to PSI centers has been proposed to be controlled by the
plastoquinone pool redox state (49, 50). cytb6f is also thought to
be the sensor for the up-regulation of the nuclear chlorophyll
a/b binding protein genes in oxidizing conditions (51, 52). The
cytb6f-associated suV is a reasonable candidate for these various signal transduction processes that control gene expression.
In particular, its stromal C-terminal domain is highly basic and
might interact with negatively charged mRNAs or proteins. A
search for mutants showing altered expression of the PETO
gene products should provide an answer as to the possible role
of suV in signal transduction.
Acknowledgments—We thank D. Bernard for immunodetection experiments, D. Drapier for RNA analysis, L.-G. Franzén for the cDNA
library, T. Kallas for comments on the manuscript, J.-L. Popot for his
interest and support during the early phase of the project, and O. Vallon
for anti-cytf monoclonal antibody. We are greatly indebted to S. Merchant (UCLA) for her financial support to P. H.
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