From the Developmental, Cell and Molecular Biology Group, Botany Department, Duke University, Durham, North Carolina 27708
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ABSTRACT |
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The phosphorylation of proteins within the
eukaryotic photosynthetic membrane is thought to regulate a number of
photosynthetic processes in land plants and algae. Both light quality
and intensity influence protein kinase activity via the levels of
reductants produced by the thylakoid electron transport chain. We have
isolated a family of proteins called TAKs, Arabidopsis
thylakoid membrane threonine kinases that phosphorylate the light
harvesting complex proteins. TAK activity is enhanced by reductant and
is associated with the photosynthetic reaction center II and the
cytochrome b6f complex. TAKs
are encoded by a gene family that has striking similarity to
transforming growth factor Protein phosphorylation in the photosynthetic membrane was first
observed over 20 years ago (1), and since then threonine phosphorylation has been linked to a number of regulatory cascades that
modulate photosynthetic rates (2). The phosphorylation of the light
harvesting complex has been implicated in the modulation of light
energy that is transmitted to the photosynthetic reaction center (3),
either by separation of the macromolecular complexes (4) or controlled
protein degradation (5). The phosphorylation is coupled to the
association of the reduced plastoquinone and the cytochrome
b6f complex (6) and is influenced in
different ways by both the quality and the amount of light (7). The D1 protein of reaction center II is also phosphorylated in a regulated fashion such that its turnover is linked to light quality and intensity
via reductants produced from the photosynthetic electron transport
chain (7, 8). Thus phosphorylation of thylakoid membrane proteins is an
important regulative process in land plants and algae. Protein kinase
activities that can phosphorylate thylakoid proteins have been
described (9, 10), yet these proteins have not been purified or cloned.
We report here on the isolation of a thylakoid protein kinase that is
associated with the cytochrome b6f
complex and photosystem II (PS
II)1 and that can
phosphorylate the LHCP complex.
Construction of pGem-Tak1 and in Vitro
Translation--
Tak1 was PCR amplified using BAC clone
T10P11 (Arabidopsis Ohio stock center) as a template using
the following primers: 5'-CGG GAA TTC AGC AGA ACA AAA GCA-3' and 5'-CGG
GAA TTC CAC ACA AGA AAA AAC-3'; introducing EcoRI sites in
both the 5' and 3' regions. This PCR product was digested, gel
purified, and ligated into an EcoRI linearized, calf
alkaline phosphotase-treated pGem4Z (Amersham Pharmacia Biotech)
vector. The correct orientation of the insert was determined by
restriction analysis. pGem-Tak1 DNA was linearized with KpnI
(Promega) and used as a template for in vitro transcription
(Sp6 RNA Polymerase, Amersham Pharmacia Biotech) and in
vitro translation in a wheat germ extract (Amersham Pharmacia
Biotech) using [35S]methionine (ICN Biochemicals) as
described earlier (11).
TAK Purification and Kinase Assay--
5 mg of purified
thylakoids (11) were resuspended in 10 ml of IB (50 mM
HEPES, pH 7.5 (KOH) and 330 mM Sorbitol) and incubated with
5 M urea, 125 mM NaCl, and 10 mM
MnCl2 at 4 °C for 30 min. The sample was centrifuged at
100,000 × g at 4 °C in a Ti 70 rotor (Beckman) for
3 h. After centrifugation the supernatant was removed, dialyzed
overnight at 4 °C against two changes of 1 liter of 50 mM HEPES, pH 7.5 (KOH), 10 mM EDTA, pH 8, 1 mM MnCl2, and 1 mM phenylmethylsulfonyl fluoride, and then applied to a Sephadex G200
column at 4 °C (40 ml of packed volume, 27 × 1.5 cm,
equilibrated and swollen in 50 mM HEPES, pH 7.5 (KOH), 10 mM EDTA, pH 8, and 1 mM MnCl2). The
sample was eluted with 50 mM HEPES, pH 7.5 (KOH), 10 mM EDTA, pH 8 in 0.5-ml fractions, subjected to SDS-PAGE
electrophoresis followed by silver staining (Bio-Rad) and transfer
(MilliblotTM, according to manufacturer's instructions) to
nitrocellulose, and probed with TAK antiserum. Approximately 0.8 µg
of TAK was present in this fraction. Fractions containing proteins that
bound TAK antiserum were then subjected to kinase assays to determine whether they would phosphorylate bovine serum albumin (BSA). The kinase
assay included 10 µCi·ml In Vitro Light Harvesting Complex Phosphorylation and TAK
Autophosphorylation--
Thylakoid membranes, equivalent to 2 µg of
chlorophyll, resuspended in 50 mM HEPES, pH 7.6 (KOH), 10 mM EDTA, pH 8, and 1 mM MnCl2, with
or without 25 µM 3-(3,4-dichlorophenyl)-1,1-dimethyl urea
(DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), respectively, were preincubated in the light (120 µmol photons/m2 s) or dark for 2 h at 25 °C; 10 µCi·ml Peptide Sequencing--
Arabidopsis purified TAK,
resuspended in 50 mM HEPES, pH 7.5 (KOH), 10 mM
EDTA, pH 8, 1 mM MnCl2, was subjected to
electrophoresis on a 10% SDS-PAGE gel, followed by transfer to
polyvinylidene difluoride membrane (Westran, Schleicher & Schuell) in
10% methanol, 10 mM CAPS, pH 11, for 2 h at 400 mA.
The membrane was rinsed with filtered double distilled H2O
and stained with 0.1% Amido Black (Sigma) in 40% methanol and 1%
acetic acid followed by destaining in filtered double distilled
H2O. The appropriate bands were excised, subjected to CNBr
treatment, and sequenced. We expected the cleaved peptides to have
amino-terminal methionines, but because this was not the case there may
have been additional nonspecific degradation during analysis.
To identify proteins that interact with the major light harvesting
protein (LHCP), we developed a yeast assay that allowed selection of
cDNAs encoding activities that bind a known target (12). We used as
target the LHCP amino terminus, which contains the site of threonine
phosphorylation, and we reported on the isolation of an
Arabidopsis cDNA that encoded a 68-kDa protein kinase,
WAK1, that could interact with LHCP in yeast (13). However, WAK1 in
plants is responsible for interaction between the plant cell wall and
plasma membrane (14), and we reasoned that its isolation was due to
reduced substrate specificity when expressed in yeast. We expressed the
kinase domain of WAK1 in Escherichia coli (14) and used this
purified protein as denatured antigen in rabbits or renatured antigen
in mice. The rabbit serum recognized WAK1 protein in
Arabidopsis (14), but the mouse serum specifically identified a 55-kDa protein in leaf extracts (Fig.
1A, lane T). Leaves
were separated into soluble, microsomal, nuclear, and chloroplast fractions, and the 55-kDa protein was found exclusively in the chloroplast. Further fractionation (11) revealed that the 55-kDa protein was on the thylakoid membrane (Fig. 1A, lane
Th) and not in the envelope (Fig. 1A, lane
E) or stroma (Fig. 1A, lane S) of the
chloroplast. We tentatively named the 55-kDa protein TAK for
thylakoid-associated kinase. TAK
can be extracted by NaOH into a low speed supernatant (Fig.
1A, lanes S versus lanes P) but not by
NaHCO3 (lanes P versus lanes S), indicating that
it is integral or internal to the thylakoid membrane (11). TAK is
sensitive to proteinase K treatment of intact thylakoid membranes (Fig.
1A, lane PRTK). Because these levels of
proteinase K do not disrupt thylakoid membranes (11), we conclude that
TAK is integral to the membrane and has a large portion exposed to the chloroplast stroma. This stromal portion reacts with antiserum raised
to the kinase, and so we predict that TAK contains a stromal kinase
domain, a necessary location for a LHCP-specific kinase.
receptors of metazoans. Thus thylakoid
protein phosphorylation may be regulated by a cascade of
reductant-controlled membrane-bound protein kinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 [32P]ATP
(ICN), kinase buffer (50 mM HEPES, pH 7.6 (KOH), 100 mM NaCl, 10 mM MgOAc, 5 mM
MnCl2, 50 µM ATP, pH 7 (KOH)) and 0.05 mM DTT (Sigma), and 2-4 µg BSA (Sigma). Multiple
purifications were combined to provide sufficient sample for kinase assays.
1 [32P]ATP and kinase buffer
components to the amounts indicated above (see "TAK Purification and
Kinase Assay") were added along with 0.5 µg·ml
1
purified TAK (in 50 mM HEPES, pH 7.5 (KOH), 10 mM EDTA, pH 8, 1 mM MnCl2, and 1 mM phenylmethylsulfonyl fluoride). The reactions were
incubated for 1 h with agitation every 15 min at 25 °C in the dark.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
A, TAK is a thylakoid membrane protein.
Arabidopsis leaves were separated into total (T),
envelope (E), stromal (S), thylakoid
(Th) fractions. Thylakoids were then further incubated with
either 5 M NaOH or NaHCO3, centrifuged at
100,000 × g to make a pellet (P) or
supernatant (S), or treated with proteinase K
(PRTK). Samples were analyzed by 12.5% SDS-PAGE and
transferred to nitrocellulose, followed by Westerns with TAK antiserum.
B, purified TAK phosphorylates LHCP II. Thylakoid membranes
were incubated with [32P]ATP in combination with DTT,
DCMU/DBMIB, and purified TAK as indicated above the gel.
TAK was first identified by an antiserum to a kinase domain, and we were interested to see whether TAK was indeed a protein kinase. We purified TAK from Arabidopsis leaves by first isolating chloroplasts and then fractionating the thylakoid complexes by sucrose gradient centrifugation (11, 15). TAK was enriched in a PS II particle containing the cytochrome b6f complex (6). The PS II-enriched membrane preparation was incubated with urea and centrifuged, and the dialyzed supernatant was separated on a Sephadex sizing column. The eluted fractions were tested for kinase activity, and a sample containing only a 55-kDa protein band that also reacted with the TAK antiserum was able to phosphorylate BSA in vitro (see "Experimental Procedures"). Silver staining of this active fraction reveals no other proteins in addition to the 55-kDA band.
We next determined whether the purified TAK preparation would phosphorylate thylakoid membrane proteins. When thylakoid membranes are incubated in the light with [32P]ATP and analyzed by denaturing gel electrophoresis and autoradiography, the 28-kDa LHCP, 31-kDa D1, 43-kDa cp43, and a 9-kDa protein are the most abundant phosphorylated species (Fig. 1B, lane 6) (16). The thylakoid phosphorylation activity can be greatly diminished if thylakoids are first incubated in the dark in the presence of both a PS II (DCMU) and b6f electron acceptor (DBMIB), which leads to the oxidization of the PQ pool and thus inhibits kinase activity (as shown in Fig. 1B, lane 1) (16). Purified TAK was incubated with the inhibitor treated thylakoids along with [32P]ATP in the dark, and the reaction was separated on denaturing polyacrylamide gels followed by autoradiography. The results are shown in Fig. 1B (lanes 4 and 5). TAK phosphorylated the 28-kDa LHCP in the presence (lane 5) but not the absence (lane 4) of the reductant DTT. Phosphorylation of other thylakoid proteins is also detected under these conditions, but these levels are significantly lower than the LHCP phosphorylation. This lower level of phosphorylation is also detected using BSA as a substrate (data not shown), indicating that it might represent nonspecific activity. Incubation of the TAK preparation with only [32P]ATP in the presence or absence of DTT did not lead to phosphorylation (Fig. 1B, lanes 2 and 3), indicating that TAK is not capable of phosphorylating itself under these conditions. Incubation of TAK with stromal proteins also does not lead to phosphorylation (data not shown), and thus we conclude that TAK preferentially phosphorylates LHCP.
To obtain cDNAs for TAK we attempted to screen an
Arabidopsis cDNA expression library with TAK antiserum,
but surprisingly we isolated only WAK1 clones. We therefore obtained
peptide sequence from the TAK preparation. Four distinct sequences were
obtained from a cyanogen bromide-degraded preparation, and these were
each used in four separate searches of the Arabidopsis data
base. One gene sequence was common to each of the four independent
searches, and this was named Tak1. The predicted amino acid
sequence of the encoded 55-kDa protein is shown in Fig.
2A where the four peptides are
lightly underlined. The amino-terminal 26 residues are
predicted to serve as a chloroplast targeting sequence (17), followed
by a 20-amino acid hydrophobic region that could serve as a
transmembrane domain (bold dashed line above sequence). No signal cleavage site is predicted (18). Amino acids 167-190 may form a
distinct ATP binding P-loop (19), and the remaining sequence includes
the 11 conserved domains (amino acids 200-450) characteristic of
protein serine/threonine kinases (20). Amino acid analysis for membrane
topology predicts that the amino-terminal regions would lie in the
thylakoid lumen and the kinase would be stromal (21). A search for
related proteins identifies numerous protein kinases, including members
of the metazoan TGF receptor family, and their identities are shown
in Fig. 2A where identical amino acids are in
bold and similar sequences in gray. The sequence shown is ALK5, a subclass of human TGF
-I receptors, but identities are also found with Caenorhabditis elegans and
Drosophila receptors (22). These similarities lie both
within and outside of the kinase domains, but the reason this receptor
class is chosen above the other kinases families is the presence of the
SGSGSG box (bold underline) in both TAK1 and the TGF
-I
receptor. This region is the TGF
-I receptor family signature and
acts as the recognition sequence for the partner receptor TGF
-II,
which phosphorylates the TGF
-I receptor upon ligand binding
(23).
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The similarity of TAK1 to the TGF receptor suggested that there
might be a kinase cascade in the thylakoid. We also predicted that
there could be multiple TAK proteins and that TAK1 would be
phosphorylated by another TAK. A search of the Arabidopsis DNA data base indeed identifies two additional Tak
sequences, Tak2 and Tak3, and predictions of both
encoded proteins show that they contain the four sequenced peptides.
These genes were not identified in the initial search because they both
contain introns. The TAKs are 90% identical except TAK3 lacks the
carboxyl-terminal 41 amino acids of TAK1 and TAK2 (Fig. 2B),
but the significance of this is unknown. Tak1, but not TAK2 and TAK3,
contains the SGSGSG motif (Fig. 2C), and because the
TGF
-II receptor phosphorylates the TGF
-I receptor by identifying
the SGSGSG box, we draw a parallel between TGF
-I receptor and TAK1
and the TGF
-II receptor and TAK2 or TAK3. To explore this similarity
we determined whether isolated TAK was indeed phosphorylated and
whether the 55-kDa protein band represented multiple TAK proteins. Fig.
3A shows that the 55-kDa
Coommassie Blue-stained purified TAK fraction can indeed be resolved
into two bands of 55 and 56 kDa upon higher resolution gel
electrophoresis (lane Coom), and both proteins react with
the TAK serum (Fig. 3A, TAK). The 56-kDa and to a
much lesser degree the 55-kDa TAK react with anti-phosphothreonine and
anti-phosphoserine antiserum (Fig. 3A), indicating that the larger TAK is preferentially phosphorylated on threonine and serine residues. Anti-phosphotyrosine serum does not react with TAK (data not
shown). These observations strengthen but do not confirm the parallel
between TAKs and the TGF
receptors, and additional analysis is
required to explore these similarities. Although the TAK preparation does not phosphorylate itself (Fig. 1B), TAK phosphorylation
may be context-specific and require the
b6f complex.
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To confirm that the TAK genes indeed encode thylakoid proteins, we imported an in vitro translated TAK1 into isolated chloroplasts. The TAK1 gene does not contain any introns, and the transcribed region was amplified by PCR, cloned, and expressed in vitro as a 35S-labeled protein (Fig. 3B, lane Trl) that migrates as a 55.5-kDa protein, between the TAK doublet isolated from tissue (Fig. 3B, lane Coom). The in vivo phosphorylation of the Arabidopsis TAK may alter the protein mobility relative to the in vitro translated protein. Southern analysis indicates that only three Tak genes are present in Arabidopsis (data not shown). Thus at present we can not conclude which gene encodes which TAK band. The in vitro synthesized TAK1 (Fig. 3B, lane Trl) was incubated with isolated Arabidopsis chloroplasts (11), and the intact organelles were treated with protease to remove material not imported. TAK1 appears as a protease-resistant 55.5-kDa protein (Fig. 3B, lane Chl) and thus is imported and as predicted is not cleaved by the stromal protease. The 55.5-kDa labeled protein is located in the thylakoid but not stromal fraction (Fig. 3B, lane Thy versus Str). Because TAK1 is imported into the thylakoid and the predicted amino acid sequence contains the four sequenced peptides, we conclude that TAK1 and probably the 90% identical TAK2 and 3 are indeed thylakoid membrane proteins and encode at least one of the proteins identified as TAK in plant tissue.
The biochemical analysis of native TAKs, recombinant TAK1, and the
predicted amino acid sequence all indicate that TAKs are anchored in
the thylakoid membrane such that the protein kinase domain is stromal
and can phosphorylate the amino terminus of LHCP. The data are
consistent with a membrane topology where the 26 amino-terminal
residues lie in the thylakoid lumen, and this raises the possibility
that this domain not only serves as an uncleaved signal sequence but
also as a sensor of the lumen. It has been demonstrated that kinase
activation depends upon the binding of reduced plastoquinone to the
b6f complex (16), and it was
predicted that the kinase is intimately associated with the
b6f complex. TAK indeed co-purifies
with both the b6f complex and PS II.
Although we do observe reductant dependence of TAK activity in
vitro, it is neither known how this is effected nor how the
transmembrane domain or the lumenal tail may sense the reduced form of
the b6f complex as models predict.
Our experiments assayed the activity of an exogenously added TAK, and
the analysis of a kinase activity that is correctly associated with the
b6f complex and PS II awaits the
generation of TAK null alleles. The confirmation of a TAK cascade
requires the analysis of recombinant protein and mutant plant analysis,
and the similarities with the TGF receptor cascade remain suggestive
but predictive. TAK antiserum detects a 55/56-kDa protein band in the
thylakoids of pea and Chlamydomonas (data not shown),
indicating that TAK is of general significance to land plants and
algae. Future analysis can now employ these novel organelle kinases to
probe the mechanism of redox-controlled phosphorylation of membrane proteins.
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ACKNOWLEDGEMENTS |
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We thank Zheng Hui He and Masaaki Fujiki for help in the initial phase of the project and Karen Bernd, Mireille Perret, Tanya Wagner, Cathy Anderson, and Bill Zerges for helpful discussions.
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FOOTNOTES |
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* This work was supported by the Pew Charitable Trust and U. S. Department of Agriculture Grant 9701608 (to B. D. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Developmental, Cell,
and Molecular Biology Group, Botany Dept., Box 91000, Duke University,
Durham, NC 27708. Tel.: 919-613-8182; Fax: 919-613-8177; E-mail:
kohorn{at}acpub.duke.edu.
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ABBREVIATIONS |
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The abbreviations used are: PS II, photosystem II; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; DTT, dithiothreitol; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; CAPS, 3-(cyclohexylamino)propanesulfonic acid; LHCP, light harvesting protein; TGF, transforming growth factor.
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