The plastid transcription kinase from mustard Sinapis alba L.A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function Karsten Ogrzewalla1, Markus Piotrowski2, Steffen
Trang 1The plastid transcription kinase from mustard ( Sinapis alba L.)
A nuclear-encoded CK2-type chloroplast enzyme with redox-sensitive function
Karsten Ogrzewalla1, Markus Piotrowski2, Steffen Reinbothe2,* and Gerhard Link1
1
Plant Cell Physiology & Molecular Biology and2Plant 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 organellochloroplast 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 vitrotranscription 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: gerhard.link@ruhr-uni-bochum.de
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)
Trang 2be 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
M A T E R I A L S A N D M E T H O D S
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¢) and 3 (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 SmaI site of
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 mMTris/HCl, pH 7.5, 50 mMKCl, 10 mMMgCl2and
40 lM[c-32P]GTP or [c-32P]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 30C for
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
1 mM and 25C 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 37C 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 4C for 12 h, followed by incubation with anti-(rabbit IgG) Ig (whole molecule) as an alkaline phosphatase conjugate (Sigma) for
1 h at 25C 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 theMAXENT3 algorithm andBIOLYNXsoftware (Micromass)
In organello chloroplast import
35S-Labeled translation products were synthesized in vitro using the wheat germ TNT quick coupled transcription/
Trang 3translation 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 vitrousing 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-StarTM
(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
R E S U L T S
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).
Trang 4typical 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 the35S-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 colias 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-32P]ATP (lane 1) or [c-32P]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 chloroplast import assays Left panel: 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).
Trang 5To 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-32P]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 50C 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; the a 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 andred ox characteristics of recombinant
cpCK2a andauthentic 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-32P]GTP as in (A)–(C).
Trang 6multiple 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 centrifugprepar-ation 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 mustardchloroplasts 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.
Trang 7D I S C U S S I O N
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 b subunit 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 on purified plastid RNA 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 bandof mustardPEP-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:
Fig 6 Genomic andtranscript analyses (A) Southern blot hybridiza-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 hidden break fragments [1,2].
Trang 8suggest 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
and cloned (for a recent review, see [45]), and in the case
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 free
and a minor bound 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-scription kinase, which in turn might affect both the activity 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
A C K N O W L E D G E M E N T S
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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