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Plant Physiol. (1999) 119: 1557-1566
Characterization of Mutants with Alterations of the
Phosphorylation Site in the D2 Photosystem II Polypeptide of
Chlamydomonas reinhardtii1
Mark M. Fleischmann and
Jean-David Rochaix*
Departments of Molecular Biology and Plant Biology, University of
Geneva, 1211 Geneva 4, Switzerland
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ABSTRACT |
We have changed the potential
phosphorylation site, a threonine residue at position 2 of the D2
polypeptide of the photosystem II complex of Chlamydomonas
reinhardtii, to alanine, valine, aspartate, proline, glycine,
or glutamate. Mutants with neutral amino acid changes did not display
any phenotype with regard to photoautotrophic growth, light
sensitivity, fluorescence transients, or photoinhibition. Pulse
labeling of these mutants with 32P indicated that a
phosphorylated protein of the same size as D2 is absent in these
mutants, suggesting that threonine-2 is indeed the unique
phosphorylation site of D2. In contrast, mutants in which threonine-2
has been replaced with acidic residues are deficient in photosystem II.
Use of chimeric genes containing the psbD
5 -untranslated region revealed that the initiation of translation was
not affected in these mutants, but the mutations interfered with a
later step of D2 synthesis and accumulation.
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INTRODUCTION |
Several polypeptides of the PSII complex can be phosphorylated in
higher plants. They include the D1 and D2 proteins encoded by the
psbA and psbD genes, as well as the products of
the psbC and psbH genes. In spinach, the
phosphorylated residue of all four proteins has been identified as an
O-phosphothreonine near the N terminus, which is exposed to
the stromal face of thylakoid membranes (Michel and Bennett, 1987 ;
Michel et al., 1988).
Phosphorylation of the PSII complex has also been reported in the
unicellular alga Chlamydomonas reinhardtii (de Vitry et al.,
1987, 1991). The products of the psbC and psbH
genes have been found to be phosphorylated (Dedner et al., 1988; de
Vitry et al., 1991). Two different sites of phosphorylation exist in the 9-kD PsbH subunit and give rise to two bands, L5 and L6, as detected by PAGE. An additional small protein of approximately 5 kD is
also phosphorylated and has been identified as the product of
psbI or psbF (de Vitry et al., 1987). The
detection of phosphorylated D1 and D2 proteins proved difficult in
C. reinhardtii because of contamination of PSII particles
with LHCII proteins. In this organism, LHCII proteins are difficult to
resolve from D1 and D2 by PAGE and can be heavily labeled with
radioactive phosphate (Owens and Ohad, 1982; Wollman and
Delepelaire, 1984). For this reason, the labeled band first believed to
originate from the phosphorylated form of the D1 protein was found to
represent a member of the LHCII protein family (de Vitry et al., 1991).
Thus, despite its sequence similarity with higher plants (Erickson et al., 1984), the N-terminal part of the D1 protein is not a target for
phosphorylation in C. reinhardtii.
In contrast to the D1 protein, phosphorylation of the D2 protein does
occur in C. reinhardtii. In vivo protein pulse labeling of
cells with [14C]acetate revealed the presence
of a new band, D2.1, corresponding to the expected size of the
phosphorylated D2 protein (Delepelaire, 1984). This band disappeared
after phosphatase treatment, with a concomitant increase in band D2.2,
the unphosphorylated form of the D2 protein (de Vitry et al., 1987).
Moreover, proteolytic treatment of the isolated D2.1 and D2.2 bands
generated similar fragments, confirming the presence of two forms of
the same protein (Delepelaire, 1984). The exact site of phosphorylation
of the D2 protein in C. reinhardtii has not been determined.
Because the N-terminal region of D2 is conserved between C. reinhardtii and higher plants, it is likely that the same Thr
residue at position 2 is phosphorylated in this alga (Erickson et al.,
1986). However, some doubts about the existence of this phosphorylation
site were raised in a recent report (Andronis et al.,
1998).
As in higher plants, the PSII complex of C. reinhardtii
appears to be assembled in the unappressed region of the thylakoid membranes (Schuster et al., 1988; de Vitry et al., 1989) before it
migrates to the grana region, where it accumulates (Vallon et al.,
1985; de Vitry et al., 1989). Mutants of C. reinhardtii deficient in the expression of the PsbC or the D1 protein are unable to
assemble the PSII complex. In these mutants, the synthesis of D2 can
still be detected by [14C]acetate pulse
labeling. However, the band corresponding to the phosphorylated form
never appears (de Vitry et al., 1987, 1989), suggesting that
phosphorylation of the D2 protein occurs only in fully assembled
complexes, which is in agreement with the delayed appearance of D2.1 in
[14C]acetate pulse-labeled wild-type cells
(Delepelaire, 1984). Phosphorylation of PSII proteins is still observed
in mutants of C. reinhardtii lacking PSI, ATPase, or the Cyt
b6/f complex (de Vitry et al., 1987; Wollman and Lemaire, 1988). The independence of phosphorylation of PSII proteins from a functional Cyt
b6/f complex has also been reported in maize (Bennett et al., 1988). This feature distinguishes PSII phosphorylation from the well-studied phenomenon of state transition, which relies on Cyt
b6/f-dependent LHCII protein
phosphorylation (Wollman and Lemaire, 1988; Allen, 1992). LHCII and
PSII protein phosphorylation are dependent on the reduction of the
plastoquinone pool (Allen, 1992; Aro et al., 1993; Ebbert and Godde,
1996; Rintamäki et al., 1996). However, one remarkable exception
concerns the phosphorylation of D2 in C. reinhardtii, which
is induced by the oxidation of the plastoquinone pool (Delepelaire,
1984; Delepelaire and Wollman, 1985).
Many different roles have been suggested for PSII phosphorylation,
including spatial separation of PSII complexes between grana and
stromal lamellae (Mattoo et al., 1989) and influence on the biogenesis,
stability, and dimerization of the PSII complex (de Vitry et al., 1989;
Kruse et al., 1997; Summer et al., 1997). However, the
best-characterized function of the phosphorylation of the D1 and D2
proteins appears to be related to photoinhibition. In higher plants,
phosphorylation of these proteins protects against proteolytic
degradation, indicating a role in the degradation-repair cycle of D1
and D2 during photoinhibition (Schuster et al., 1988; Aro et al., 1993;
Koivuniemi et al., 1995; Ebbert and Godde, 1996).
To gain new insights into the role of D2 phosphorylation in C. reinhardtii, the putative phosphorylation site was changed and the
properties of the mutants were analyzed. Substitution of Thr-2 with
acidic residues (either Asp or Glu) interfered drastically with the
expression of the D2 protein at a step following the initiation of
translation. Substitution with neutral amino acids (Ala, Val, Pro, or
Gly) affected phosphorylation of the D2 protein. However, no detectable
change in phenotype was observed with these mutant cells.
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MATERIALS AND METHODS |
Strains and Media
The Chlamydomonas reinhardtii mutants FuD7
and nac2-26 have been described previously (Bennoun et al.,
1986; Kuchka et al., 1989; Nickelsen et al., 1994). FuD7
contains a deletion of approximately 9 kb, which removes the
chloroplast psbA gene; nac2-26 is a nuclear mutant that specifically lacks the psbD mRNA. Wild-type and
mutant strains were grown as described by Harris (1989). If necessary, TAP medium and HSM were solidified with 2% Bacto-agar (Difco, Detroit, MI) and supplemented with spectinomycin (Sigma).
DNA Constructs
Thr-2 Mutants
Preparation of plasmids was as described by Sambrook et al.
(1989). The chloroplast EcoRI fragment R3 (Rochaix et al.,
1984) was cloned into the EcoRI site of pBluescript
SK (Stratagene) in which both PvuII
sites had been destroyed. The unique NsiI site was blunt
ended, and a 1.8-kb EcoRV-SmaI fragment of
pUC-atpX-AAD (Goldschmidt-Clermont, 1991) was
inserted at this site to obtain plasmid pSK-108#14. This plasmid
contains the 5 part of psbD and the aadA-rbcL
cassette (Goldschmidt-Clermont, 1991) upstream of psbD
oriented in the opposite direction.
Mutagenesis of psbD was performed according to the
megaprimer method (Sarkar and Sommer, 1990). A PCR reaction
using the oligonucleotides 1963 (5-AGAAACAGCTGCTGTTAA-3) and 1965 (5-TTTGGAGATACACGCCATGG[A/G/C/T][A/T]ATTGCGAT-3) and pSK-108#14
as a template was performed first. The product of this reaction, the
megaprimer, was used with oligonucleotide 1365 (5-CCATCGATAAGCTTGATTTTTTATATCATAATAATAAA-3) on the same template in
a second PCR reaction. The PCR product was cloned into pBluescript
SK (Stratagene) by the T-vector method (Marchuk
et al., 1990) and sequenced. Finally, fragments containing different
mutations at the second codon of psbD were recloned into
pSK-108#14 using the unique ClaI and PvuII sites
of the vector. The resulting plasmids were called pSK-140(T2A),
pSK-141(T2V), pSK-142(T2D), pSK-143(T2P), pSK-144(T2G), and
pSK-145(T2E), and each contained a different substitution of the second
psbD codon (Fig. 1). These plasmids were used to transform
C. reinhardtii. pSK-146(T2T) was constructed similarly
except that oligonucleotide 146 (5-TTGGAGATACACGCCATGACAATTGCG-3) was
used instead of oligonucleotide 1965.
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| Figure 1.
Sequences of the psbD codon 2 mutations and the corresponding D2 sequences. Mutated nucleotides and
amino acids are indicated in boldface. When present, the
NcoI restriction site is underlined. WT, Wild type.
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Fusion Proteins
PCR fragments of 399 bp were obtained using
oligonucleotides 1365 and psbD-BspHI
(5-TACTGAACGAGTCATGACAAACTGACG-3) on plasmids pSK-108#14, pSK-142,
pSK-144, pSK-145, and pSK-146. After they were cloned in pBluescript
SK and sequenced, each plasmid was cut with
ClaI and BspHI and the resulting fragment was
used to replace the promoter-containing atpA
ClaI-NcoI fragment of plasmid
pUC-atpX-AAD. The ClaI-SacI fragments containing psbD-aadA-rcbL
were then used to replace the 1.8-kb ClaI-SacI
fragment in pBS-5.8-aadA (Fischer et al., 1997). This
yielded plasmids pBS-61(WT), pBS-62(T2D), pBS-63(T2G), pBS-64(T2E), and
pBS-65(T2T) (see Fig. 6), which were used to introduce the
psbD-aadA reporter genes near psaC by
chloroplast transformation.
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| Figure 6.
Structure and sequence of the chimeric
psbD-aadA genes. The chimeric psbD-aadA
gene and the psaC gene are shown. psbD,
Promoter, 5-untranslated region, and coding sequence for the 35 N-terminal codons of psbD. aadA, Coding
sequence for the aminoglycoside adenine transferase.
rbcL, 3-untranslated region of rbcL. The
sequence around the start of translation is shown. Mutations are
indicated in boldface. WT, Wild type.
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Chloroplast Transformation
Chloroplast transformation was carried out as described previously
(Fischer et al., 1997). The plasmids pSK-108#14, pSK-140, pSK-141,
pSK-142, pSK-143, pSK-144, pSK-145, and pSK-146 were used
to transform C. reinhardtii wild-type cells
(mt+) by selecting for growth at low light (6 µE m2 s1) on TAP plates
supplemented with 150 µg/mL spectinomycin. The corresponding
transformants were WT-aadA, 140(T2A), 141(T2V), 142(T2D),
143(T2P), 144(T2G), 145(T2E), and 146(T2T), respectively.
Plasmids pBS-61, pBS-62, pBS-63, pBS-64, and pBS-65 were
used to transform the mutant psaC Spcs (Fischer et al.,
1996) and selected on TAP plates in the presence of light (60 µE
m2 s1) to obtain mutants 61(WT), 62(T2D),
63(T2G), 64(T2E), and 65(T2T), respectively.
Fluorescence Transients
Fluorescence transients were measured on whole cells grown on TAP
plates, as described previously (Fenton and Crofts, 1990). The curves
were then normalized to their maximal values using the Excel program
(Microsoft, Redmond, WA).
Immunoblot Analysis
Proteins from whole cells or subcellular fractions were separated
by SDS-PAGE, as described previously (Sambrook et al., 1989). For
LDS-PAGE, SDS was replaced with LDS in the gel and in the buffers, and
electrophoresis was performed at 4°C instead of at room temperature
(Ebbert and Godde, 1996).
Immunoblotting and enhanced chemiluminescence detection were carried
out according to the manufacturer's protocol (Amersham). Detection
with an antibody raised against the D2 protein was modified as follows.
The nitrocellulose membranes were saturated with 5% yeast extract
instead of nonfat milk and incubated with the antibody overnight at
4°C. It was necessary to double the number of washings with this
antibody.
35S Pulse Labeling
Approximately 107 cells in exponential
growth were inoculated in 100 mL of low-S TAP medium (TAP without
NH4Cl, CaCl2, or
MgSO4) and grown overnight. Cells were pelleted,
washed once with minus-S TAP medium (20 mM Tris-acetate-OH,
pH 7.0, and 1 mM
K2HPO4/KH2PO4, pH 7.0), and resuspended in 20 mL of the same medium. The cell suspension was then incubated at 25°C with agitation for 2 to 4 h, during which time the chlorophyll concentration was measured according to the method of Porra et al. (1989). Cells corresponding to
40 µg of chlorophyll were pelleted, resuspended in 1 mL of minus-S
TAP medium, and incubated for 5 min at room temperature with agitation.
After the addition of cycloheximide (6 µg/mL final concentration),
the cell suspension was incubated for 10 min at room temperature before
100 µCi of
Na235SO4
was added. Labeling was carried out at room temperature with agitation
for different times. Aliquots were withdrawn, microcentrifuged for 2 min, and quickly frozen. For analysis, thawed cells were washed with TE
buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) containing 1 mM PMSF, resuspended in loading buffer, and
fractionated by SDS-PAGE (Sambrook et al., 1989). The gel was then
stained with Coomassie blue, dried, and subjected to autoradiography
(Sambrook et al., 1989).
RNA Analysis
Fifty milliliters of an exponentially growing cell
culture was centrifuged at 4000g for 5 min. The pellet was
washed once with 5 mL of 10 mM Tris-HCl, pH 7.5, and frozen in dry ice. Two milliliters of TEN-SDS buffer (200 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 10 mM EDTA, and 0.2% SDS) and 2 mL of
phenol:chloroform (1:1, v/v) were added to the frozen pellet before it
was resuspended by homogenization in a Polytron (Brinkmann). The
solution was centrifuged at 8000g for 5 min at 4°C. The
aqueous phase was retrieved and extracted for a second time before the
nucleic acids were precipitated with 5 mL of ethanol and washed with
70% ethanol. Finally, the nucleic acids were resuspended in 400 µL
of 1 mM EDTA with 0.1% diethylpyrocarbonate. RNA
was quantified by spectrophotometry (Sambrook et al., 1989) and kept
frozen. Northern-blot analysis was carried out as described previously
(Sambrook et al., 1989; Stampacchia et al., 1997).
32P Pulse Labeling
The protocol for in vivo pulse labeling with radioactive phosphate
was adapted from the method of Wollman and Delepelaire (1984).
Approximately 5 × 108 cells were pelleted
and resuspended in 20 mL of minus-P TAP medium (TAP without
K2HPO4 or
KH2PO4). The chlorophyll
concentration of the cell suspension was determined according to the
method of Porra et al. (1989), and the equivalent of 200 µg of
chlorophyll was pelleted. The pellet was resuspended in minus-P TAP
medium to a final concentration of 25 µg chlorophyll
mL1 followed by the addition of
K2HPO4 and
KH2PO4, pH 7.0 (5 mM final concentration), and 32Pi (1 mCi/mL final concentration). Labeling was carried out at room
temperature with agitation in the presence of medium light (approximately 30 µE m2
s1) for 2 h, with the addition of DCMU
(ICN) after 1 h. Labeled cells were pelleted and washed with 5 mL
of minus-P TAP medium containing 10 mM NaF.
Thylakoid membrane isolation has been described (Chua and Bennoun,
1975). The labeled cells were resuspended in 500 µL of buffer H1 (25 mM Hepes-KOH, pH 7.5, 5 mM
MgCl2, and 0.3 M Suc) with 1 mM PMSF. Cells were broken by the addition of 700 mg of glass beads and vortexing at maximum speed for 2 min. Membrane fractions were recovered in 1 mL of buffer H1 and pelleted by microcentrifugation for 3 min. The pellet was washed with 1 mL of
buffer H2 (5 mM Hepes-KOH, pH 7.5, 10 mM EDTA,
and 0.3 M Suc) and resuspended in 1 mL of buffer H3 (5 mM Hepes-KOH, pH 7.5, 10 mM EDTA, and 1.8 M Suc). Thylakoid membranes were then purified on a
discontinuous Suc gradient consisting of 1 mL of buffer H3 (containing
the membrane fraction), 1 mL of buffer H4 (5 mM Hepes-KOH, pH 7.5, 10 mM EDTA, and 1.3 M Suc), and 1 mL of
buffer H5 (5 mM Hepes-KOH, pH 7.5, and 0.5 M
Suc) in an ultracentrifugation tube (SW60, Beckman). The gradient was
centrifuged at 24,000 rpm for 1 h at 4°C. The upper green band
containing the thylakoid membranes was retrieved and washed with 1 mL
of buffer H6 (5 mM Hepes-KOH, pH 7.5, and 10 mM
EDTA). Finally, thylakoid membranes were resuspended in 100 µL of
buffer H6 with 10% glycerol and kept at  70°C. Analysis of labeled
proteins was carried out as described for
35S-labeled samples.
Photoinhibition Measurement
Exponentially growing cells were diluted to a final concentration
of 0.5 µg chlorophyll mL1 in TAP medium. For
photoinhibition, cell cultures were exposed to high light (1500 µE
m2 s1) at room
temperature with agitation. After 1.5 h, light was reduced to
approximately 5 µE m2
s1 to allow for recovery of PSII activity.
Fluorescence transients of the cell cultures were measured at different
times using the Plant Efficiency Analyser (Hansatech, King's Lynn,
UK), and the ratio of variable to maximal chlorophyll fluorescence was
used as an indicator of the efficiency of light capture by PSII (Kyle et al., 1984; Schuster et al., 1988; Krause and Weis, 1991).
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RESULTS |
The second codon of the psbD gene of C. reinhardtii, coding for Thr, was changed to Ala, Val, Asp, Pro,
Gly, or Glu by site-directed PCR mutagenesis (Fig.
1). For practical reasons, these
mutations also convert the A preceding the AUG into a C to create a new NcoI site. Mutant T2T, which contains only this last
substitution, was generated as a control (Fig. 1). The psbD
mutations were introduced in a transformation vector carrying the
2.7-kb EcoRI chloroplast fragment R3 containing
psbD (Rochaix et al., 1984), thus replacing the endogenous
psbD gene. As a selectable marker, the aadA
cassette (Goldschmidt-Clermont, 1991) was inserted in the opposite
direction 261 bp upstream of the translation start site of
psbD. The constructs were introduced into C. reinhardtii wild-type (mt+) cells by
biolistic transformation, and transformants were selected on
spectinomycin-containing plates. Correct integration of the transforming DNA and the homoplasmicity of the transformants were confirmed by Southern blotting, and the DNA regions that had been amplified by PCR for mutagenesis were verified by sequencing (data not
shown).
Table I summarizes the growth properties
of the transformants on TAP medium and HSM under different light
intensities. Mutants T2A, T2V, T2P, T2G, T2T, and WT-aadA
were indistinguishable from the wild type under all conditions tested.
Mutant T2D did not grow on minimal medium, indicating that it is
deficient in photosynthetic activity. Reduced photosynthetic activity
probably explains the slow growth of mutant T2E on minimal medium. In
addition, mutants T2D and T2E were sensitive to high light (1000 µE
m2 s1) on TAP medium or
HSM (Table I). This was also observed with nac2-26, a
nuclear mutant that does not express the D2 protein (Kuchka et al.,
1989).
As expected for psbD mutants, the PSII activity of T2D and
T2E was affected. Fluorescence transients of T2D cells displayed a flat
curve characteristic of PSII-deficient mutants (Fig.
2). The fluorescence transient of mutant
T2E was intermediate between that of the wild type and
nac2-26, indicating partial PSII activity. The fluorescence
transients of the other transformants were indistinguishable from that
of the wild-type control. Therefore, despite its strong conservation
across species, Thr-2 can be replaced by Ala, Val, Pro, or Gly without
any apparent effect on PSII activity. We noticed, however, a small but
significant difference in the stationary fluorescence level, which was
consistently higher in the strains containing the aadA
cassette than in the wild-type strain. The significance of this
difference is not clear.
The reduction of PSII activity in the mutants T2D and T2E can be
attributed to a low level of PSII complex, because less D2 protein
accumulated in these mutants than in the wild type (Fig. 3A). No D2 protein was seen in mutant
T2D, and it was barely detectable in mutant T2E (Fig. 3A). The
accumulation of the D2 and D1 proteins followed the same pattern in all
mutants examined, i.e. the amount of D1 in mutants T2D and T2E was
reduced to 10% and 30%, respectively, of wild-type levels (Fig. 3C),
and D1 and D2 accumulated to wild-type levels in the other mutants
(Fig. 3B). It is well established that these two proteins can
accumulate only in the presence of each other. In the absence of D1 in
FuD7, the D2 protein is synthesized but rapidly degraded
(deVitry et al., 1989), and in the absence of D2 in the
nac2-26 mutant, the D1 protein is synthesized and turns over
rapidly (Kuchka et al., 1989). Overexposure of the immunoblot in Figure
3A reveals trace amounts of D2 in FuD7. However, under the
same conditions D2 was undetectable in the T2D and nac2-26 mutants. Taken together, these results suggest that the presence of Asp
at position 2 affects a step of the synthesis or assembly process of
the D1 and D2 proteins. The accumulation of the PsaA reaction center
subunit of PSI was not affected in these mutants, indicating that the
mutations act specifically on PSII (Fig. 3B).
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| Figure 3.
Accumulation of the PSII proteins. A, The
equivalent of 2.5 µg of chlorophyll from each sample was analyzed by
immunoblotting using antibodies directed against the D2 protein. B, The
equivalent of 2 µg of chlorophyll from each sample was analyzed by
immunoblotting using antibodies directed against the D1 or PsaA
proteins. C, Thirty micrograms of protein extract from each sample was
analyzed by immunoblotting using antibodies directed against the D1
protein. Dilutions of the wild type (WT) were done in
FuD7 extracts to keep total protein concentration
constant. D, The equivalent of 1.5 µg of chlorophyll from each sample
was analyzed by 12.5% LDS-PAGE at 4°C. Immunoblotting was performed
with antibodies directed against the D1 and D2 proteins.
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To examine the synthesis of the D2 protein in mutants T2D and T2E, the
chloroplast proteins of these cells were pulse labeled with
35S (Fig. 4). It
can be seen that the synthesis of D2 was strongly diminished in mutant
T2D compared with the other mutants or the control strains. D2 protein
synthesis was also affected in mutant T2E, but to a lesser extent.
Synthesis of the D2 protein in mutant T2E was estimated at
approximately 30% of the wild-type level by quantification of Figure 4
(not shown).
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| Figure 4.
35S pulse labeling of wild-type (WT)
and mutant cells. Different strains were labeled in vivo with
35S in the presence of cycloheximide for 5, 10, or 15 min.
Labeled chloroplast proteins were analyzed on a 7.5% to 15% PAGE
gradient gel and subjected to autoradiography.
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Reduction of D2 protein synthesis in mutants T2D and T2E cannot be
explained by an effect on the transcription of the psbD gene
or on stabilization of its mRNA, because the psbD mRNA
accumulated normally in these mutants (Fig.
5). Nor can reduction of the D2 protein
be explained by an indirect effect of low PSII activity in these
mutants, because D2 protein synthesis is unaffected in mutants lacking
D1 (de Vitry et al., 1989). This implies that the mutations in strains
T2D and T2E diminish the expression of psbD at the level of
mRNA translation or protein stability.
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| Figure 5.
Accumulation of psbD mRNA in
wild-type (WT) and mutant cells. Total RNA of the different strains was
analyzed by northern blotting. PsbD probe, 917-bp
DpnI-EcoRI fragment of
psbD (nucleotides 13 to 930 relative to the translation
start site). PsbA probe, 1.8-kb
BamHI-XbaI fragment of
psbA (nucleotides 50 to1750 relative to the
translation start site).
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To determine whether translation initiation was affected, chimeric
genes were constructed between psbD and aadA
(Fig. 6). These genes encode proteins
containing the N-terminal 35 amino acids of the D2 protein fused to the
aadA reporter protein and are driven by the promotor and the
5-untranslated region of psbD. These chimeric genes were
introduced into the chloroplast genome near the psaC gene
(see ``Materials and Methods''). Correct insertion and homoplasmicity of these constructs in the transformants were confirmed by Southern blotting (data not shown).
Expression of the fusion protein was monitored by growth of the
transformants with increasing amounts of spectinomycin (Table II). All transformants tested were
resistant to a low concentration of spectinomycin (50 µg/mL), in
contrast to the wild type or psaCSpcs, the
recipient strain used for transformation. No correlation could be found
between the negative charge on residue 2 of D2 and the reduction in
expression of the chimeric gene. Instead, a clear increase in
spectinomycin resistance was observed with the T2D mutant. This
increase must have been caused by enhanced translation or stabilization
of the fusion protein, because the level of the chimeric mRNA was the
same in all transformants (data not shown). Comparison of the
spectinomycin-resistance levels showed that the negative charge at the
N terminus did not interfere with the initiation of translation.
Therefore, the reduced levels of the D2 protein in mutants T2D and T2E
must be attributed to a step subsequent to translation initiation.
The protein pulse-labeling patterns in Figure 4 show that, in addition
to the reduction in the rate of synthesis of D2, D1 synthesis was also
considerably diminished in mutants T2D and T2E. To determine whether
this occurred at the level of initiation of translation, a chimeric
gene consisting of the psbA 5-untranslated region fused to
aadA was inserted into the chloroplast genome. However, the
expression of this chimeric gene could not be tested properly because
its transcript was present in considerably lower amounts in the
transformed wild-type strain than in the transformed nac2-26
and FuD7 strains, for reasons that are not clear (data not
shown).
Figure 3A reveals small differences in the size of the protein detected
by the D2 antibody. In samples from mutants T2A and T2G, the D2 protein
had a slightly smaller apparent molecular mass than in samples from T2T
and the wild type. An improved gel resolution was obtained with
LDS-PAGE at 4°C (Fig. 3D). Under these conditions, the D2 band of
lower mobility, labeled D2.1, represents the phosphorylated form of D2,
whereas the faster-migrating band, D2.2, represents the
unphosphorylated form (Delepelaire, 1984). Although D2.1 was absent in
T2A, this band could be detected in both the wild type and
WT-aadA (Fig. 3D), suggesting that Thr-2 is the unique
phosphorylation site of D2.
To test directly whether phosphorylation is impeded by substitutions of
Thr-2, mutant cells were pulse labeled in vivo with Pi (Fig. 7). The
labeling was performed under conditions that lead to the oxidation of
the plastoquinone pool, and hence favor phosphorylation of the D2
protein, and that reduce phosphorylation of the LHCII proteins
(Delepelaire and Wollman, 1985). Analysis of purified thylakoid
membranes from these cells indicated the presence of a phosphorylated
protein the size of D2 in both the wild type and T2T. This band was not
detectable in the mutants T2V (Fig. 7), T2G (Fig. 7), or T2A (data not
shown). Quantification of the radioactive signals on this gel excluded
the possibility that in mutants T2V and T2G the D2 signal was shifted
and hidden by one of the LHCII protein bands (Fig. 7). These
observations are compatible with the idea that the D2 protein is
phosphorylated on Thr-2 in C. reinhardtii, as it is in
spinach (Michel et al., 1988).
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| Figure 7.
32P pulse labeling. Mutant strains
were labeled in vivo with 32Pi for 2 h under oxidizing
conditions. Purified thylakoid membranes were analyzed on a 7.5% to
15% PAGE gradient gel and subjected to autoradiography. A scan of the
autoradiogram in the region of D2 is shown in the lower part of the
figure. WT, Wild type.
|
|
Because strains T2V and T2G accumulate PSII, in which D2 does not
appear to be phosphorylated, we tested whether phenotypic differences
could be detected in these cells. Strains T2V and T2G grew as well as
the wild type on minimal medium (Table I), indicating that
photosynthesis was not appreciably affected. In higher plants,
phosphorylation of the D1 and D2 proteins has been shown to play a role
in the turnover of the proteins under high light (Koivuniemi et al.,
1995; Ebbert and Godde, 1996). In C. reinhardtii, the mutant
cells T2V and T2G grew normally at light intensities as high as 1000 µE m2 s1 (Table I),
indicating that phosphorylation of the D2 protein has no obvious role
in the protection against photoinhibition.
To measure photoinhibition more quantitatively, the ratio of variable
to maximal chlorophyll fluorescence was determined for mutant and
control cells during photoinhibition and the subsequent recovery period
(Fig. 8). The overall kinetics of
photoinhibition and recovery were identical for mutants and control
cells. The small differences in absolute values were not reproducible
from one experiment to another, so they are not significant. Because mutants T2A and T2G prevented phosphorylation of D2, these data show
that this posttranslational modification is not important for the
protection of PSII during photoinhibition and the recovery of PSII
activity. Therefore, the function ascribed to D2 protein phosphorylation in higher plants is not conserved in C. reinhardtii.
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| Figure 8.
Kinetics of photoinhibition and recovery. Cell
suspensions at a concentration of 2 µg chlorophyll mL1
were subjected to high light for 90 min, followed by an incubation in
low light for 2 h. The ratio of variable to maximal chlorophyll
fluorescence (Fv/Fm), calculated from fluorescence measurements, is
plotted versus time for each mutant.
|
|
| |
DISCUSSION |
The Phosphorylation Site of D2 Is Not Essential for PSII
Function
The N-terminal region of the D2 protein is highly conserved among
different photosynthetic organisms. In particular, Thr-2, which has
been shown to be phosphorylated in spinach (Michel et al., 1988), is
present in all D2 sequences that have been determined. It is
surprising, therefore, that this residue can be changed to Ala, Val,
Pro, or Gly without any apparent effect on PSII activity. The same
observation was reported recently with Ala and Ser substitutions at the
same site (Andronis et al., 1998). In that report, photoautotrophic growth rates, fluorescence induction and decay, thermoluminescence, and
photoinhibition were not significantly different in the D2 mutants
compared with the wild type. Although Andronis et al. (1998) noticed
differences in the migration of the D2 protein, they favored the idea
that this protein is not phosphorylated in C. reinhardtii.
Our data indicate that mutations of Thr-2 of D2 abolish specifically
the phosphorylation of a polypeptide of the same size as D2 (Fig. 7).
This implies that the mutations of Thr-2 affect the only
phosphorylation site of the protein. It could still be argued that the
modifications introduced into the D2 protein affect phosphorylation
indirectly on other residues. However, because these modifications have
no apparent effect on PSII activity, any structural perturbation of the
protein seems unlikely. Nevertheless, unequivocal identification of the
phosphorylation site on the D2 protein still awaits MS analysis as
performed for higher plants (Michel et al., 1988).
It appears that phosphorylation of the PSII proteins differs between
C. reinhardtii and higher plants. The D1 protein, which is
most prominently phosphorylated in higher plants, is not phosphorylated in this unicellular alga (de Vitry et al., 1991). Phosphorylation of
the D2 protein is regulated in an opposite manner than phosphorylation of LHCII with respect to the plastoquinone redox state (Delepelaire, 1984; Delepelaire and Wollman 1985). Phosphorylation of D2 in C. reinhardtii was also demonstrated by analysis of purified PSII complexes after labeling of cells in vivo or thylakoids in vitro (Ikeuchi et al., 1987). However, we and others (Andronis et al., 1998)
were not able to detect phosphorylation of the D2 protein by in vitro
phosphorylation of thylakoid membranes (data not shown). One
possibility is that the kinase associated with this process is only
loosely associated with the thylakoid membranes, in contrast to the
situation in higher plants (Koivuniemi et al., 1995). Finally, the
relationship between PSII phosphorylation and photoinhibition does not
seem to hold for C. reinhardtii (Andronis et al., 1998; this
work).
The D2 Polypeptide Is Destabilized by Thr-2 Asp/Glu Changes
Substitution of Thr-2 of the D2 protein with acidic residues had a
drastic effect on its synthesis and, consequently, on PSII stability.
By using chimeric genes containing the first 35 residues of the D2
protein fused to the aadA sequence, no reduction in expression of the chimeric genes carrying the T2D and T2E mutations was
observed compared with genes containing the wild-type residue. It is
highly unlikely, therefore, that the loss of D2 in these mutants is
attributable to an inhibition of initiation of translation. In these
mutants, the effect of the mutations could occur at the level of
translation elongation or at the level of stability of D2. Acidic
residues near the N-terminal end of D2 could induce a conformational
change that makes the protein very susceptible to proteases. One
possibility is that the negative charge at the N terminus interferes
with the insertion of the protein into the thylakoid membranes. Because
the D2 protein inserts into the membranes before it interacts with the
D1 protein (Jensen et al., 1986; de Vitry et al., 1989), this last
model would explain the more pronounced destabilization of D2 in the
T2D mutant compared with FuD7, which lacks D1 but is still
able to synthesize D2. The fact that the presence of Asp and Glu
reduced D2 to different levels indicates that, in addition to its
negative charge, the nature of the residue is also important.
The Absence of D2 Synthesis Leads to the Absence of D1
Synthesis
Our protein pulse-labeling studies have shown that substitution of
Thr-2 with neutral amino acids has no effect on the rate of synthesis
and accumulation of D2 and D1. In contrast, in the T2D and T2E mutants,
in addition to D2, D1 was also strongly destabilized. Similar effects
on D1 have been observed in other mutants in which D2 is no
longer synthesized or in which D2 is rapidly degraded (Erickson et al.,
1986; de Vitry et al., 1989). It is not yet known through which step of
gene expression this effect is mediated.
| |
FOOTNOTES |
1
This work was supported by grant no.
3100-050895.97 from the Swiss National Science Foundation.
*
Corresponding author; e-mail jean-david.rochaix{at}molbio.unige.ch;
fax 41-22-702-68-68.
Received October 5, 1998;
accepted January 12, 1999.
| |
ABBREVIATIONS |
Abbreviations:
HSM, high-salt minimal medium.
LDS, lithium
dodecyl sulfate.
LHCII, light-harvesting antenna of PSII.
TAP, Tris-acetate-phosphate.
| |
ACKNOWLEDGMENTS |
We thank P. Nixon for antibodies against D2, A. Auchincloss, M. Goldschmidt-Clermont, and M. Hippler for helpful comments, and N. Roggli for drawings and photography.
| |
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Abstract
Introduction
