Plant Physiol. (1998) 117: 515-524
Mutation of Residue Threonine-2 of the D2 Polypeptide and Its
Effect on Photosystem II Function in
Chlamydomonas
reinhardtii1
Christos Andronis2, *,
Olaf Kruse3,
Zsuzsanna Deák,
Imre Vass,
Bruce A. Diner, and
Peter J. Nixon
Department of Biochemistry, Imperial College of Science, Technology
and Medicine, London SW7 2AY, United Kingdom (C.A., O.K., P.J.N.); Institute of Plant Biology, Biological Research Centre, Hungarian
Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary (Z.D., I.V.); and Central Research and Development Department, Experimental Station,
P.O. Box 80173, E.I. du Pont de Nemours & Co., Wilmington, Delaware
80173-0173 (B.A.D.)
 |
ABSTRACT |
The
D2 polypeptide of the photosystem II (PSII) complex in the green alga
Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site
is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which
residue Thr-2 has been replaced by either alanine or serine. Both
mutants grew photoautotrophically at wild-type rates, and noninvasive
biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and
rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The
susceptibility of mutant PSII to photoinactivation as
measured by the light-induced loss of PSII activity in whole cells in
the presence of the protein-synthesis inhibitors chloramphenicol or
lincomycin was similar to that of wild type. These results indicate
that phosphorylation at Thr-2 is not required for PSII function or for
protection from photoinactivation. In control experiments the
phosphorylation of D2 in wild-type C. reinhardtii was
examined by 32P labeling in vivo and in vitro. No evidence
for the phosphorylation of D2 in the wild type could be obtained.
[14C]Acetate-labeling experiments in the presence of an
inhibitor of cytoplasmic protein synthesis also failed to identify
phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results
suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.
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INTRODUCTION |
The existence of phosphoproteins in thylakoid membranes from
higher plants was first reported by Bennett (1977)
, who described a
number of polypeptides from pea (Pisum sativum) that were
reversibly phosphorylated in the presence of
32Pi. The most conspicuously phosphorylated bands
belong to LHCII (Bennett, 1977). In addition to LHCII it is
now known that four PSII proteins, the PsbH polypeptide, CP43, D1, and
D2, are also reversibly phosphorylated in pea and spinach
(Spinacia oleracea) (Ikeuchi et al., 1987a; Michel and
Bennett, 1987; Telfer et al., 1987; Michel et al., 1988; Elich et al.,
1992). For both the D1 and D2 proteins, the site of phosphorylation has
been identified using tandem MS as the N-terminal Thr at position 2 (Michel et al., 1988).
Phosphorylation of the D1 and D2 subunits does not appear to be
ubiquitous. In cyanobacteria neither D1 nor D2 are phosphorylated, and
in the green alga Chlamydomonas reinhardtii only the D2
protein appears to be phosphorylated (Delepelaire, 1984). The analogous amino acid residue to D2 Thr-2 of higher plants is also a Thr residue
(Erickson et al., 1986). Phosphorylation of D2 in C. reinhardtii was first suggested by Delepelaire (1984) after
carrying out pulse-chase-labeling experiments with
[14C]acetate in the presence of
protein-synthesis inhibitors. After electrophoresis (using a urea-SDS
gel system) and autoradiography, Delepelaire observed a doublet that he
attributed to the phosphorylated and nonphosphorylated forms of D2
(D2.1 and D2.2, respectively). Based largely on the work of
Delepelaire, various reports have appeared in the literature assigning
a band with an apparent molecular mass of approximately 34 kD, labeled
with 32P both in vivo and in vitro, and migrating
between polypeptides 9 and 10 and 11 and 13 of the LHCII complex as the
phosphorylated form of D2 (D2.1) (Delepelaire and Wollman, 1985; de
Vitry et al., 1987, 1991; Ikeuchi et al., 1987b; de Vitry and Wollman, 1988). However, conclusive evidence indicating phosphorylation of D2
(e.g. by immunoprecipitation or amino acid sequencing) has not appeared
in the literature to date.
It is now well established that LHCII phosphorylation is involved in
the regulation of excitation energy distribution between PSI and PSII
(Bennett et al., 1980; Allen et al., 1981; Bennett, 1991; Allen, 1992,
1995). In contrast to our knowledge concerning LHCII phosphorylation,
very little is known about the role of phosphorylation of the PSII core
proteins. Possible roles that have been suggested include the spatial
separation of the two populations of PSII centers found in grana and
stroma lamellae (PSII-
and PSII-
) (Mattoo et al., 1989), a role
in biogenesis and assembly of PSII (Owens and Ohad, 1983; de Vitry et
al., 1989; Summer et al., 1997), regulation of D1 and D2 degradation
during photoinhibition (Elich et al., 1992; Aro et al., 1992, 1993;
Giardi, 1993; Rintamaki et al., 1995, 1996), and stabilization of
dimeric PSII complexes (Kruse et al., 1997).
In this paper we describe the characterization of two mutants of
C. reinhardtii that were constructed to investigate the role of D2 phosphorylation based on the reasonable assumption that the
D2-phosphorylation site in C. reinhardtii, like that in
higher plants, is at residue Thr-2 of D2. Mutant D2 Thr2Ala lacks the hydroxyl group required for attachment of the phosphate group and
should provide information on the function of D2 phosphorylation. Mutant D2 Thr2Ser, on the other hand, contains a hydroxyl group and was
constructed to investigate the substrate specificity of the kinase.
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MATERIALS AND METHODS |
Strains and Growth Conditions
Chlamydomonas reinhardtii strain CC125 (mating type +)
was obtained from the Chlamydomonas Genetic Center (Department of
Botany, Duke University, Durham, NC). This strain was used as the host for chloroplast-transformation experiments and as the reference wild-type strain. C. reinhardtii wild-type and mutant
strains were grown in TAP medium or HSM and handled as described by
Harris (1989). Solid media were supplemented with spectinomycin (Sigma) at 100 µg mL
1, ampicillin (Sigma) at 50 µg
mL1, and 10 µM DCMU (British
Greyhound, Birkenhead, UK). The addition of DCMU at 10 µM
should inhibit PSII activity and prevent competition from wild-type
copies of the psbD gene and from PSII revertants. Liquid
cultures were grown at 25 to 30°C in an orbital shaker/incubator, either at an incident light intensity of 30 to 50 µmol
m2 s1 or in the dark.
Recombinant Plasmids and in Vitro Mutagenesis
Plasmid pH3 (Erickson et al., 1986) was digested with
HindIII to yield a 7.5-kb fragment containing the
psbD gene, and was then partially digested with
HpaI to give a 4.7-kb HpaI/HindIII fragment. The plasmid vector pTZ19U (Bio-Rad) was digested with HindIII and SmaI and ligated with the 4.7-kb
HpaI/HindIII fragment to yield the plasmid pCA1.
To select for transformed C. reinhardtii cells the
aadA-selectable marker (Goldschmidt-Clermont, 1991), which
confers resistance to spectinomycin and streptomycin, was inserted into
pCA1 into a NsiI restriction site 261 bp upstream of the
translation initiation codon of psbD. Plasmid pUC-atpX-AAD, which contains the aadA-selectable marker, was digested with
EcoRV and SmaI to yield a 1.9-kb blunt-end
fragment containing the spectinomycin-resistance cassette. pCA1 was
digested with NsiI. Ends were blunt-ended with T4 DNA
polymerase and then ligated with the 1.9-kb
EcoRV/SmaI fragment, creating plasmid pNsi16.
Site-directed mutagenesis was carried out as described by Kunkel et al.
(1987). Single-stranded DNA was prepared from pNsi16 and was annealed
to the synthetic oligonucleotides
5
-ATACACGCAATGGCTATTGCGATCGGT-3 and
5-ATACACGCAATGTCTATTGCGATCCGT-3 to create mutants Thr2Ala and Thr2Ser, respectively. Both mutagenic oligonucleotides were designed to delete a restriction site for the enzyme MunI
along with the Thr-2 mutation (Fig. 1).
Clones exhibiting a restriction pattern indicative of the presence of
the desired mutation were subjected to sequence analysis using a
double-stranded DNA-sequencing protocol (Sequenase 2.0, United States
Biochemical). The whole psbD gene was sequenced in these
clones to ensure that unwanted sequence alterations were not present in
the gene and, subsequently, that the generated mutant plasmids were
transformed into the chloroplast genome of C. reinhardtii.
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| Figure 1.
Restriction map of a 7.5-kb HindIII
fragment from the chloroplast genome of C. reinhardtii
showing the mutations made to the psbD gene. The
positions and orientations of psbD,
psaA-2, and the spectinomycin-resistance cassette
(specR) are indicated. Restriction sites are
HindIII (H), EcoRI (R), NsiI (N), HpaI (Hp), and
MunI (M). The MunI site eliminated along with the creation of the Thr-2 mutations is shown in bold.
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Chloroplast Transformation
Plasmid DNA precipitation onto M10 tungsten particles (Bio-Rad)
and chloroplast transformation were performed essentially as described
by Boynton and Gillham (1993) and Sanford et al. (1993). The
particle-delivery instrument used was the gunpowder-driven model Mk2
from Shearline (Cambridge, UK).
Genetic Analysis of Site-Directed Mutants
Total cellular DNA was isolated using a slight modification of the
procedure described by Newman et al. (1990). Instead of precipitating
the nucleic acids after phenol/chloroform extraction, the top aqueous
phase was mixed with 1 mL of resin (Wizard, Promega), and the DNA was
recovered using a miniprep DNA-purification system (Wizard, Promega).
ctDNA was prepared according to the method of Roffey et al. (1991).
Southern-blot hybridizations were performed following standard
protocols. Sequencing of algal DNA was performed after
amplification of miniprep DNA with primers C10
(5-ctcggatccAAATACACAATGATTAAAAT-3) and C70
(5-tggaagcttAAAAATATATTATAGAGCGT-3), which incorporate into the PCR
product restriction sites for the enzymes BamHI and HindIII, respectively. The amplified DNA was digested with
BamHI and HindIII and then cloned into the
corresponding restriction sites of a pBluescript KS vector
(Stratagene). The altered nucleotides in the Thr2Ala and Thr2Ser
mutants were identified using primer C8 (5-CAAGGAATAGTAATAAACC-3).
Phenotypic Analysis of Site-Directed Mutants
The growth pattern of transformants was studied by growing liquid
cultures photoautotrophically in 100 mL of HSM at 25°C and at an
incident light intensity of 50 to 70 µmol m2
s1. The cultures were bubbled with air and
stirred continuously. The optical density at 750 nm was monitored in a
spectrophotometer (model MPS-2000, Shimadzu, Kyoto, Japan) at regular
intervals until the cultures had reached the stationary phase.
Oxygen evolution of whole cells was measured with an oxygen
electrode (model DW, Hansatech, Kings Lynn, UK). The measurements were
performed in HSM at 25°C and at saturating light
intensities (4000-6000 µmol m2
s1).
Fluorescence and Thermoluminescence Measurements
For the measurements of the thermoluminescence characteristics in
whole cells of wild-type and transformant strains, cells were grown in
TAP medium at 25°C and at an incident light intensity of 10 µmol
m2 s1. Samples were
dark adapted for 3 min at 20°C in the absence or presence of 10 µM DCMU, and were then excited by a single saturating flash at 
10°C, followed by fast cooling to 40°C. The
warming-ramp speed for these measurements was 10°C/min. Fluorescence
induction was measured with a pulse-amplitude-modulated fluorometer
(PAM 101, Walz, Effeltrich, Germany) for up to 3 s in the absence
or presence of 10 µM DCMU. Measurements of the decay of
Fv were performed according to the method
of Whitelegge et al. (1995).
Photoinhibition Measurements
C. reinhardtii cells were grown with air bubbling and
stirring in HSM until their middle- or late-exponential phase. The
cultures were subjected to heat-filtered high-light illumination of
1000 µmol m2 s1
(provided by an apparatus equipped with a 1-kW halogen lamp). CAP at
200 µg/mL or lincomycin at 100 µg/mL (both from Sigma) was added to
some of the samples to inhibit chloroplast-protein synthesis. One- to
2-mL samples were taken at set intervals during a time course of 4 to
5 h, and light-saturated oxygen evolution was measured.
32P Labeling of Whole Cells and Thylakoid Membranes
32P labeling of C. reinhardtii
whole cells was performed according to the method of de Vitry et al.
(1991). Cells grown in TAP medium until the late-exponential phase were
incubated with 1 mCi of 32Pi for 16 h at
room temperature under low light. Thylakoid membranes were isolated
according to the method of Diner and Wollman (1980), except that all
buffers contained 20 mM NaF to inhibit phosphatase. PSII-RC
particles were prepared as described by Alizadeh et al. (1995). All
buffers until the stage of the column wash contained 20 mM
NaF.
In vitro 32P labeling of thylakoid membrane
proteins was carried out in 100 mM Suc, 50 mM
Hepes-KOH, pH 8.0, 10 mM MgCl2, 0.2 mM ATP, and 10 mM NaF at a chlorophyll
concentration of 0.2 mg mL1.
[
-32P]ATP (specific activity, approximately
3000 Ci mmol1; Amersham) at a concentration of
10 µCi for every 100 µg of chlorophyll was added and the
suspension was incubated for 15 min at 50 to 70 µmol
m2 s1 of heat-filtered
light at 25°C. The reaction was stopped by centrifugation for 10 min
in a microcentrifuge.
Pulse-Chase Labeling of Whole Cells Using
[14C]Acetate
The following protocol was based on the procedure of Kuras and
Wollman (1994). Cells grown in TAP medium to an optical density (750 nm) of 0.2 to 0.5 were harvested, washed twice in HSM, and resuspended
in HSM to a chlorophyll concentration of 25 µg
mL1. Cells were depleted of acetate by
incubating them at 25°C in HSM for 1 h at an incident light
intensity of 100 µmol m2
s1. Ten minutes before the addition of the
label, the cytoplasmic protein-synthesis inhibitor cycloheximide was
added at a concentration of 10 µg mL1.
[1-14C]Sodium acetate (specific activity, 56 mCi/mmol; Amersham) was then added at 5 µCi
mL1, and the incubation was continued for 5 min. For the chase, the labeled cells were washed twice in HSM
containing 25 mM cold sodium acetate to deplete them of the
radioactive label, and then resuspended in the same medium plus 10 µg
mL1 cycloheximide. The cell suspension was then
incubated under the same conditions (100 µmol
m2 s1 at 25°C) for an
additional 90 min. Ten-milliliter aliquots were withdrawn at 0, 45, and
90 min, and thylakoid membranes were isolated for SDS-PAGE and
autoradiography.
SDS-PAGE and Immunoblotting
SDS-PAGE was carried out according to the method of Hankamer et
al. (1997). Before loading, protein samples were solubilized in the
dark by the addition of an equal volume of solubilization buffer (187 mM Tris, pH 6.8, 30% glycerol, 9% SDS, 15%
-mercaptoethanol). Proteins from whole cells were solubilized by
boiling the cell suspension (at a chlorophyll concentration of 0.5 µg/µL) for 1 min in an equal volume of solubilization buffer.
Pigmented samples were loaded on the gel on an equal-chlorophyll basis.
Protein samples resolved by SDS-PAGE were transferred to a
nitrocellulose membrane according to the method of Dunn (1986).
Immunodecoration of protein was achieved by incubating membranes with
one of the following antisera: a D2 antibody raised against a synthetic
peptide corresponding to the last 12 amino acids of PsbD (Nixon et al., 1990), a spinach LHCII antibody (Hilditch, 1986), or a monospecific antibody to phospho-Thr (Sigma). The specificity of the latter was
tested by adding phospho-Thr or phospho-Tyr (Sigma) during incubation
with the primary antibody. Cross-reacting proteins were detected using
an anti-rabbit IgG-alkaline phosphatase conjugate (Sigma) and
5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and
nitroblue tetrazolium chloride (Sigma).
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RESULTS |
Construction of D2 Thr2Ala and D2 Thr2Ser Mutants
The mutants D2 Thr2Ala and D2 Thr2Ser were constructed in C. reinhardtii using the biolistic technique pioneered by Boynton and
co-workers (1988). To select for transformants, a
spectinomycin-resistance cassette was inserted upstream of the
psbD gene (Fig. 1). After bombardment of wild-type cells
with a plasmid carrying the mutant psbD gene, the
site-directed mutation was incorporated into the psbD gene
on the chloroplast genome through homologous recombination. Figure
2 shows a DNA gel blot of ctDNA isolated
from mutants Thr2Ser and Thr2Ala, the wild-type control strain Nsi16,
which contains the spectinomycin-resistance cassette upstream of a
wild-type copy of psbD, and the wild type. Figure 2 confirms
that the transformants were homoplasmic.
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| Figure 2.
Southern-blot analysis of the wild type (lanes 1 and 5), Nsi16 (lanes 2 and 6), and mutants Thr2Ser (lanes 3 and 7) and
Thr2Ala (lanes 4 and 8). ctDNA was digested with HindIII
(lanes 1-4) and MunI (lanes 5-8), and hybridized to a
0.6-kb psbD probe.
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The 7.5-kb HindIII fragment in the wild type, which contains
psbD, is replaced by a 3.8-kb fragment in the transformants
(Fig. 2, lanes 2-4). The presence of the aadA cassette in
the transformants was confirmed in DNA gel blots by probing with an
aadA-specific probe (data not shown). In addition, mutants
Thr2Ser and Thr2Ala have lost a MunI site because of their
mutations, so a 0.4-kb MunI fragment found in the wild type
and in Nsi16 is replaced by a 2.8-kb fragment in the mutants (Fig. 2,
lanes 7 and 8). The psbD gene in the transformants was
recloned and sequenced to confirm the existence of the desired
mutations at Thr-2. DNA sequencing also confirmed that no other
spontaneous mutations were incorporated into the psbD gene
during construction of the mutants (data not shown).
Biophysical Characterization of the Thr-2 Mutants
The photoautotrophic growth rates of the wild type, Nsi16, and the
Thr-2 mutants in HSM were indistinguishable, with a doubling time of
approximately 10 h at a light intensity of 50 to 70 µmol m2 s1 (data not shown).
Table I confirms that PSII activity,
assayed by the rate of light-saturated oxygen evolution in the presence of the artificial electron acceptors ferricyanide and DCBQ, was largely
unaffected in the mutants, although a slight reduction was observed in
Thr2Ala. A number of noninvasive techniques were therefore used to
determine if substitution of the Thr-2 residue had subtle effects on
the electron-transfer reactions within PSII. The fluorescence-induction
characteristics of the mutants were similar to those of the wild type,
suggesting little perturbation to photosynthetic electron flow (data
not shown). The
Fv/Fm value in
the presence of DCMU was approximately 0.69 for both the wild type and
the mutants. The
Fv/Fm ratio is
a measure of the efficiency of light capture by PSII (Krause and Weis,
1991).
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Table I.
Phenotypic characteristics of the D2-Thr2 mutants
Measurements were carried out in HSM in the presence of 1 mM potassium ferricyanide and 1 mM DCBQ. Data
presented are means of four independent experiments.
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Thermoluminescence measurements confirmed that the peak positions of
the Q and B bands were also unaffected in the mutants (Table I). The Q
and B bands are associated with charge recombination between
S2QA and
S2QB,
respectively, with the peak position indicative of the degree of charge
stabilization (Vass et al., 1981).
The rate of decay of chlorophyll fluorescence from whole cells after a
saturating flash of light is indicative of the rate of electron
transfer between QA and QB
(Whitelegge et al., 1995). From the fluorescence-decay measurements
shown in Figure 3A, recorded after each
of five saturating flashes spaced 0.6 s apart, the rate of
electron transfer between QA and
QB appears similar in the wild type and the
mutants. Figure 3A also shows that the
QA-to-QB electron transfer
is faster on the odd flashes than on the even. Such an oscillation of
period two is consistent with the normal operation of electron transfer
on the acceptor side of PSII (Robinson and Crofts, 1983). Figure 3B
shows the decay of chlorophyll fluorescence in intact cells in the
presence of DCMU, an herbicide that blocks electron transfer between
QA and QB. After a single
saturating flash, the decay in fluorescence is now a measure of the
rate of charge recombination between the S2 state
of the water-oxidizing complex and
QA. No differences were
observed between the different strains. These analyses therefore
suggested that electron transfer within PSII and the redox properties
of the various cofactors are largely unaffected in the Thr-2 mutants.
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| Figure 3.
Relaxation of Fv in the
Thr-2 mutants. A, Relaxation of the Fv after
each of a series (1.67 Hz) of five saturating 2-µs flashes in whole
cells of the C. reinhardtii mutants Thr2Ala and Thr2Ser. Shown is the 5-ms range after each flash starting at 50 µs. The 50-µs values of Fv are normalized to
Fo [(F Fo)/Fo]. B,
Relaxation of Fv resulting from charge
recombination between QA and the PSII donor
side after a single, saturating 2-µs flash excitation of whole cells
of C. reinhardtii. Wild type ( ) is compared with
transformant Nsi16 (×) and with the mutants Thr2Ala ( )
and Thr2Ser ( ).
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Photoinhibition of the Thr-2 Mutants
To determine whether the Thr-2 residue played a role in protecting
PSII from photoinactivation, cells of the wild type and the Thr-2
mutants were exposed to a high-light irradiance (1000 µmol
m2 s1), and PSII
activity was monitored as a function of time. PSII activity in both the
wild type and Thr2Ala, measured as the rate of light-saturated oxygen
evolution in the presence of the artificial electron acceptors DCBQ (1 mM) and ferricyanide (1 mM), was stable over
the time period of the experiment (data not shown). When chloroplast-protein synthesis was blocked by the addition of CAP to the
medium, the loss of PSII activity could be used to monitor the rate of
photoinactivation of PSII, since repair of PSII by de novo protein
synthesis was unable to proceed. Both the wild type and Thr2Ala showed
similar rates of photoinactivation (t1/2 approximately 80 min; Fig. 4), indicating
that the wild-type and mutant PSII complexes were similarly susceptible
to photodamage. Photoinactivation of PSII activity in the Thr2Ser
mutant was slightly faster (t1/2
approximately 50-60 min; Fig. 4). Similar results were obtained using
lincomycin instead of CAP to inhibit chloroplast-protein synthesis
(data not shown). Immunoblotting experiments confirmed that the
steady-state levels of D2 in photoinhibited cells were similar between
the wild type and Thr2Ala (data not shown).
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| Figure 4.
Photoinhibition measurements. Effect of high-light
treatment (1000 µmol m2 s1) on the
light-saturated rate of oxygen evolution from whole cells of the wild
type, Nsi16, and the mutants Thr2Ala and Thr2Ser in the presence of 200 µg/mL CAP. Cells were suspended in HSM at 25°C to a chlorophyll
concentration of 25 µg/mL. Oxygen evolution was measured using 1 mM DCBQ and 1 mM potassium ferricyanide. The
100% rates of oxygen evolution were approximately 251 µmol O2 mg1 chlorophyll h1 for the
wild type (), 356 µmol O2 mg1
chlorophyll h1 for Nsi16 (×), 222 µmol
O2 mg1 chlorophyll h1 for
Thr2Ala ( ), and 333 µmol O2 mg1
chlorophyll h1 for Thr2Ser ().
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In Vitro-Phosphorylation Experiments
The lack of any phenotypic difference between the wild type and
the Thr-2 mutants led us to reinvestigate the assignment of D2 as a
phosphorylated protein in C. reinhardtii. In
vitro-phosphorylation experiments using
[32P]ATP were performed to determine if a
phosphorylated form of D2 could be detected. The experimental
conditions were similar to those that had previously been shown to give
rise to a labeled band, which was assigned to phosphorylated D2
(Wollman and Delepelaire, 1984; Delepelaire and Wollman, 1985). The
labeling reaction contained 10 mM NaF to inhibit
phosphatases and approximately 0.2 mM ATP at a specific
activity of approximately 3000 Ci mmol1.
Preliminary experiments in the wild type showed that the best labeling
of thylakoid membrane proteins occurred using thylakoids isolated from
cultures grown in the dark and labeled in the light (Fig.
5A). Light stimulates the labeling of all
of the phosphorylated polypeptides and, in agreement with a study by
Wollman and Delepelaire (1984), some phosphorylation occurred in the
absence of light.
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| Figure 5.
In vitro labeling of wild-type thylakoid membranes
with [-32P]ATP. A, Autoradiogram of a 12%, 6 M urea-SDS gel. B and C, Immunoblotting with -LHCII (B)
and -D2 (C) antibodies. Lanes 1, Cells grown in the light and
thylakoids labeled in the light; lanes 2, cells grown in the
light and thylakoids labeled in the dark; lanes 3, cells grown in the
dark and thylakoids labeled in the light; and lanes 4, cells grown in
the dark and thylakoids labeled in the dark. Arrows indicate positions
of main LHCII bands picked up by the -LHCII antibody. The triangle
represents the position of CP43. Dots indicate unidentified
phosphoproteins. Low-molecular-mass polypeptides have been run off the
bottom of the gel. Numbers next to the -D2 blot represent the bands
of the molecular-mass marker in kilodaltons (prestained LMW,
Bio-Rad).
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In broad detail the pattern of phosphorylation was similar to
previously published autoradiograms (Owens and Ohad, 1982; Wollman and
Delepelaire, 1984; Delepelaire and Wollman, 1985; Gans and Wollman,
1995). Immunoblotting experiments using antisera specific for
higher-plant LHCII and D2 indicated that all of the labeled polypeptides could be assigned to LHCII components (Fig. 5, B and C).
The uppermost, heavily labeled band can be assigned to CP29 (Bassi and
Wollman, 1991; Allen and Staehelin, 1994). The labeled band migrating
below CP29 is probably an LHCII polypeptide, number 11 or 13 (Wollman
and Delepelaire, 1984; Allen and Staehelin, 1994). This band migrated
in proximity to the upper part of D2 (Fig. 5, compare B and C).
Therefore, its identification as the phosphorylated form of D2 cannot
be completely excluded. The lightly labeled band (designated with a
triangle) in Figure 5A is most probably the phosphorylated form of
CP43.
The profile obtained using thylakoids isolated from the Thr2Ala mutant
was very similar to the one obtained with wild-type membranes (Fig.
6A). However, immunoblotting using an
-D2 antibody, shown in Figure 6B, reveals differences between
wild-type and Thr2Ala samples. The Thr2Ala samples appear to lack the
upper part of the immunodecorated D2 band seen in the wild type,
regardless of the growth or labeling conditions. This part of D2 does
not correspond to any labeled band in the autoradiogram and therefore cannot be attributed to the phosphorylated form of D2. In the experiment shown in Figure 6B, the reduced level of D2 in the Thr2Ala
thylakoids may reflect fewer PSII centers compared with the wild type.
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| Figure 6.
In vitro labeling of wild-type (lanes 1-4) and
Thr2Ala (lanes 5-8) thylakoid membranes with
[-32P]ATP. A, Autoradiogram of a 12%, 6 M
urea-SDS gel. B, Immunoblotting with -D2 antibody. Lanes 1 and 5, Cells grown in the light and thylakoids labeled in the light; lanes 2 and 6, cells grown in the light and thylakoids labeled in the dark;
lanes 3 and 7, cells grown in the dark and thylakoids labeled in the
light; lanes 4 and 8, cells grown in the dark and thylakoids labeled in
the dark. Arrows indicate positions of main LHCII bands picked up by
the -LHCII antibody as shown in Figure 5. The triangle represents the position of CP43. Dots indicate unidentified phosphoproteins. Numbers next to the -D2 blot represent the bands of the
molecular-mass marker in kilodaltons (prestained LMW, Bio-Rad).
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In Vivo Phosphorylation Experiments
One of the main lines of evidence supporting D2 phosphorylation in
C. reinhardtii comes from the work of de Vitry and co-workers (1987, 1991), who used the double mutant F54-14 (strain CC2655) in in vivo 32P-labeling experiments.
After labeling whole cells with 32Pi, these
investigators isolated PSII-containing particles, which they then
examined by SDS-PAGE and autoradiography. The upper part of a protein
band, assigned to D2 (D2.1), comigrated with a phosphorylated band.
However, LHCII contamination within the core complex makes it difficult
to rule out LHCII as the cause of the phosphorylated band. Recently the
PSI ATPase strain used
by de Vitry and co-workers was used for the preparation of a PSII RC
containing D1, D2, Cyt b559, and PsbI
(Alizadeh et al., 1995). Therefore, by labeling thylakoid membrane
proteins with 32Pi and subsequently isolating
PSII RCs, one should be able to detect phosphorylated D2 in a
preparation containing minimal LHCII. Analysis of phosphorylated PSII
RCs has been previously used successfully to show that D1 and D2 are
both phosphorylated in vitro in pea (Pisum sativum) (Telfer
et al., 1987).
Figure 7 shows the SDS-PAGE analysis and
autoradiogram of thylakoid proteins and a PSII-RC preparation isolated
from C. reinhardtii strain CC2655 after in vivo
phosphorylation. Because of the small amounts involved, the PSII-RC
preparation from C. reinhardtii contained some protein
contamination not seen in the pea PSII RC. The labeling pattern of the
thylakoid membranes was similar to that observed after in vitro
labeling of wild-type thylakoids (Fig. 5). The four labeled bands with
apparent molecular masses between 25 and 35 kD (Fig. 7B) were similar
in size to corresponding in vivo-labeled phosphoproteins reported in
the literature using either wild-type cells (Delepelaire and Wollman,
1985; Gans and Wollman, 1995) or a PSI mutant
of C. reinhardtii (Delosme et al., 1996). These bands are
probably the LHC polypeptides designated in C. reinhardtii as 10, 9, 13/11, and 17 (in descending order of molecular mass, starting from approximately 35 kD). No phosphorylation of D2 or any
other PSII subunit was detected in the PSII-RC preparation (Fig. 7B,
lane 2).
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| Figure 7.
In vivo labeling of C. reinhardtii
CC2655 cells with 32Pi using 10 to 17% Coomassie
blue-stained, 6 M urea-SDS gel (A) and autoradiography (B).
Lane M, Molecular-mass marker in kilodaltons (prestained LMW, Bio-Rad);
lanes 1, thylakoid membranes; lanes 2, isolated PSII RCs; lane 3, PSII
RCs from pea. The autoradiogram was obtained by exposing the gel to a
Phosphor Imager screen (Molecular Dynamics) for 7 d. The amount of
chlorophyll was 5 µg for the CC2655 thylakoids, 1 µg for the CC2655
PSII RCs, and 0.2 µg for pea PSII RCs.
|
|
Anti-phospho-Thr antibodies were used to probe PSII-RC preparations
isolated from pea and C. reinhardtii. Although the signals were weak, only D1 and D2 in the higher-plant preparation gave a
cross-reaction (data not shown).
In Vivo [14C]Acetate-Labeling Experiments
Cells of the wild type, the control strain Nsi16, and the mutants
Thr2Ala and Thr2Ser were radiolabeled with
[14C]acetate as described by Kuras and Wollman
(1994) and analyzed by urea-SDS-PAGE under conditions that have been
reported to resolve D2 into the nonphosphorylated (D2.2) and
phosphorylated (D2.1) forms. As shown in Figure
8, both the immunodetectable D2 band and
the radiolabeled D2 band migrated as diffuse bands.
View larger version (91K):
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| Figure 8.
Pulse-chase labeling of whole cells with
[14C]acetate. Autoradiogram (A) and -D2 immunoblot of
a 14%, 8 M urea-SDS gel (B) of thylakoid membranes
isolated from wild-type, Nsi16, Thr2Ala, and Thr2Ser cells pulse
labeled for 5 min with [14C]acetate at an incident light
intensity of 100 µmol m2 s1 and then
chased for 45 and 90 min in the absence of label. Both pulse and chase
were carried out in the presence of 10 µg/mL cycloheximide.
|
|
| |
DISCUSSION |
Our results show that the replacement of Thr-2 of the D2 protein
with either Ala or Ser does not have any dramatic effect on PSII
function. Photoautotrophic growth rates, fluorescence decay,
fluorescence-induction measurements, and thermoluminescence measurements all indicate that the Thr2Ala and Thr2Ser mutants behave
similarly to the wild type. Wild-type levels of steady-state oxygen
evolution were also obtained for the Thr2Ser mutant, whereas the
Thr2Ala mutant exhibited rates of oxygen evolution around 55 to 100%
of those of the wild type, possibly because of the variability of PSII
content in different cultures.
It was speculated by Michel et al. (1988) that phosphorylation of the N
terminus of D1 or D2 in higher plants may lead to an interaction
between the phosphoryl group and the nonheme iron atom located between
the QA- and QB-binding
sites. Such an interaction may provide pH buffering, which would affect
protonation of D1 and D2 and rates of
QA-to-QB electron transfer
(Michel et al., 1988). However, no significant perturbation to electron
transfer between QA and QB
was detected in the Thr-2 mutants described here (Fig. 3A).
One of the aims of the construction of the two Thr mutants was to study
the relationship between the phosphorylation of PSII proteins and
photoinhibition (for review, see Allen, 1992). Our results indicate
that Thr-2 of D2 does not have a crucial role either in protecting PSII
from photoinactivation or in regulating the degradation of the D2
polypeptide (data not shown). Therefore, phosphorylation of Thr-2, if
it exists, plays only a minor role in these processes.
Attempts to interpret the results obtained from the phenotypic analysis
of the mutants have been based on the assumption that D2 can be
phosphorylated in wild-type C. reinhardtii. The evidence for
D2 phosphorylation in C. reinhardtii in the literature
is based on autoradiograms of 14C- and
32P-labeled cells and thylakoid membranes
(Delepelaire and Wollman, 1985; de Vitry et al., 1987, 1991; de Vitry
and Wollman, 1988), which were not accompanied by immunoblots to
identify D2. In an effort to obtain satisfactory data concerning the
state of D2 phosphorylation in the wild type and in mutant strains,
different approaches were followed, including in vitro and in vivo
32P labeling of thylakoid membrane proteins,
identification of labeled bands using a monoclonal antibody against
phospho-Thr, and pulse-chase labeling of whole cells with
[14C]acetate.
The in vitro-phosphorylation pattern of thylakoid membrane polypeptides
from the wild type was similar to previous results (Owens and Ohad,
1982; Wollman and Delepelaire, 1984; Delepelaire and Wollman, 1985;
Gans and Wollman, 1995). However, all of the radiolabeled bands could
be assigned to LHC polypeptides using a specific antibody. This is
particularly relevant to the assignment of the radiolabeled band that
migrates close to the upper part of D2, since this band has most
commonly been attributed to phosphorylated D2 in previous studies
(Lemaire et al., 1987; de Vitry and Wollman, 1988; de Vitry et al.,
1991; Gans and Wollman, 1995).
The profile obtained in the in vitro-phosphorylation experiments for
the Thr2Ala mutant was similar to that for the wild type. At first
sight this would indicate that D2 is not phosphorylated in C. reinhardtii. However, other possible explanations are that (a) D2
can still be phosphorylated in the Thr2Ala mutant, possibly at
alternative Thr residues such as those present at positions 7 and 13 (Erickson et al., 1986); (b) phosphorylated D2 migrates as a very
diffuse band (Ikeuchi et al., 1987b), making identification difficult;
(c) D2 cannot be labeled in the wild type in in vitro-labeling experiments because D2 is already phosphorylated or because the kinase
has been inactivated; or (d) D2 is readily dephosphorylated by
phosphatases during sample preparation. Although the latter three
explanations cannot be ruled out, the experimental conditions used were
identical to conditions in other studies that assigned a phosphorylated
D2 band.
Immunoblotting with D2-specific antibodies revealed that the upper part
of the D2 band is apparently missing in the Thr2Ala mutant (Fig. 6).
Although suggestive of the absence of the slower-migrating, phosphorylated form of D2 in this mutant, it is possible that the
Thr-to-Ala change alters the mobility of the protein on SDS-PAGE, as in
the case of the Pro161Leu mutation of D2 in Synechocystis PCC 6803 (Tommos et al., 1993).
A more revealing experiment regarding D2 phosphorylation came from the
in vivo labeling of whole cells with 32Pi and the
subsequent isolation of PSII RCs. The labeling pattern obtained for the
thylakoid membranes was quite similar to the in vitro-labeling profile.
No labeled band could be observed in the PSII RC, implying that none of
the PSII-RC proteins in C. reinhardtii was phosphorylated.
Immunoblots using a monoclonal antibody specific for phospho-Thr also
suggested that D1 and D2 were unphosphorylated in the PSII-RC sample.
Again, these results cannot be interpreted unambiguously, for the
reasons outlined above.
Attempts to identify the two bands that constitute the D2 doublet (D2.1
and D2.2) (Delepelaire, 1984) were also carried out by pulse labeling
with [14C]acetate and chasing in the absence of
radioactive label. In these experiments, D2 always appeared as one
diffuse band. Some broadening of the immunodetected D2 band during the
chase is ascribed to D2 oxidation upon exposure to high light, as has
been observed in in vitro systems (e.g. Ponticos et al., 1993). It
should also be noted that in higher plants phosphorylated and
nonphosphorylated D2 cannot be separated by SDS-PAGE (Telfer et al.,
1987).
In summary, replacement of Thr-2 of D2 with either Ala or Ser did not
result in any dramatic changes in PSII function. We cannot, however,
conclude that phosphorylation of D2 is not important for PSII function,
because our data did not provide us with conclusive evidence concerning
D2 phosphorylation in either the wild type or the mutant strains. Most
of the labeled bands observed after in vitro and in vivo labeling with
32P could be attributed to polypeptides belonging
to the LHCII complex or to minor antenna polypeptides (CP29). We must
therefore seriously consider the possibility that the band that has
been reported in the literature as the phosphorylated form of D2
(designated as D2.1) is in fact a phosphorylated form of a LHCII
polypeptide. This possibility is particularly pertinent to the
assignment of 32P-labeled bands in PSII core
complexes that contain trace amounts of LHC polypeptides (de Vitry et
al., 1987, 1991). Therefore, final confirmation that D2 is indeed
phosphorylated in C. reinhardtii awaits the application of
more refined techniques such as MS.
| |
FOOTNOTES |
1
This research was supported by The Royal Society
(P.J.N.) and the Hungarian granting agency OTKA (nos. F017454 and
T017049). C.A. was supported by a fellowship from the State Scholarship Foundation of Greece.
2
Present address: Department of Molecular, Cell
and Developmental Biology, University of California, Los Angeles, CA
90095-1606.
3
Present address: Fakultaet Biologie,
Universität Bielefeld, 33501 Bielefeld, Germany.
*
Corresponding author; e-mail andronis{at}ucla.edu; fax
1-310-206-4386.
Received December 2, 1997;
accepted February 19, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CAP, chloramphenicol.
DCBQ, 1 mM
2,6-dichloro-p-benzoquinone.
Fm, maximal level of chlorophyll
fluorescence when all PSII centers are closed.
Fo, minimal level of chlorophyll
fluorescence when all PSII centers are open.
Fv, variable chlorophyll fluorescence
(Fm Fo).
HSM, high-salt minimal medium.
LHCII, light-harvesting antenna of PSII.
QA and QB, primary and secondary
electron-accepting plastoquinones of PSII.
RC, reaction center.
TAP, Tris-acetate phosphate.
| |
ACKNOWLEDGMENTS |
We thank Jean-David Rochaix (University of Geneva, Switzerland)
for plasmid pH3, Michel Goldschmidt-Clermont (University of Geneva) for
plasmid pUC-atpX-aadA, and Howard Thomas (Institute of Grassland and
Environmental Research, Aberystwyth, UK) for the LHCII antiserum.
| |
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Top
Abstract
Introduction