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: 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) 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. 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