Báo cáo Y học: A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function docx

The plastid transcription kinase from mustard (
Sinapis alba
L.)
A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function
Karsten Ogrzewalla
1
, Markus Piotrowski
2
, Steffen Reinbothe
2,
* and Gerhard Link
1
1
Plant Cell Physiology & Molecular Biology and
2
Plant Physiology, University of Bochum, Germany
The plastid transcription kinase (PTK), a component of the
major RNA polymerase complex from mustard chloro-
plasts, has been implicated in redox-mediated regulation of
plastid gene expression. A cloning strategy to define the PTK
gene(s) resulted in the isolation of a full-length cDNA for a
protein with overall high homology with the a subunit of
cytosolic casein kinase (CK2) that contained an N-terminal
extension for a putative plastid transit peptide. Using
in organello chloroplast import studies, immunodetection
and MS, we found that the corresponding protein, termed
cpCK2a, is targeted to the chloroplast and is associated with
the plastid RNA polymerase PEP-A. The bacterially over-
expressed protein shows CK2 kinase activity and is subject to
glutathione inhibition in the same way as authentic chloro-
plast PTK. Furthermore, it readily phosphorylates compo-

nents of the plastid transcription apparatus in vitro with a
substrate specificity similar to that of PTK.
Keywords: chloroplast transcription factor; phosphorylation
control; plant nuclear gene; protein kinase CK2; redox
regulation.
Chloroplasts, the vital organelles of green plant cells,
contain the photosynthetic apparatus responsible for most
life on earth [1]. In addition, and in close physical proximity
to the photosynthetic apparatus, they have a functional gene
expression machinery different from that of the
nucleo-cytosolic compartment [2].
It has become increasingly clear that signaling mecha-
nisms exist that connect photosynthetic electron flow with
gene expression responses [3–5]. These mechanisms include
both phosphorylation/dephosphorylation and reversible
changes in redox state, and they operate at more than one
level of gene expression [6]. For instance, SH-group redox
regulation has been shown to control initiation of chloro-
plast translation in the case of the green alga Chlamydo-
monas reinhardtii, in which a redox-responsive oligomeric
protein complex capable of binding to the 5¢-untranslated
region of chloroplast mRNA has been shown to be a critical
component in this process [7–9]. In addition, several other
post-translational steps in chloroplast gene expression,
including translation elongation [10], RNA degradation
[11,12] and RNA splicing [13], have been shown to be
subject to redox regulation as well.
Several lines of evidence suggest that, in higher plant
chloroplasts, processes at the transcriptional level can also
be controlled by photosynthetic electron transport via the

reduced or oxidised state of signal-transmitting proteins.
The transcription rate of isolated chloroplasts has been
shown to be affected by both the spectral quality [14,15] and
intensity [16] of photosynthetic light, and this has further
been substantianted by the use of electron-transfer inhibi-
tors and redox-reactive reagents (for a recent review, see
[17]).
Chloroplasts, and possibly all plastid types, contain dual-
transcription machinery consisting of two different RNA
polymerases named nuclear-encoded phage-type plastid
RNA polymerase and bacterial-type plastid RNA polym-
erase (PEP) [18]. The former is a single-subunit (phage-type)
enzyme of nuclear origin, whereas the latter is a multisub-
unit (bacterial-type) polymerase with chloroplast-encoded
core subunits. Depending on the plastid type, the PEP
enzyme can have a variable number of accessory polypep-
tides, most of which seem to have a regulatory role in
transcription. For instance, the major chloroplast RNA
polymerase (PEP-A) from mustard (Sinapis alba L.) has at
least 15 subunits, including polypeptides sequence-related to
iron superoxide dismutase, RNA-binding proteins, and
annexins [19].
One of the polymerase-associated components has been
functionally identified on the basis of its in vitro activity as a
serine-specific protein kinase [20]. It was shown to affect
in vitro transcription in a reversible manner depending on its
own phosphorylation state. Furthermore, its activity varies
with its SH-group redox state, as operationally defined by
the extent of thiol/disulfide exchange at vicinal cysteine
residues [21]. This protein kinase was named plastid

transcription kinase (PTK) because of its association and
functional interaction with the PEP-A RNA polymerase.
Biochemical characterization [20,21] revealed that PTK can
Correspondence to G. Link, Plant Cell Physiology & Molecular
Biology, University of Bochum, Universitaetsstr. 150,
D-44780 Bochum, Germany.
Fax: + 49 234 3214 188, Tel.: + 49 234 322 5495,
E-mail:
Abbreviations: CK, casein kinase; PEP, bacterial-type plastid RNA
polymerase with core subunits encoded by organellar genes; pSSU,
small subunit precursor; PTK, plastid transcription kinase; Rubisco,
ribulose-1,5-bisphosphate carboxylase/oxygenase.
Enzymes: DNA-dependent RNA polymerase (EC 2.7.7.6); protein
kinase (EC 2.7.1.37); ribulose-1,5-bisphosphate carboxylase/oxygenase
(EC 4.1.1.39); superoxide dismutase (EC 1.15.1.1).
*Present address: Plant Molecular Genetics, University of Grenoble,
38041 Grenoble, France.
(Received 26 March 2002, revised 17 May 2002,
accepted 23 May 2002)
Eur. J. Biochem. 269, 3329–3337 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03017.x
be best classified into the so-called CMGC group of protein
kinases [22]. This group includes mostly nucleo-cytosolic
members that often represent terminal components of
signaling chains acting on, for example, (nuclear) transcrip-
tion factors [23]. This is particularly true for casein kinase II
(CK2), which is a well-known transcriptional regulator both
in animal and yeast [24] as well as in plant systems [25].
CK2-type kinase activity has also been reported in
chloroplasts [26], and among the known substrates are
photosynthetic proteins such as CP29 [27] and the b subunit

of the ATP synthase [28]. Considering the biochemical
similarity of PTK to (nucleo-cytosolic) CK2 kinases noted
in our previous studies [20,21], we set out to clone the gene
for the catalytic PTK component, and to study the
recombinant protein in relation to the authentic chloroplast
transcription kinase.
MATERIALS AND METHODS
PCR cloning and library screening
Primer 1 (5¢-CCATTGAACAGCAAGGGACTCG-3¢)was
derived from Arabidopsis thaliana EST sequence 11926,
GenBank accession number T88230 (now assigned to
gi17065109 for a putative Ck2a gene). It was used in
combination with a vector primer (5¢-AGGGATGTTTA
ATACCACTAC-3¢) for PCR amplification from a mustard
cDNA library. This HybriZAP (Stratagene) library had
previously been generated using RNA from 5-day-old light-
grown mustard seedlings [29]. Resulting PCR fragments
were purified using the QIAquick kit (Qiagen), cloned into
the EcoRV site of pBluescript (Stratagene), and then
sequenced. A positive clone, pBS/CK2A-0.3 containing an
 300-bp insert with Ck2a homology, was used as a probe for
rescreening of the cDNA library by plaque-filter hybridiza-
tion. Sequencing identified clone pAD/CK2A-1.5, which
contains the full-length Cpck2a cDNA sequence. This clone
served as a template for further PCR amplification. Primers 2
(5¢-TCATTGGGCACGCGGGGTGGA-3¢)and3(5¢-GC
ACAGAAGATCGGTAAATCC-3¢) resulted in amplifica-
tion of an  1-kb fragment containing the coding region
without the transit peptide region. This PCR product was
purified as described above and cloned into the SmaIsiteof

pBluescript. The insert was subsequently excised with
BamHI and KpnI and cloned into the expression vector
pQE30 (Qiagen). Primers 2 and 4 (5¢-ATGGCCTTTAG
GCCTATCGGA-3¢) were used for amplification of the
1.2-kb full-length coding region of Cpck2a. After purification
(see above) the fragment was cloned into the EcoRV site of
pBluescript vector, resulting in clone pBS/CK2A-1.2.
Protein kinase assays
Kinase activity was assayed in a reaction mixture containing
20 m
M
Tris/HCl, pH 7.5, 50 m
M
KCl, 10 m
M
MgCl
2
and
40 l
M
[c-
32
P]GTP or [c-
32
P]ATP (10 lCiÆmmol
)1
). Where
indicated, 2 lg hydrolyzed and partially dephosphorylated
casein (Sigma, C4765) was added to the reaction mixture as
substrate for phosphorylation. After incubation at 30 °Cfor

30 min, reactions were stopped by the addition of SDS
sample buffer, and the polypeptides were then separated by
SDS/PAGE (10% or 12% gels) [30]. The gels were
subsequently dried and exposed to a phosphoimaging plate
(Fuji BAS 2040) or autoradiographed using Kodak
X-Omat films and Dupont Quanta-II screens.
Bacterial expression of cpCK2a
Recombinant cpCK2a lacking the transit peptide and
containing an N-terminal hexahistidine tag was expressed
in Escherichia coli strain M15 using the pQE system
(Qiagen). After isopropyl thio-b-
D
-galactoside induction at
1m
M
and 25 °C for 2 h, cells were lysed and soluble
cpCK2a protein was purified on a Ni-nitrilotriacetatic acid–
agarose (Qiagen) column according to the manufacturer’s
instructions. Inclusion bodies were isolated from cultures
after incubation at 37 °C for 4 h, and recombinant protein
was solubilized with SDS [31].
Antibodies and immunoblot analysis
Rabbit antisera directed against the recombinant cpCK2a
protein after solubilization from bacterial inclusion bodies
were generated at Eurogentech using their standard
immunization protocol. Antibodies were purified from
whole sera using antigen-affinity chromatography after
coupling of cpCK2a to CNBr-activated Sepharose 4B
(Amersham Biosciences). For immunodetection, protein
samples were separated by SDS/PAGE and transferred to

nitrocellulose membranes. They were then probed with
purified cpCK2a primary antibody at 4 °C for 12 h,
followed by incubation with anti-(rabbit IgG) Ig (whole
molecule) as an alkaline phosphatase conjugate (Sigma) for
1hat25°C. Signals were detected using nitroblue tetrazo-
lium/5-bromo-4-chloro-3-indolyl phosphate.
Purification of PTK and PEP-A RNA polymerase
The chloroplast transcriptional complex (PEP-A and PTK)
was purified from 5-day-old light-grown mustard seedlings
as described [21]. Fractions were assayed for protein kinase
activity as outlined above, and for RNA polymerase activity
[32]. In brief, chloroplast lysates were chromatographed on
heparin–Sepharose CL6B (Amersham Biosciences). Frac-
tions containing both the RNA polymerase and PTK
activity were pooled and either used directly or further
purified by centrifugation on linear 15–30% (v/v) glycerol
gradients.
In-gel protein digestion and MS
Coomassie-stained protein bands were in-gel digested with
sequencing-grade modified trypsin (Promega) [33]. After
extraction from the gel, peptides were desalted using
ZipTips C18 (Millipore). MS measurements were carried
out in a Q-TOF2 (Micromass). The nanospray sample was
introduced in positive ion mode and spectra were recorded
at m/z 400–1600 and 2.4 s integration time. Doubly or triply
charged molecules were selected for fragmentation in MS/
MS mode, and spectra were analysed using the
MAXENT
3
algorithm and

BIOLYNX
software (Micromass).
In organello
chloroplast import
35
S-Labeled translation products were synthesized in vitro
using the wheat germ TNT quick coupled transcription/
3330 K. Ogrzewalla et al.(Eur. J. Biochem. 269) Ó FEBS 2002
translation system (Promega). Chloroplast isolation from
rosette leaves of 3-week-old Arabidopsis plants by differen-
tial centrifugation, followed by Percoll (Amersham Bio-
sciences) density gradient centrifugation, and subsequent
import assays were carried out as described previously [34].
In brief, the translation reaction mixture was incubated with
intact chloroplasts, which were then treated with thermo-
lysin to remove adhering proteins. Membrane and stroma
fractions were prepared by differential centrifugation and
polypeptides were analysed by SDS/PAGE and subsequent
autoradiography. The mRNA-directed translation products
representing the precursor of the small subunit of ribulose-
1,5-bisphosphate carboxylase/oxygenase (Rubisco pSSU)
from barley served as a control [34].
Southern and Northern blot analysis
Total DNA was isolated from 5-day-old light-grown
mustard seedlings using the CTAB method as described
[35]. After restriction enzyme digestion of the DNA, 20-lg
samples were separated on 1% (w/v) agarose gels and
blotted on to positively charged nylon membrane (Roche).
The 300-bp cDNA insert of pBS/CK2A-0.3 was transcribed
in vitro using digoxigenin (DIG) labeling (Roche). Blotted

DNA fragments were probed with the DIG-labeled RNA in
50% (v/v) formamide and 5 · NaCl/Cit at 50 °C for 12 h.
Washing was in 0.1% (w/v) SDS in either 0.5 · NaCl/Cit
or, at higher stringency, in 0.1 · NaCl/Cit, and chemilumi-
nescent bands were then detected using CDP-Star
TM
(Roche).
Total RNA was prepared from 5-day-old mustard
seedlings grown in the light or in the dark as described
[36]. Samples of 10 lg were separated in 1.5% (w/v) agarose
gels containing 6.7% (v/v) formaldehyde and transferred to
positively charged nylon membranes. The blots were probed
with the DIG-labeled 300-bp cpCK2a transcript in
5 · NaCl/Cit at 68 °C for 12 h. They were then washed
and treated with CDP-Star following the Roche users’
guide.
RESULTS
Cloning of the cDNA for a putative chloroplast
CK2 kinase
A nuclear gene for a plastid-localized CK2-type protein
kinase would be expected to give rise to a precursor protein
that reveals both an N-terminal transit peptide and
conserved CK2 elements. Database searches identified an
Arabidopsis EST (11926; GenBank accession number
T88230) that could potentially specify a protein that fulfils
these criteria. By using a primer derived from the 5¢ end of
this sequence (primer 1) in combination with a vector
primer, we were able to amplify a 300-bp PCR product from
a mustard cDNA library. The derived amino-acid sequence
showed a conserved stretch of residues reminiscent of

(nucleo-cytosolic) CK2a subunits, which was preceded by a
region assigned both by PSORT [37] and ChloroP [38] as a
potential plastid transit peptide (data not shown).
We next used the 300-bp fragment as a probe to screen
the mustard cDNA library by plaque-filter hybridization,
which led to the isolation of an  1.5-kb cDNA insert
with an ORF coding for 414 amino acids (clone pAD/
CK2A-1.5).
BLAST
[39] searches (not shown) and multiple
alignments [40] with amino-acid sequences from A. thaliana
(gi585349), maize (gi3318993), rice (gi12697577), human
(gi11421546) and mouse (gi3413816) (Fig. 1) suggested that
this mustard cDNA clone contained the complete coding
region for a mature CK2a protein. In addition, the derived
mustard protein was found to have an N-terminal exten-
sion, which was subsequently analysed for features consis-
tent with a possible role as a transit peptide.
Chloroplast import
As shown in Fig. 2A, the N-terminal extension of the
putative CK2a precursor is rich in serine and threonine
residues and contains many positively charged amino acids
but only a few acidic amino acids, which are considered
Fig. 1. Alignment of the putative chloroplast trancription kinase with
nucleo-cytosolic CK2a proteins. The derived protein of the mustard
cDNA clone (Sin; EMBL accession number AJ420786) is shown on
top, followed by CK2a from Arabidopsis (Ara; gi585349), rice (Ory;
gi12697577), maize (Mai; gi3318993), human (Hom; gi11421546), and
mouse (Mus; gi3413816). Marked residues in this ClustalW alignment
[40] include identical positions (*) as well as conservative (:) and

semiconservative substitutions (.). Also indicated are the four cysteines
mentioned in the Discussion as well as the putative cleavage site of the
predicted transit peptide (Fig. 2).
Ó FEBS 2002 Cloned redox-responsive chloroplast transcription kinase (Eur. J. Biochem. 269) 3331
typical features of chloroplast transit peptides [41]. ChloroP
[38], PSORT [37] and PCLR [42] all predicted a significant
(at least 60%) probability of chloroplast import. As a
conserved cleavage-site motif according to [43] could not be
detected within the N-terminal extension, we tentatively
assigned the potential site to the location predicted by
ChloroP, i.e. between residues 66 (leucine) and 67 (alanine).
To demonstrate the chloroplast targeting of the CK2a-
like mustard protein, we carried out in organello import
experiments (Fig. 2B). As a control, an in vitro-synthesized
small subunit precursor of ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco pSSU; not shown) was used
[34], which is a prototype nuclear-encoded protein localized
to the chloroplast stroma [41]. After coupled transcription-
translation of clone pBS/CK2A-1.2 in the wheat germ TNT
system (Promega), SDS/PAGE of the
35
S-labeled reaction
products revealed the presence of a major 48-kDa polypep-
tide corresponding to the expected full-length size (Fig. 2B,
lane 1). This 48-kDa product was found to be largely absent
after incubation with chloroplasts and instead a smaller
band of 38–40-kDa appeared (lane 2) which was resistant to
thermolysin treatment of the chloroplasts (lane 3). After
fractionation of the organelles into membrane (lane 4) and
stroma (lane 5) fractions, the putative processed polypep-

tide was predominantly found in the stroma. This directly
reflects the situation observed for the 20-kDa Rubisco
pSSU polypeptide, which likewise was converted into a
smaller ( 15 kDa) thermolysin-resistant product and was
localized to the stroma (not shown). These results streng-
thened the conclusions from the sequence analyses (Fig. 2A)
that the mustard CK2a-like protein is synthesized as a
precursor (Fig. 2B), which is imported post-translationally
into the chloroplast and processed to mature size. To
indicate the plastid localization, the protein represented by
cDNA clone pAD/CK2A-1.5 hence was named cpCK2a
and the corresponding cDNA sequence Cpck2a.
Bacterial expression and functional analysis of cpCK2a
To study functional properties of the gene product in vitro,
we constructed a truncated version of cpCK2a lacking the
66-amino-acid putative transit peptide. It was expressed in
E. coli as a fusion protein with an N-terminal hexahistidine
tag and, after nickel-chelate affinity purification, the
recombinant protein was tested for protein kinase activity
(Fig. 3). The His-tagged cpCK2a protein phosphorylates
casein in the presence of either [c-
32
P]ATP (lane 1) or
[c-
32
P]GTP (lane 2), and it is inhibited by the polyanion
heparin (lane 3). The same enzymatic characteristics, which
are typical CK2 features [23], were also observed with the
authentic PTK from mustard chloroplasts. As is shown in
Fig. 3B for a partially purified preparation (heparin–

Sepharose stage), and in Fig. 3C for the more highly
purified enzyme (glycerol gradient stage), both PTK prep-
arations shared the ability to phosphorylate casein using
ATP (lanes 1) or GTP (lanes 2) as phosphate donor, and in
both cases this activity was inhibited in the presence of
heparin (lanes 3).
Another approach to test the biochemical similarity
between recombinant cpCK2a and authentic PTK was
based on findings that the latter is selectively inhibited by
GSH, but not by either the oxidized form (GSSG) or other
reductants such as dithiothreitol and 2-mercaptoethanol
[21]. As shown in Fig. 3, lower panel, the recombinant
cpCK2a protein was inhibited by GSH (Fig. 3D) but not by
GSSG (Fig. 3E), and neither dithiothreitol nor 2-mercapto-
ethanol had any effect on its kinase activity (not shown).
Hence, these data suggest an essentially similar in vitro
behaviour of cpCK2a and PTK activity in response to
SH-group redox state.
We next asked whether both the authentic chloroplast
PTK and the recombinant cpCK2a protein were capable of
using the same set of transcription-associated proteins as
phosphorylation targets. As chloroplast PTK had previ-
ously been shown to phosphorylate sigma-like transcription
factors [20], the same may be true also for cpCK2a.With
recombinant sigma factor 1 (SIG1) from mustard [29] as a
substrate (Fig. 4A), neither cpCK2a (lane 2) nor SIG1
(lane 4) alone showed any phosphorylated polypeptides in
the kinase assay. Mixing the two recombinant proteins,
however, resulted in a single phosphorylation signal at
43 kDa (lane 3), i.e. the size of the sigma factor [29].

Fig. 2. Transit peptide and in organello chloroplast import of cpCK2a.
(A) The 66-amino acid N-terminal region of the cloned full-length
protein shows features of plastid transit peptides, i.e. high contents of
serine and threonine residues (shaded) and positively charged amino
acids (+). (B) In organello chloroplastimportassays.Leftpanel:The
48-kDa cpCK2a translation product (lane 1) was incubated with
Arabidopsis chloroplasts, resulting in a processed 38- to 40-kDa
polypeptide detectable before (lane 2) and after (lane 3) thermolysin
treatment. After import, the organelles were lysed and separated into
membrane (lane 4) and stroma fractions (lane 5). Reactions were
analysed by SDS/PAGE, followed by autoradiography. Numbers in
margins: sizes of precursor and processed polypeptides (kDa).
3332 K. Ogrzewalla et al.(Eur. J. Biochem. 269) Ó FEBS 2002
To test further the possible relation of the recombinant
cpCK2a polypeptide to the plastid transcription apparatus
from mustard, we took advantage of the known phos-
phorylation pattern of a partially purified PEP-A RNA
polymerase that contains associated PTK activity (kinase–
polymerase complex; heparin–Sepharose stage) [20]. As
shown in Fig. 4B, lane 1, incubation of this fraction in the
presence of [c-
32
P]GTP resulted in a number of labeled
polypeptides. None of these signals were detected when
cpCK2a was incubated alone in the absence of the
chloroplast protein substrates; neither did we observe any
qualitative changes in the phosphorylation pattern when the
recombinant protein was added to the latter (data not
shown). However, when the endogenous chloroplast kinase
was first heat-inactivated at 50 °C for 10 min (preventing

phosphorylation; Fig. 4B, lane 3), subsequent addition of
cpCK2a restored the phosphorylation signals (lane 2) in a
pattern very similar to that with active PTK (lane 1). To
substantiate this, we used highly purified PEP-A RNA
polymerase that had retained only weak endogenous PTK
activity (see Fig. 3C) [20]. Incubation of neither PEP-A
alone (Fig. 4B, lane 5) nor cpCK2a alone (see Fig. 4A,
lane 2) gave any significant phosphorylation signals. As
shown in Fig. 4B lane 4, however, the full reaction mixture
containing both the polymerase and recombinant kinase
produced a pattern of labeled PEP-A polypeptides similar
to that previously observed after phosphorylation by
chloroplast PTK, i.e. major bands at 72–76 kDa and
30 kDa [19,20].
Together, the data presented in Fig. 4A,B therefore
support the notion that recombinant cpCK2a has a
substrate specificity similar to that of PTK with regard to
phosphorylation of chloroplast proteins.
Detection of cpCK2a in mustard chloroplast
preparations
If recombinant cpCK2a was equivalent to the catalytic
subunit of PTK, it should be possible to demonstrate the
direct physical existence of a CK2a-type subunit as a
functional constituent of the chloroplast transcription
apparatus in vivo. This was addressed by immunodetection
(Fig. 5) and MS (Table 1).
Both partially (heparin–Sepharose stage; Fig. 5A) and
highly purified (glycerol gradient stage; Fig. 5B) PEP-A
preparations were probed using an antibody to recombinant
cpCK2a. In either experiment, after SDS/PAGE and

Western blotting, subsequent immunodetection revealed a
signal at 38–40 kDa (Fig. 5A,B, lane 2), i.e. the estimated
size of cpCK2a polypeptide lacking the transit peptide
(Fig. 2). That this signal appears as a double band in
Fig. 5A, lane 2 (and less so in Fig. 5B, lane 2) is probably
the result of limited proteolysis, as has been observed for
nucleo-cytosolic CK2a from animal sources [23].
To confirm the presence of a PEP-A constituent that is
immunochemically related to cpCK2a by an independent
technique, PEP-A fractions were also analysed by electro-
spray ionization-MS. Initial attempts using highly purified
preparations after glycerol gradient centrifugation (Fig. 5B)
did not give consistent results, because of limited amounts of
material and variations from one preparation to another
(data not shown; see Discussion). Using partially purified
PEP-A after heparin–Sepharose chromatography (Fig. 5A),
the prominent stained band at 38–40 kDa was found to
contain three similar-sized but different polypeptides
(Table 1): cpCK2a;thea core subunit (rpoA gene product)
of the plastid RNA polymerase; an RNA-binding protein
that had been previously identified as part of the polymerase
complex [19]. The cpCK2a polypeptide in this triple band
was present in substochiometric amounts, which explains
why it was previously difficult to detect this minor
component in the more highly purified PEP-A preparation
by electrospray ionization-MS.
Southern and Northern blot analyses
Mustard total genomic DNA was digested and hybridized
to a DIG-labeled RNA probe generated by transcription of
the 300-bp insert of pBS/CK2A-0.3. Washing under stand-

ard conditions (see Materials and methods) resulted in
Fig. 3. Phosphorylation and redox characteristics of recombinant
cpCK2a and authentic PTK. Upper panel: the bacterially expressed
cpCK2a protein (A) and chloroplast PTK after heparin–Sepharose
chromatography (B) or after additional gycerol gradient centrifugation
(C) were assayed for kinase activity. Reaction mixtures containing
casein as substrate were carried out with [c-
32
P]ATP (lane 1) or
[c-
32
P]GTP in the absence (lane 2) or presence (lane 3) of heparin.
Samples were subjected to SDS/PAGE, followed by autoradiography.
Lower panel: recombinant cpCK2a was incubated with increasing
concentrations of reduced (D) or oxidized glutathione (E) (lanes 1–4),
followed by activity assays using [c-
32
P]GTP as in (A)–(C).
Ó FEBS 2002 Cloned redox-responsive chloroplast transcription kinase (Eur. J. Biochem. 269) 3333
multiple signals, the majority of which were thought to be
due to detection of nucleo-cytosolic CK2A-type sequences
by this probe (data not shown). At higher stringency,
however, only a few signals per lane were visible (Fig. 6A),
suggesting the possible existence of a single-copy gene or a
small gene family for cpCK2a in mustard.
For Northern blot transcript analysis, total RNA was
isolated from 5-day-old mustard seedlings grown in either
the light or dark, and gel blot hybridizations were carried
out at equal loading per lane (Fig. 6B, lower panel) using
the same probe as described above. As shown in Fig. 6B,

upper panel, this revealed a single RNA signal at  1.5 kb,
i.e. matching the size of the full-length cpCK2a cDNA
(Fig. 2). The labeled hybridization band was visible with
RNA from either dark-grown or light-grown seedlings.
Unlike for the b-tubulin transcript [44] used as a constitutive
control (not shown), the signal intensity was higher under
light growth conditions, suggesting that cpCK2a gene
expression at RNA level is not completely constitutive but
may be under moderate light control.
Fig. 4. Substrate recognition of cpCK2a. (A) Phosphorylation of
recombinant sigma factor 1 (SIG1) from mustard by cpCK2a.Purified
SIG1 (lane 1, silver-stained) was incubated in the presence (lane 3) or
absence (lane 4) of cpCK2a under phosphorylation conditions. A
control reaction mixture contained only cpCK2a (lane 2). (B)Phos-
phorylation of chloroplast polypeptides. A partially (heparin–
Sepharose) purified RNA polymerase preparation with associated
PTK activity [20] showed phosphorylation of endogenous substrates
(lane 1). The same fraction did not show any kinase activity after heat
treatment at 50 °C for 10 min (lane 3). When the heat-treated fraction
was supplemented with recombinant cpCK2a and again tested for
kinase activity (lane 2), a phosphorylation pattern comparable to that
in lane 1 was observed. A highly purified PEP-A polymerase prepar-
ation after glycerol gradient centrifugation showed little, if any,
phosphorylation activity in the absence of cpCK2a (lane 5). In its
presence, effective labeling of the endogenous substrates was noticeable
(lane 4), with a pattern that closely resembled that for PEP-A
phoshorylation by PTK [20]. All phosphorylation assays were
performed using [c-
32
P]GTP.

Fig. 5. Immunodetection of cpCK2a in transcriptionally active fractions
from mustard chloroplasts. PEP-A RNA polymerase preparations were
analysed by silver-staining (lane 1) and by immunoblotting using
antibodies raised against the recombinant cpCK2a polypeptide
(lane 2). (A) Partially purified fraction after heparin–Sepharose chro-
matography; (B) highly purified PEP-A after subsequent glycerol
gradient centrifugation.
3334 K. Ogrzewalla et al.(Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
In this study we have obtained evidence for the existence of
a nuclear-encoded chloroplast protein from mustard
(S. alba L.) which can be assigned as a CK2a-type protein
kinase on the basis of the following criteria. (a) The cloned
protein shows overall high homology with nucleo-cytosolic
CK2a sequences from other organisms. (b) In addition, it
has an N-terminal extension typical of chloroplast transit
sequences. (c) The gene product synthesized in vitro by
coupled transcription–translation was found to be imported
into isolated chloroplasts as a precursor, followed by
processing to a size expected for the mature protein. (d)
The bacterially overexpressed and purified recombinant
protein had biochemical characteristics typical of the
catalytic subunit of protein kinase CK2 [23]. (e) The
authentic plastid protein was detected as a component of
the chloroplast transcription apparatus by both antibodies
raised against the recombinant protein and MS.
The existence of a plastid CK2 activity was initially
demonstrated by Kanekatsu and coworkers [26], who were
able to biochemically characterize such an enzyme from
spinach chloroplasts. In addition, chloroplast proteins were

identified that could serve as potential substrates for CK2-
type kinases, including the chlorophyll a/b-binding PSII
protein CP29 [27] and the bsubunit of chloroplast ATP
synthase [28].
That a protein kinase with biochemical properties similar
to nucleo-cytosolic CK2 could be a component of the
chloroplast transcription apparatus was initially borne out
by in vitro studies onpurified plastidRNA polymerase PEP-A
[20,21]. It was shown that this polymerase contains an
associated serine/threonine kinase activity named PTK. The
cloned recombinant cpCK2a protein described in the
present work resembles the authentic PTK by several
criteria. (a) Both enzyme preparations are capable of using
ATP as well as GTP as a phospho donor. (b) They both are
inhibited by heparin (this work) and 5,6-dichloro-1-b-
D
-
ribofuranosylbenzimidazole [20], and the latter was found
also to severely affect run-on transcription in isolated
chloroplasts (T. Pfannschmidt, K. Ogrzewalla & G. Link,
unpublished data). (c) Both PTK and recombinant cpCK2a
seem to act independently of second-messenger molecules
[20] (data not shown), and both are capable of using plastid
sigma factor(s) and other RNA polymerase-associated
proteins as phosphorylation substrates (Fig. 4). (d) Finally,
both PTK and cpCK2a activity is negatively affected in vitro
by the presence of GSH, whereas other reducing reagents
such as 2-mercaptoethanol and dithiothreitol seem to have
little effect [21] (Fig. 3, this work). Together, these data
Table 1. MS assignment of polypeptides within the 38-kDa band of mustard PEP-A RNA polymerase. Chloroplast RNA polymerase preparations

after heparin–Sepharose chromatography [20] were subjected to SDS/PAGE, followed by electrospray ionization-QTOF MS and database peptide
analyses as described in Materials and methods. Each component was identified by two peptides. L, note that leucine and isoleucine cannot be
distinguished by Q-TOF MS. M*, oxidized methionine.
Protein
(plant species)
GenBank
identifier
Identified by peptide:
m/z Charge Sequence
RNA polymerase a subunit gi7388101 456.7 2
+
EALHEASR
(S. alba) 574.7 2
+
GQADTLGLAM*R
RNA-binding protein gi2765081 604.3 2
+
DQHFFASVEK
(A. thaliana) 885.9 2
+
QLPGESDQDFADFSSK
cpCK2a gi17977867 720.9 2
+
VLYPTLSDYDVR
(S. alba) 754.4 2
+
VLGTDELNTYLNR
Fig. 6. Genomic and transcript analyses. (A)Southernblothybridiza-
tion of mustard total DNA digested with EcoRI (E, lane 1), BamHI
(B, lane 2) and HindIII (H, lane 3) and probed with DIG-labeled

cpCK2A-RNA. (B) RNA gel blot hybridization using total RNA from
dark-grown (lane 1) and light-grown (lane 2) mustard seedlings.
Upper panel: autoradiograph. Lower panel: ethidium bromide-stained
samples (10 lg each). The heavily stained bands contain 25S rRNA
(top; 3.7 kb), 18S rRNA (second; 2.0 kb), and large chloroplast
rRNAs, including the 23S Ôhidden breakÕ fragments [1,2].
Ó FEBS 2002 Cloned redox-responsive chloroplast transcription kinase (Eur. J. Biochem. 269) 3335
suggest that the cloned recombinant protein representing
cpCK2a closely mimics the catalytic component of PTK
that is associated with the PEP-A polymerase.
These findings raise a number of intriguing questions
about the role of a CK2-type chloroplast kinase as a
potential mediator of both phosphorylation and redox
signaling, its own regulation, and the identity of its
interaction partners. In this context, it seems appropriate
to compare the chloroplast enzyme with plant
nucleo-cytosolic CK2, which has long been characterized
andcloned(forarecentreview,see[45]),andinthecase
of CK2a from Zea mays even the crystal structure is
available [46]. These studies have provided detailed
insights into the domain structure of the a subunit [46],
but the role of reversible disulfide bond formation was
not addressed. Furthermore, available evidence in animal
cells does not support a role for nucleo-cytosolic CK2 in
redox signaling [47]. It is interesting to note, however,
that the mustard cpCK2a sequence in Fig. 1 has four
cysteine residues (C139, C163, C221, and C294), the
C-terminal pair of which is conserved in all aligned
species (both animal and plant), whereas the N-terminal
pair seems to be plant-specific. Considering the known

differences between plant and animal CK2a (such as
different length, stability and interaction properties) [45],
it is conceivable that redox regulation may be another
distinguishing feature.
In addition, other polypeptides that interact with the
a subunit could be expected to modulate the catalytic
properties of the kinase. In the case of nucleo-cytosolic
CK2a, a prototype interaction protein is the regulatory
a subunit [23], although it is interesting to note that plant
CK2 preparations lacking the a polypeptide have been
described [45]. Our preliminary evidence from immunose-
lection studies suggests that several proteins of the organ-
ellar transcription machinery specifically interact with
cpCK2a (K. Ogrzewalla, D. Scharlau & G. Link, unpub-
lished data). This is consistent with our previous findings
that the (PTK) kinase activity can be biochemically purified
as a more than 100-kDa subcomplex of the chloroplast PEP
transcription apparatus containing several polypeptides
[20]. Work is in progress to investigate the contribution of
these additional components to the activity and specificity of
the complex.
Part of the work reported here was directed towards the
question of whether cpCK2a sequences can be detected in
chloroplasts, and more specifically, in purified PEP-A
preparations. Both the immunodetection experiments
(Fig. 4) and the results of MS (Fig. 5) support this notion,
although the latter technique detected CK2-related peptides
in partially purified PEP-A preparations, but not the most
highly purified preparations after glycerol gradient centrif-
ugation. This apparent failure is most likely related to the

presence of the cpCK2a polypeptide in a band that contains
two additional polypeptides, i.e. the a core subunit of the
RNA polymerase (rpoA gene product) and an RNA-
binding protein previously described [19] (Table 1). We note
that the PTK activity was found to be loosely associated
with the chloroplast polymerase activity, with a major ÔfreeÕ
and a minor ÔboundÕ form of the kinase detected throughout
the purification [20]. This is reminiscent of the situation
reported for mammalian (nucleo-cytosolic) CK2 in prepa-
rations of nuclear RNA polymerase I [48], where the kinase
is also loosely associated and present in lower than expected
amounts. The reason for this behaviour is not clear, but
could reflect different conformational states of the tran-
scriptionkinase,whichinturnmightaffectboththeactivity
and interaction with other components of the transcription
complex.
In view of the close physical and functional similarity
between the cloned cpCK2a polypeptide and the authentic
PTK kinase moiety of chloroplast RNA polymerase PEP-
A, it seems reasonable to suggest that it is this CK2a-type
activity that is directly involved in the phosphorylation and
redox control of the PEP-A transcription system [20,21].
Available in cloned and overexpressed form, this gene
product now provides an opportunity to investigate its
role in protein–protein interaction studies as well as a target
for mutagenesis and functional analysis of chloroplast
transcription.
ACKNOWLEDGEMENTS
We thank Professor E. W. Weiler for guidance and support during
mass spectrometry, and Anke Homann for critical reading of the

manuscript and discussion. This work was funded by the Deutsche
Forschungsgemeinschaft (Li 261/18-1; FOR 387/1-1).
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