doi:10.1016/j.jmb.2005.12.079 J. Mol. Biol. (2006) 357, 535–549

TCP34, a Nuclear-encoded Response Regulator-like TPR of Higher

P. Weber1†, H. Fulgosi1†, I. Piven1,L.Mu¨ ller2, K. Krupinska2 V. H. Duong1, R. G. Herrmann1 and A. Sokolenko1*

1Department fu¨r Biologie I We describe the identification of a novel protein, designated Bereich Botanik, Ludwig- TCP34 (tetratricopeptide-containing chloroplast protein of 34 kDa) due to Maximilians-Universita¨t the presence of three tandemly arranged tetratricopeptide repeat (TPR) Menzingerstr. 67, 80638 arrays. The presence of the genes encoding this protein only in the Mu¨nchen, Germany of higher but not in photosynthetic cyanobacterial prokaryotes suggests that TCP34 evolved after the separation of the higher plant 2Institut fu¨r Botanik, lineage. The in vitro translated precursor could be imported into intact Universita¨t Kiel, spinach chloroplasts and the processed products showed stable association Olshausenstr. 40, with thylakoid membranes. Using a specific polyclonal antiserum raised 24098 Kiel, Germany 1 2 against TCP34, three protein variants were detected. Two forms, T and T , were associated with the thylakoid membranes and one, S1, was found released in the stroma. TCP34 protein was not present in etioplasts and appeared only in developing chloroplasts. The ratio of membrane-bound and soluble forms was maximal at the onset of photosynthesis. The high molecular mass thylakoid TCP34 variant was found in association with a transcriptionally active protein/DNA complex (TAC) from chloroplasts and recombinant TCP34 showed specific binding to Spinacia oleracea chloroplast DNA. Two TCP34 forms, T1 and S1, were found to be phosphorylated. An as yet unidentified phosphorelay signal may modulate its capability for plastid DNA binding through the state of the putative response regulator-like domain. Based on the structural properties and biochemical analyses, we discuss the putative regulatory function of TCP34 in plastid gene expression. q 2005 Elsevier Ltd. All rights reserved. Keywords: DNA-binding; post-transcriptional regulation; signal trans- *Corresponding author duction; TAC; TPR array

Introduction are synthesized in the cytosol and subsequently imported into the organelle.1–6 Both, genetic and The expression of chloroplast genes coding for biochemical data suggest that these factors act components of the photosynthetic machinery either as constituents of the organellar transcrip- depends largely on nucleus-encoded factors that tion/translation machinery or are involved in various post-transcriptional processes, such as RNA stabilization and processing.7–10 Recent work † P.W. and H.F. contributed equally to this work. indicates that several chloroplast involved Present addresses: P. Weber, Institut fu¨ r Neuropatho- in post-transcriptional steps of chloroplast gene logie, Ludwig-Maximilians-Universita¨t, Marchioninistr. expression contain tetratricopeptide repeat (TPR) or 17, 81377 Mu¨ nchen, Germany; H. Fulgosi, Department of TPR-like motifs, which usually interact with large Molecular Genetics, Ruder Bosˇkovic´ Institute, Zagreb, protein complexes.11,12 TPR arrays consist of a Croatia; V. H. Duong, Centre of Biotechnology, Vietnam repeated, degenerated stretch of 34 amino acid National University, Hanoi, Vietnam. residues and are presumed to form helix-turn Abbreviations used: TAC, transcriptionally active chromosomes; TCP34, tetratricopeptide-containing poly- structures acting as scaffolds to mediate protein– protein interactions and often in the assembly/ peptide; TPR, tetratricopeptide repeat; PEP, encoded 13,14 RNA polymerase. disassembly of multiprotein complexes. E-mail address of the corresponding author: The level of expression of chloroplast genes [email protected] varies considerably during plastid development

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. 536 TCP34, a Response Regulator-like TPR Protein and differentiation, and is substantially influenced reported, i. e. in rhodoplasts from the raphidophytic by changes in light intensity and quality. Protein alga Heterosigma akashiwo.26 phosphorylation and redox control are considered Here, we report the identification and characteri- to provide regulatory feed-back connections zation of a novel nucleus-encoded, plastid-located between photosynthesis and organellar gene polypeptide of 37.2 kDa, designated TCP34 (tetra- expression. Activation and coordination of these tricopeptide-containing chloroplast protein of mechanisms require a precise sensing and regu- 34 kDa). The protein possesses remarkable struc- lation which can, in principle, be managed by tural features and specifically binds to plastid “classical” two-component systems consisting of a protein–DNA complexes, suggesting a regulatory sensor kinase and its cognate response regulator.15–17 role in plastid gene expression by means of protein– The sensor component recognizes environmental protein and/or protein–DNA interactions. stimuli through phosphorylation of a conserved residue within its kinase domain. Sub- sequently, the phosphoryl group is transferred to a Results conserved aspartate residue in the receiver domain of the response regulator component. As a con- Isolation and characterization of a spinach cDNA sequence of the phosphorylation-induced confor- encoding a novel chloroplast precursor protein mational change, the output activity of the response regulator is modulated. Several proteins similar to During the course of studies aimed at isolating components involved in and fungal signal thylakoid protein kinases and molecular compo- transduction pathways have been identified in nents involved in regulation of photosynthesis and various compartments of the plant .18–21 To chloroplast gene expression, a spinach cDNA elucidate an entire plant signal transduction path- encoding a novel tetratricopeptide-containing poly- way incorporating a two-component regulatory peptide, designated TCP34, was identified. The full- system, the output activity of plant response length cDNA contained a single open reading frame regulator proteins must be known. Whereas various of 1237 bp that coded for a polypeptide of 339 bacterial response regulator proteins are known, amino acid residues (Figure 1(a)) with a predicted and function as factors in the regu- molecular mass of 38.2 kDa. The protein-coding lation of gene expression, only a limited number of sequence starts from ATG at position 61, which is plant counterparts with DNA-binding activities embedded in a typical plant translation initiation have been isolated so far. Among 14 Arabidopsis consensus sequence AACAATGGC.27 The ATG thaliana response regulator homologues identified codon is preceded by an in-frame upstream stop up to now,20,22–24 two, ARR1 and ARR2, have codon, implying that it represents the codon for the been found to bind double-stranded DNA in a potential initiator methionine. The 30 end of the sequence-specific manner. They both appear to cDNA carried a poly(A) sequence. operate as transcriptional activators in the The most recent protein databases were searched expression of nuclear genes.22,25 For plastids, only with the deduced 339 amino acid sequence as the oneresponseregulator-likeproteinhasbeen query using the BLAST and FASTA3 programs at

Figure 1. (a) Schematic presentation of functional domains and amino acid sequence of spinach TCP34 precursor protein. The hydrophilic protein contains a single predicted membrane-anchoring (MA) domain located at the C terminus (italics in the sequence). The putative response regulator-like domain (RR) is in boldface. Asp95, the putative phosphorylation site in that domain, is presented in italic boldface. The two serine-rich regions S56–S73, and S268–S298 are in grey italic boldface. The TPR domain is underlined. Two histidine residues within the second TPR unit are in grey boldface. A single cysteine residue is indicated with an asterisk, a putative processing site of the transit peptide (TP) by an arrow. HLH, helix-loop-helix. The numbers below the protein scheme indicate the amino acid range of each domain. (b) Predicted secondary structure of TCP34. The smaller cylinder depicts six TPR amphipatic helices forming a bulky moiety with exposed charged residues. Larger cylinders represent two helices forming a putative helix-loop-helix domain. The separating loop is highly negatively charged. Regions rich in serine are indicated with S; interchanging charged residues are represented by C or K. The protein is presumed to be anchored monotopically in the thylakoid membrane with its carboxy-terminal hydrophobic stretch. TCP34, a Response Regulator-like TPR Protein 537 the NCBI and GenomeNet www-services. A search factors29 and the catalytic domain of PPZ1 phos- of the A. thaliana databases revealed only a single phatase.30 The charged region, integrating the helix- homologous gene (At3g26580) encoding a protein loop-helix motif, and the serine-rich segment of unknown function with 67% identity. Northern together form an w140 residue long domain similar analysis of total RNA isolated from spinach and to the CheY superfamily of two-component A. thaliana seedlings revealed a single transcript of response regulators (Figure 2(b)). A crucial feature 2.0 kb (data not shown), which argues for an of these components is the conservation of D34, D95 absence of alternative splicing of the TCP34 and K147 (corresponding to D12, D57 and K109 of transcript. Two cyanobacterial proteins, encoded CheY), which are invariant in all response regulator by the hypothetical open reading frame slr1052 and proteins and indispensable for their phosphoryl- the stress-inducible gene ycf37 (slr0171)were ation-induced activation.31 identified with region-confined similarity scores of 36% and 30%, respectively, notably in the TPR array. Chloroplast localization and topological studies The search also revealed a regional similarity of 49% of TCP34 to MOM72, a 72 kDa mitochondrial outer mem- brane import receptor for the ADP/ATP carrier Hydropathy analysis of the deduced TCP34 from Neurospora crassa (P23231). The domain amino acid sequence as described32 suggested homology was found mainly in TPR regions. No a predominantly hydrophilic protein with a C- homology in other domains, like response regulator terminally located transmembrane domain of 17 or DNA-binding helix-loop-helix motifs, could be amino acid residues (Figure 1). Its predicted pre- observed with cyanobacterial homologues. The sequence displays all attributes of stroma targeting sequence analysis demonstrated the presence of chloroplast transit peptide.33 This peptide contains both a response regulator motive and an Asp a high percentage of leucine residues (w24%) but is residue, in the TCP34 molecule that seems to be not particularly abundant in serine and threonine unique for higher plant chloroplasts. residues. The processing site is ascribed to position P17 (Figure 1(a)) resulting in a mature protein of Sequence and structural analysis of TCP34, a w34 kDa. TPR-containing protein with unique molecular In organello experiments were conducted to verify features that the novel polypeptide resides indeed in the chloroplast. Following cDNA transcription and Inspection of the aforementioned local simi- translation of the resulting mRNA in vitro in the larities and application of algorithms for secondary presence of [35S]methionine, the labelled TCP34 structure predictions led to the identification of polypeptide (Figure 3(a), lane TP) was incubated three tandemly arranged, imperfect 34 amino acid with isolated intact spinach chloroplasts. Trans- residue repeat motifs, known as TPR arrays lation of TCP34 in vitro resulted in a highly labelled (Figure 1(a)).28 Because of the presence of these band of 40 kDa, which slightly varied from the structural motifs and the predicted chloroplast theoretically predicted size of 38.2 kDa, and other location, the novel protein was designated TCP34. polypeptides of lower molecular masses. The latter The TPR arrays in TCP34 are centrally positioned, ones could result from translation starting from spanning residues A163 to S275 within the large, each methionine in vitro. Since these products lack mostly hydrophilic segment (Figure 1(a) and (b)). the transit peptide sequences they cannot be Based on analogy with other TPR-containing imported into chloroplasts. After washing and proteins, each of the three units consists of a pair thermolysin treatment, chloroplasts were sub- of amphipatic, antiparallel a-helices of nearly equal fractionated into stroma (lane S) and thylakoids length (domains A and B, respectively), punctuated (lane T). Figure 3(a) illustrates that the in vitro- by proline-induced turns (Figure 2(a)). made, labelled precursor (arrowhead in Figure 3(a)) Another conspicuous feature of TCP34 is an was efficiently imported after 30 min of reaction N-terminal response regulator-like domain integra- into the organelle, processed to the expected mature ting a predicted helix-loop-helix motif. The large size of the protein and inserted into the thylakoid segment directly preceding the TPR array (from E83 membrane in two forms that were designated T1 to R160) contains 45 clustered charged residues and and T2 (lane T). A minute signal of unprocessed shows a substantial net negative charge precursor has also been consistently noted in the (Figure 2(b)). The two amphipatic a-helices (wE97 thylakoid fraction (marked with an arrowhead). No to wI124, and wE136 to wT163, respectively), and signal was detected in the stroma fraction. The two the loop between them represented by a negatively TCP34 forms remained stably associated with charged spacer region with predicted b-sheet thylakoid membranes even in the presence of features that can generate a structure known as chaotropic salts or alkaline solutions (lanes M). To the helix-loop-helix or EF-hand motif. Furthermore, analyse whether the C-terminal hydrophobic two serine-rich domains with intervening charged domain is responsible for the anchoring of the residues (segments S56–S73 and S268–S298) are polypeptide to the thylakoid membrane the present in the TCP34 sequence. Similar serine-rich, construct lacking the hydrophobic C terminus (the but more positively charged, domains precede the last 27 amino acid residues) was translated leucine zipper motif in some bZip transcription in vitro as well and imported into intact spinach 538 TCP34, a Response Regulator-like TPR Protein

Figure 2. (a) Sequence alignment of TCP34 TPR arrays and (b) putative response regulator receiver domain (b) in TCP34 with the similar motifs found in other sequence related proteins. (a) The TPR motif of TCP34 was compared with homologous motifs from other TPR-containing proteins. Asterisks indicate residues used by Sikorski et al.28 to define TPR motifs. The TPR consensus sequence usually comprises the highly conserved amino acid residues L7, G8, Y11, A20, F24, and A27, as well as P32.14 These residues have been proposed to interact with each other and thereby to mediate intra- or intermolecular protein–protein interactions. TPR consensus 1 and 2 are modified from Sikorski et al.,28 Becker et al.71 and Chen et al.72 The sign § represents any residue with a large hydrophobic side-chain. Aligned are the TPRs of the following proteins: serine/threonine PP5 from Rattus norvegicus (P53042) and PPT1 from TCP34, a Response Regulator-like TPR Protein 539

To understand the nature of these two TCP34 forms and to check whether one of them could be the result of processing of the higher molecular mass forms, import kinetics were performed with radiolabelled TCP34 precursor. Radiolabelled pre- cursor was incubated with intact spinach chlor- oplasts for 10, 20, 30 and 60 min (Figure 3(b)). Three TCP34 forms including the protein precursor were found in approximately equal quantities after the first 10 min of incubation of the assay. After import, the precursor protein was quite stable, but pro- cessed with increasing incubation time and dis- appeared after 60 min of import. TCP34-T1 and -T2 were present as well after 10 min and increased slightly in amount after 60 min of import reaction. The T1 and T2 doublets were not interconverted within our experimental time-range and probably were processed within the first 10 min of precursor Figure 3. (a) Import of pre-TCP34 into intact spinach translocation into chloroplasts. Consistent with our chloroplasts. Pre-TCP34 and truncated TCP34 lacking the observations above, no processing forms were C-terminal hydrophobic domain (-C-term) obtained found in the stroma. Treatment of thylakoid through in vitro translation (TP and TPt, respectively; C 35 membranes with thermolysin (Figure 3(b), lane, ) arrowheads) in the presence of [ S]methionine were completely removed all three thylakoid membrane- incubated with intact chloroplasts for 30 min. After associated TCP34 variants. This confirmed that incubation, chloroplasts were sub-fractionated into stroma (S) and thylakoid proteins (T). Thylakoid samples most part of the protein is exposed to the stroma face. were treated with various chaotropic salts (NaBr, Na2CO3 or NaSCN). After salt treatment thylakoid membranes To further characterize the TCP34 protein, poly- were centrifuged and proteins were separated into clonal, monospecific antisera were raised against membrane (M) and soluble (S) fractions (containing the recombinant precursor molecule overexpressed peripheral membrane proteins). Autoradiography of an in cells. The overexpression of TCP34 SDS 10%–17% polyacrylamide gel. Two thylakoid mem- in E. coli cells transformed with the plasmid was brane associated TCP34 forms, designated as TCP34-T1 2 induced by addition of IPTG. Cells were fractio- and -T , were apparent. (b) Import kinetics of pre-TCP34 nated into soluble and insoluble proteins, which into spinach chloroplasts. Radiolabelled pre-TCP34 was could contain inclusion bodies as well, and the incubated with intact spinach chloroplasts for 10, 20, 30 and 60 min. After incubation chloroplasts were treated overexpressed TCP34 was found in the insoluble with thermolysin and fractionated into stroma (S) and fraction of IPTG-induced cells (Figure 4(a)). This thylakoid. The latter were treated and non-treated with protein was used for immunisation of rabbits and thermolysin (lanes, C and K). Protein molecular mass antiserum specificity was tested on E. coli cells with markers are indicated at the left (in kDa). overexpressed TCP34 (Figure 4(b)). The antisera reacted specifically with only one band, the over- expressed protein after IPTG induction. chloroplasts. The translation product was approxi- The TCP34 antisera were then used for probing mately 3 kDa smaller than the entire precursor chloroplast subfractions. Unexpectedly, the protein, protein. Its import resulted in three TCP34 variants, probed by immunological analysis of fractionated which were targeted to the stroma (Figure 3(a)). chloroplast proteins from 14 day-old pea plants, This suggests that the predicted C-terminal hydro- was found in the stroma fraction, but not in phobic domain is indeed involved in the association thylakoid or in the envelope membranes of TCP34 with the membrane and that the doublet (Figure 5(a)). Comparison of TCP34 localisation in TCP34-T1 and -T2 is not the result of processing the pea, chosen because of its standardized envelope transmembrane domain. preparations, and spinach chloroplast fractions did

Saccharomyces cerevisiae (P53043), heat shock protein STII from S. cerevisiae (P15705), mitochondrial import receptor MOM72 from Neurospora crassa (P23231), FKB506-binding immunophiline FKBP52 from Oryctolagus cuniculus (M84988), cyclophilin CYP40 from Bos taurus (P26882), and protein kinase PKR inhibitor P58, also from B. taurus (A56534). (b) Sequence alignment of the TCP34 N-terminal region with regulatory domains of selected CheY superfamily members, and the putative regulatory domain of the ethylene response receptor, ETR1, from Arabidopsis. In consensus 1 conserved residues in both the CheYand the TCP34 are indicated in capital letters, asterisks represent residues which are conserved in at least one of the 78 other superfamily members listed by Volz.31 Consensus 2 according to Volz;31 in pairwise comparison individual members may show as little as 6% identity. The three residues that are highly conserved in the receiver domains are boxed. Aligned are response regulators; CheY from E. coli (NP_416396), RegA from Rhodobacter capsulatus (S41451), OmpR from E. coli (NP_417864), Spo0A from Bacillus subtilis (P06534), NRI from E. coli (NP_290493), ArcB from E. coli (NP_418818), and ETR1 from A. thaliana (NP_176808). D34, D95 and K147 indicate the amino acid residues in spinach TCP34 that are conserved in response regulator domains of other proteins. 540 TCP34, a Response Regulator-like TPR Protein

immunological detection the previously observed signals were not detectable any more, except of a weak signal with overexpressed protein (Figure 5(b), lower panel). To understand the cause for the different locali- sation and protein processing noted from the in organello approach and immunologically by in vivo experiments, the TCP34 protein was studied immunologically in etiolated spinach seedlings and after their exposure for 10 h, 24 h, 48 h and two or six weeks to light (Figure 5(c)). First, the amount of photosynthesis-related proteins was estimated in etioplasts and chloroplasts extracted from spinach material exposed for the aforemen- tioned periods to light (Figure 5(c)). Exposure to light increased the accumulation of the b subunit of ATP synthase, associated with thylakoid mem- branes, and of the small subunit of rubisco (SSU). On the contrary, chaperonin 60 protein (Cpn60), a protein not related to photosynthesis, decreased and could not be detected in chloroplasts after six weeks of light exposure. This is consistent with findings of a higher expression of Cpn60 protein in non-photosynthetic tissues.34 To analyse the local- isation of TCP34 forms, etioplasts (E) and chlor- oplasts (C) were purified from etiolated and light- exposed material, respectively, and fractionated Figure 4. (a) Overexpression of TCP34 in E. coli cells into prolamellar membranes (PM) and soluble and (b) analysis of specificity of polyclonal antisera raised proteins (PS) from etioplasts, or thylakoids (T) and against overexpressed TCP34 protein. (a) E. coli cells were stromal (S) proteins from chloroplasts (Figure 5(c)). transformed with a plasmid containing full-length gene encoding TCP34. The overexpression of TCP34 was No TCP34 signals were immunodetected in mem- induced by IPTG. The cellular localisation of TCP34 was brane and soluble fractions from the etiolated analysed by sub-fractionation of non-induced and material. In contrast, the anti-TCP34 serum revealed induced E. coli cells into soluble (S) and membrane three polypeptide species in developing chloro- fraction (P; pellet). (b) Proteins extracted from induced plasts that showed different accumulation patterns and non-induced E. coli cells were separated on SDS-12% with the time of exposure to light: two forms, PAGE and transferred to nitrocellulose membrane. The TCP34-T1 and -T2, were found in thylakoid overexpressed protein was immunologically detected membranes and a single product, TCP34-S1, in the with anti-TCP34 serum. The arrow indicates an over- soluble stroma fraction. The two thylakoid-bound expressed TCP34. forms accumulated in developing chloroplasts, i.e. between 48 h and two weeks of exposure to light, before decreasing drastically during the subsequent four weeks of illumination. The soluble form S1 not show any difference. The purity of the isolated appeared in small amounts at very early stages, i.e. protein subfractions was checked immunologically 10 h after exposure to light. It then accumulated with antisera raised against the large subunit of the steadily for 48 h and then remained nearly constant, stroma rubisco (LSU), the chlorophyll over six weeks of light exposure. This experiment a-binding protein of photosystem II, CP47, located demonstrated that there are at least three variants of in thylakoid membranes, and the TOC34 protein of TCP34 that can be distinguished both by their the outer envelope membrane import machinery pattern of developmental regulation, apparent (Figure 5(a)). The last mentioned component was molecular masses and localisation. To verify co- enriched in envelope membranes, although some migration of the TCP34 variants detected by in contamination was also noted in the stroma fraction. organello experiments and immunological analysis To exclude an unspecific reaction of TCP34 the samples were run on the same gel (data not antisera with pea and/or spinach fractions Western shown). This analysis demonstrated the identity of analysis was performed with anti-TCP34 in the three variants in both, the in organello assay and presence or absence of denatured overexpressed immunological analysis, respectively. TCP34 protein (Figure 5(b)). When overexpressed protein and chloroplast fractions from pea and Evidence for plastid DNA-binding activity of spinach were incubated with TCP34 antisera TCP34 expected signals corresponding to TCP34 were observed. After the incubation of TCP34 antisera The presence of a helix-loop-helix domain, with denatured overexpressed TCP34 prior to resembling the DNA-binding module of the TCP34, a Response Regulator-like TPR Protein 541

Figure 5. (a) Immunological analysis of TCP34 protein in pea sub-chloroplast fractions. Intact chloroplasts were isolated from 20 day-old pea plants and fractionated into envelope, thylakoid and soluble stroma proteins. Proteins were separated by SDS-12% PAGE and transferred onto nitrocellulose membranes. Proteins were immunodecorated with antisera raised against TCP34 protein, large subunit of rubisco (LSU) as a control for stroma proteins, the chlorophyll- binding protein CP47 of photosystem II as a control for thylakoid proteins, and TOC34, a protein of the envelope translocation machinery. (b) Competition of TCP34 antibodies immunoreaction in the absence and presence of denatured overexpressed protein. Overexpressed in E. coli TCP34 protein before (K) and after (C) addition of IPTG, spinach and pea chloroplast (C), thylakoid (T) and stroma (S) protein fractions were separated by SDS-12% PAGE and transferred onto nitrocellulose membrane. One membrane was incubated with standard anti-TCP34 and the other one with anti-TCP34 pre-incubated with denatured overexpressed TCP34 protein. (c) Kinetics of accumulation of the membrane associated and soluble forms of the TCP34 protein from etiolated seedlings and plants transferred to light for various periods. Spinach plants were germinated and grown for six days in darkness and then transferred to light for 10 h, 24 h or 48 h and two or six weeks. Etioplasts (E) and chloroplasts (C) were isolated from etiolated and light-treated plants, respectively. Prolamellar membranes (PM) and soluble fraction (PS), thylakoid (T) and stroma (S) proteins were separated from etioplasts and chloroplasts, respectively: 150 mg of protein from each fraction per lane was separated by 12% PAGE containing 6 M urea and transferred onto a nitrocellulose membrane. TCP34 was immunodetected by a polyclonal monospecific TCP34 antiserum. Thylakoid-associated TCP34 forms are indicated as T1 and T2, the stroma variant as S1. The amounts of chaperonin 60 protein (Cpn60), the b subunit of ATP synthase (b-ATPase) and the small subunit of rubisco (SSU) were analysed in etioplasts/chloroplasts, thylakoid and stroma proteins, respectively. prokaryotic response regulator OmpR,35 raised the thetic machinery, notably psaA, psbC, psbD,orrbcL- possibility that TCP34 possesses DNA-binding atpB, were incubated with the recombinant pre- activity. The recombinant precursor molecule was cursor protein. All these chloroplast DNA frag- therefore probed with plastid DNA (ptDNA) and ments could bind the recombinant protein used for South-Western analysis (Figure 6(a)). (Figure 6(c), lanes 2 and 3). Significant binding to ptDNA (Figure 6(a), lane 2) was detected. The competition experiments with Supercomplex localization of TCP34 component non-labelled ptDNA in amounts that were equal or higher than radioactively labelled plastid ptDNA The presence of three tandemly arranged TPR demonstrated its specific binding to TCP34 protein. arrays and the ability of TCP34 to bind specifically The signals reflecting the DNA-binding obtained to ptDNA raised the possibility that TCP34 is with 32P-labelled DNA decreased when the higher associated with other proteins or with multimeric amounts of non-labelled ptDNA (1 and 10 mg) were complexes. Therefore, protein–DNA complexes, the incubated together with the radioactive probes. To so-called TAC (transcriptionally active chromo- probe whether TCP34 binds specifically chloroplast somes), released from photosynthetic membranes DNA or can bind to any other DNA, competition by Triton X-100 solubilisation36,37 was checked for experiments with different amounts of genomic the presence of TCP34. Size-exclusion chromato- E. coli DNA were performed (Figure 6(b)). No graphy has shown that the TAC complex possesses changes of DNA-binding by TCP34 were observed a high molecular mass (w2 MDa) and contains 30– in the presence or absence of E. coli DNA. To 40 different polypeptide bands including subunits pinpoint the region(s) of the plastid chromosome of the plastid-encoded RNA polymerase (PEP).37 that is (are) recognized by TCP34, four chloroplast The protein pattern of the TAC complex extracted DNA fragments encoding genes for the photosyn- from spinach chloroplasts was compared with 542 TCP34, a Response Regulator-like TPR Protein

Figure 6. DNA-binding analysis of the recombinant TCP34 precursor protein. (a) and (b) Binding specificity of the TCP34 protein to chloroplast DNA. Protein gel blots were probed with 32P-labelled chloroplast DNA (ptDNA) and different amounts of added (a) non-labelled competitor ptDNA (100 ng, 1 and 10 mg; indicated vertically) or (b) E. coli genomic DNA (100 ng and 1 mg; indicated vertically). Lane 1, non-transformed E. coli M15 cells; lane 2, insoluble protein fractions from transformed E. coli M15 cells 4 h after addition of IPTG; lane 3, calf thymus histones. (c) Affinity of the TCP34 protein for chloroplast DNA fragments. A protein gel blot was probed with four 32P-labelled spinach chloroplast DNA fragments containing psaA, rbcL-atpB, psbC and psbD probes, respectively. Lanes 1–3, insoluble protein fractions from E. coli M15 cells transformed with the TCP34 expression plasmid before (lane 1) and in two different amounts after induction of protein synthesis with IPTG (lanes 2 and 3). chloroplast proteins by silver staining after SDS- and large subunit of rubisco complex were used as a PAGE (Figure 7(a)) and TCP34 was immunologi- control for TAC purity. cally checked for in both fractions (Figure 7(b)). The TCP34 protein was clearly detectable in the TAC Phosphorylation of TCP34 protein fraction immunologically but could not be visual- ized by silver staining. The presence of the In most cases the regulation of response regula- a-subunit of PEP in the same complex provides tors and factors involved in gene expression is additional evidence that this is a bona fide TAC controlled by phosphorylation/dephosphorylation complex. No reaction with the TAC fraction was mechanisms. Phosphorylation of TCP34 was ana- observed when an antiserum against cytochrome f, lysed in two ways, first immunologically with which possesses a molecular mass similar to TCP34, antisera raised against phosphothreonine residues in spinach chloroplast, stroma and thylakoid protein fractions (Figure 8(a)). The antisera recog- nized the TCP34-T1 and TCP34-S1 forms, but not TCP34-T2 among various phosphorylated proteins; the TCP34 variants separated in one gel system were identified immunologically by anti-TCP34 and phosphothreonine serum (data not shown). To analyse, whether the serological reaction of these TCP34 forms was due to phosphorylation and not due to some non-specific binding of phosphothreo- nine antisera, fractionated chloroplast proteins were treated with alkaline that hydrolyses orthophosphoric monoester groups (Figure 8(a), lanes CAP). Dephosphorylation was efficient for most phosphorylated components, although the CP43 protein of photosystem II and some stroma proteins were not completely dephos- phorylated by this approach. The incomplete dephosphorylation could have resulted from an improper ratio of enzyme and substrate(s), usage of Figure 7. Supramolecular organisation of the TCP34 a heterologous enzyme–substrate system and/or protein. (a) Silver staining of an SDS/12% polyacrylamide from a lower accessibility of the phosphorylated gel of total proteins extracted from spinach chloroplasts sites masked due to secondary/tertiary structures (C) and TAC complex. (b) Western analysis of chloroplast of the protein(s) to the alkaline phosphatase. proteins (lanes 1 and 2 correspond to 10 mg and 2 mgof chlorophyll, respectively) and TAC proteins with anti- Dephosphorylation of the stroma form resulted in a the appearance of a second, slightly lower molecu- serum raised against TCP34, -subunit of plastid- 2 encoded RNA polymerase (a-PEP), cytochrome f (cyt f) lar mass band, designated TCP34-S (Figure 8(a), 1 of the cytochrome b6f complex and the large subunit of the lower panel). The TCP34-T form was fully dephos- rubisco enzyme (LSU). phorylated in vitro in thylakoids, while TCP34-S1 TCP34, a Response Regulator-like TPR Protein 543

Figure 8. Phosphorylation of TCP34 protein variants. (a) Detection of phosphorylated proteins using anti- phosphothreonine serum. Chloroplast (C), thylakoid (T) and stroma (S) proteins (150 mg of protein of each) from two week-old spinach seedlings that were treated at room temperature (C AP, RT) and non-treated at 4 8C(K AP, 4 8C) by alkaline phosphatase were separated by denaturing 10% PAGE containing 6 M urea. Phosphorylation was visualized by immunodetection with anti-phosphothreonine antibodies (upper panel) and TCP34 by immunodetection with TCP34 antiserum (lower panel). Various forms were observed, designated as T1 and T2 (for thylakoid variants) and S1 and S2 (for stroma variants). The phosphorylated forms of TCP34 are marked by stars. Protein fractions were either treated with alkaline phosphatase at room temperature for 30 min (CAP, RT) or incubated at 4 8C as a control or 30 min at room temperature without addition of alkaline phosphatase (K AP, 4 8C and KAP, RT). Photosystem II proteins that are known to be phosphorylated in thylakoid membranes are indicated at the left (CP43, D1 and D2). (b) Phosphorylation of chloroplast, thylakoid and stroma proteins with [g-32P]ATP. Total broken chloroplast, thylakoid, stroma and TAC proteins were phosphorylated in vitro in the presence of [g-32P]ATP and protein kinase. TAC and thylakoid phosphorylated proteins were immunoprecipitated with antisera raised against TCP34 protein. was dephosphorylated only partially. This could Discussion have a similar explanation as the uncompleted dephosphorylation of CP43, which was described The TCP34 protein described here represents a above. The protein fractions treated or untreated novel response regulator-like component in chloro- with alkaline phosphatase were probed with TCP34 plasts with intriguing features. It provides a unique antisera. No visible shifts or redistribution of the example of a chloroplast TPR protein that also TCP34-T1 and -T2 forms could be noted, although displays a helix-loop-helix containing response the bands often became slightly diffuse in the latter regulator-like structure. TCP34 is both, associated case (Figure 8(a), lower panel). This indicates that with thylakoid membranes, specifically with the the TCP34-T2 variant is probably not the result of membrane-associated transcriptionally active protein dephosphorylation. We conclude that the chromosomes but also found in the stroma, doublet of thylakoid TCP34 should result from depending on the developmental stage of the some other protein modifications that may involve material. TCP34 homologues are found in other processing of the TCP34-T1 variant from the N sequenced plant genomes (Arabidopsis and rice) but terminus. not in the chloroplast ancestors, photosynthetic We also investigated the state of phosphorylation cyanobacteria. of TCP34 in vitro upon incubation of broken chloroplasts with [g-32P]ATP and protein kinase TCP34 mature forms are generated by multiple (Figure 8(b)). The radiolabelling with [g-32P]ATP processing events confirmed the phosphorylation of TCP34-T1 and -S1 forms as found with phosphothreonine antisera. Two lines of evidence establish that the cDNA we The purified TAC complex was deficient of isolated encodes the entire TCP34 polypeptide. activity, probably due to the First, the deduced translation initiation codon is lack of associated protein kinase(s), and an extrinsic embedded in a typical canonical plant consensus enzyme had to be added in order to phosphorylate sequence27 and preceded by an in-frame stop codon TAC proteins. Phosphorylation of TAC proteins implying that this ATG codon represents the actual resulted in the detection of a phosphoprotein band initiation codon. Second, the in vitro synthesized comigrating with the TCP34-T1 variant. Immuno- translation product is competent for import into precipitation of a radiolabelled TAC protein fraction isolated chloroplasts. The presence of a plastid- with antisera against TCP34 picked up only one targeting signal, in organello experiments and protein, corresponding to TCP34-T1. These data cellular subfractionation clearly demonstrate that argue for a TAC complex containing at least the TCP34 is a chloroplast protein. These experimental higher molecular mass form of TCP34, which can be data were recently proven by proteomic analysis reversibly phosphorylated. of thylakoid membrane proteins that found the 544 TCP34, a Response Regulator-like TPR Protein

TCP34 Arabidopsis homologue in association with release from the membrane could reflect an chloroplast thylakoids.38 The detection of two inactivation process, but the preservation of large processed forms of that protein that are bound to amounts of the presumably C-terminally truncated thylakoid membranes in in organello experiments product TCP34-S1 in chloroplasts from green differs from the situation in vivo where only one mature spinach leaves suggests that it also serves soluble variant in the stroma was immunologically some critical function. The processing of TCP34 detectable in chloroplasts of mature spinach plants. with its release into the chloroplast stroma could as The fact that the C-terminally truncated protein that well be an additional control mechanism for the lacks its hydrophobic domain accumulates in the activity of TCP34; for instance, in regulating the stroma of intact spinach chloroplasts when trans- efficiency of gene expression by replacing a highly lated and imported in organello suggests that the active thylakoid-bound form for a poorly active membrane interaction of TCP34 is controlled by a stromal form and/or by modulating the association post-translational cleavage of the hydrophobic C of nucleoids with thylakoid membranes. terminus. As judged from the import experiments of full-size TCP34 protein, it is conceivable that the TCP34 is a TPR protein that associates with regulatory system responsible for this C-terminal plastid DNA conversion does not operate properly in the in organello import system, therefore preserving mem- The presence of a TPR module in the central brane attachment of two mature TCP34 variants region with an N-terminally positioned potential that probably differ from each other by some response regulator-like domain that includes a additional N-terminal processing. Interestingly, putative helix-loop-helix motif could be responsible the probably C-terminally processed form and the for DNA-binding and/or to some proteins as well. mature thylakoid membrane-associated form T1 Prediction and biochemical evidence suggest that in possess similar molecular masses. This suggests TCP34 both modules are stroma-exposed, forming a that the C-terminally processed stromal S1 variant large solvent-accessible moiety. Two highly con- contains an unprocessed N terminus. It is therefore served amino acid residues appear to be of central likely that after import and maturation of the importance in TPR repeats: residue G8 in domain A precursor form there are two additional, perhaps has a small side-chain and forms a hole into which alternative processing events that operate either the bulky F24 residue, serving as a knob, fits. These from the N terminus that preserves membrane residues are conserved in the TPR arrays of the binding of TCP34, or from the C terminus that TCP34 protein as well, whereas the hydrophobic releases the product into the chloroplast stroma. residues Y11 and A20 are replaced by alanine, and Processing of various transcription factors has been serine, respectively. The replacement of Y11 appears reported in mammals and prokaryotes.39–41 Two to be a common feature of TPR-containing chloro- candidate membrane proteases homologous to plast proteins identified up to now. It clearly bacterial peptidases involved in the processing of distinguishes them from members of the PPR transcription factors have recently been found in family, which are characterized by tandemly the Arabidopsis and were predicted to be arranged 35 amino acid repeats, and are supposed targeted to chloroplasts.42,43 to mediate RNA binding rather than protein– protein interactions.45 Developmental control of TCP34 localisation In plastids, DNA exists as large protein–DNA complexes, organised in nucleoids.46,47 DNA in The membrane and soluble forms of TCP34 nucleoids is generally bound to membranes of the showed unique and differing accumulation pat- inner envelope in etioplasts. During the initial steps terns in developing chloroplasts. TCP34 was absent of chloroplast development nucleoids relocate to in etioplasts but significant protein levels were thylakoid membranes of mature chloroplasts.46,48,49 reached within two days of light exposure, a period Several DNA-binding chloroplast proteins identi- during which etioplasts turn into photosyntheti- fied up to now are associated with plastid cally active chloroplasts.44 Interestingly, whereas nucleoids, in particular the tobacco CND41 pro- the membrane-bound forms reached a maximum tein,50 the pea PEND protein, a member of the bZip level within two weeks of light exposure, before protein family,51,52 and the MFP1 DNA-binding dropping in mature leaves, the soluble form was protein.53 The MFP1 protein was found in thylakoid also light-induced but its amount remained highly membranes of developed plastids and is supposed stable in fully developed chloroplasts. This con- to represent an anchor component of nucleoids to trasting behaviour argues for a transient require- the thylakoid membrane system.52 Similarly to ment of the membrane-bound forms at the time of MFP1 protein, TCP34 was also found in association leaf and photosynthetic development. In this with transcriptionally active chromosomes, which respect, the presence of one membrane-bound represent nucleoid fractions and thylakoid mem- form in the TAC complex (see below), together branes. with its ability to bind specifically to ptDNA in vitro, Three tandemly arranged TPR arrays are prob- suggests a role of TCP34 in promoting transcription ably responsible for the association of TCP34 with a of photosynthetic genes. At the present stage, the large transcriptionally active protein–DNA role of the soluble TCP34 form, if any, is not clear. Its complex. The association of only one, the higher TCP34, a Response Regulator-like TPR Protein 545 molecular mass TCP34-T1 form, with the TAC a serine/threonine kinase. Another crucial question argues for its functionality in gene expression is whether “one-component” systems exist in which and/or cellular signalling. Since the TAC complex an “orphan” receiver or transmitter module oper- is extracted from thylakoid membranes that are ates without a partner.57 The phosphorylated amino washed to remove stroma proteins, we suggest that acid residue(s) in TCP34 have not been identified. it contains the thylakoid-associated TCP34-T1 form The reaction of TCP34 with anti-phosphothreonine and not the soluble S1 variant. serum and the stability of the of TCP34 phosphoryl- The association of TCP34 with the TAC complex ation against acid treatment argue for phosphoryl- and its DNA binding suggest involvement of TCP34 ation of threonine or serine residues. On the in the expression of plastid genes. If so, the question other hand, the limited dephosphorylation of arises how this gene expression is controlled and TCP34 by alkaline phosphatase that is specific for whether this gene expression is under redox O-phosphorylation of threonine or serine residues control. Although redox regulation has been indicates possible phosphorylation of residues reported for some plastid genes54 the mechanism other than threonine or serine. The data suggest and components that are involved have not been also that several phosphorylation sites in TCP34 identified. could be involved in the case of a one-component system grouping receiving and transmitting Phosphorylation of TCP34 variants modules. Reversible protein phosphorylation has been We found that at least two of the TCP34 variants, shown to affect DNA binding and properties of T1 and S1, can be phosphorylated. Analysis of the various transcription factors in and ani- TCP34 phosphorylation status both with phospho- mals.58–61 Relatively little is known about phos- threonine antisera and by radiolabelling with phorylation of plant transcription factors.62 [g-32P]ATP shows reversible phosphorylation of Furthermore, the absence of a phosphorylated the T1 variant. Absence of phosphorylation of the T2 TCP34 form in the TAC preparation, but its form indicates that the phosphorylation site is potential phosphorylation with externally added located closer to the N terminus and probably protein kinase, suggest a possible modulation of its cleaved off from the T2 variant. Dephosphorylation affinity for ptDNA and/or a DNA-binding protein of the stroma variant TCP34-S1 in vitro resulted in complex by post-translational modification of an electrophoretic shift of the protein band corro- distinct threonine and/or serine residues. borating the phosphorylation of the soluble TCP34- In conclusion, the discovery of TCP34 provides S1 variant. The fact that it is seen only in vitro first evidence for a possible involvement of a indicates that the dephosphorylated S2 variant is nucleus-encoded, TPR-containing response regula- rapidly degraded in vivo or can be reversibly tor-like protein in the regulation of plastid gene phosphorylated and converted to form S1. expression. It reinforces the importance of post- The presence of an N-terminally positioned translational modification, such as processing and response regulator-like domain raises the possi- protein phosphorylation, in TPR-mediated protein– bility that TCP34 is part of a regulatory chloroplast protein interactions in chloroplast signal transduc- two-component His-to-Asp phospho-relay system. tion processes. The presence of an aspartate residue in this TCP34 domain raised the possibility of its phosphory- lation. However, attempts to transfer the phos- Materials and Methods phorylated group of a histidine residue heterologously with overexpressed EnvZ histidine Plant material and growth conditions kinase from E. coli to an aspartate residue of overexpressed spinach TCP34 protein were not Spinach (Spinacia oleracea, var. Monopa, Fa. Sperling) successful (data not shown). On one hand, this and pea plants were grown in a green house at 100 mEmK2 K could mean that despite a high level of sequence s 1. For all experiments, two month-old spinach plants homology with ancient response regulators, the were used for the isolation of chloroplasts, if not mechanisms of signal transfer are modified in otherwise indicated. For kinetic experiments, spinach plants were germinated in darkness, grown for six to eukaryotic homologous components. On the other K2 K1 hand, one could suggest that the extracted protein seven days and transferred to light (100 mEm s ) for fraction as well as overexpressed protein did not 10 h, 24 h, 48 h and two and six weeks. contain the corresponding histidine sensor kinase. Regarding the evolution of plant two-component Isolation of TCP34 cDNA systems, it is of particular interest that distinct cyanobacterial histidine kinases have been pro- Heterologous serological screening of a lgt11-based posed as ancestors of plant phytochromes, which, green leaf cDNA library from spinach was performed using an antibody raised against Synechocystis sp. PCC despite their -like domains, show 55,56 6803 histidine kinase slr0311.Afterthreecyclesof serine/threonine protein kinase activity. One screening total DNA from positive phages was isolated could therefore speculate that spinach chloroplasts using standard PEG/NaCl precipitation of lambda contain a modified two-component system in particles,63 followed by a phenolisation step and ethanol which the classical histidine kinase is replaced by precipitation. After restriction with EcoRI the cDNA 546 TCP34, a Response Regulator-like TPR Protein inserts were purified and sub-cloned into pBluescript fragment. The transcription reaction was performed K SK vector for sequencing. Two inserts of different sizes in vitro with phage T7 RNA polymerase. The transcripts (350 bp and 900 bp, respectively) contained identical were then translated for 90 min at 30 8C in a cell-free sequences, which showed 68% homology to an unknown reticulocyte lysate in the presence of [35S]methionine open reading frame from A. thaliana. Expectedly, none of (Amersham Pharmacia, Freiburg, Germany). the selected cDNA fragments encoded a complete open Protein import into isolated intact chloroplasts was K K reading frame. Therefore, a second round of screening performed in light (50 mEm 2 s 1) for 30 min to 60 min at was performed using radioactively labelled cDNA 25 8C in a total volume of 240 ml containing 30 mlof fragments as probes. Five recombinant phage were translation assay and chloroplasts corresponding to selected. The largest insert of 1237 bp represented a 200 mg of chlorophyll. The chloroplasts were then full-length TCP34 cDNA. For membrane binding protease-treated to remove adhering precursor, washed studies the clone deleted in the last 78 bp of the coding and subsequently sub-fractionated into stroma and region was constructed by a PCR approach. The product thylakoids following osmotic shock as described.66 For was amplified on full-length cDNA with primers topographical studies thylakoids were incubated for TCP34precursor.fwd (50-TAGGATCCATGGCGACGGTG 30minonicewith2MNaBr,2MNaSCN,0.1M 0 0 CTCGG-3 ) and TCP34MinCterm.rev (5 -CCCAAGCTTA Na2CO3, or 0.1 M NaOH. Proteins from various sub- GCCAACAGGAGGTTTCCAC-30) and sub-cloned into fractions were then precipitated by acetone, pelleted at K pBluescript SK vector. 13,000g for 2 min and dissolved in sample buffer for electrophoresis. Sequence analysis

Analysis of protein and gene homologies were Overexpression of TCP34 in E. coli cells and antisera performed using a BLAST and FASTA search on NCBIa production and CLUSTAL W on the EMBL databaseb. Sequence analyses were obtained from the Arabidopsis and rice The DNA sequence encoding for full-length TCP34 was genome TIGR databasesc. Predictions of chloroplast cloned into pQE-30 plasmid (Qiagen GmbH, Hilden). The localisation of TCP34 were obtained by Target Pd and expression of the protein was performed in the E. coli M15 Predotare. The position of transit peptide cleavage sites host. For fractionation of E. coli cells, they were was predicted by Signal P softwaref. sedimented and lysed for 30 min in a buffer containing 0.3 M NaCl, 0.05 M NaH2PO4 (pH 8.0), 0.01 M imidazole and 1 mg/ml of lysozyme, sonicated four times for 5 s Protein gel electrophoresis and immunological each and centrifuged for 30 min at 10,000g. The over- analysis expressed protein was excised from the gel, eluted and injected into rabbits. Proteins were separated by Tris–glycine SDS-PAGE64 and denaturing 6 M urea SDS-PAGE. For Western analysis, proteins were transferred onto either nitrocellulose or Fractionation of intact spinach chloroplasts and polyvinylidene difluoride membranes (Biotrace NT; PALL 65 preparation of the plastid TAC complex Filtron, Dreieich, Germany). Immunodetection was performed using the enhanced chemiluminiscence system. Chloroplast phosphoproteins were immuno- Etioplasts from spinach were isolated according to a 67 detected by polyclonal anti-phosphothreonine antibodies described procedure. Intact chloroplasts were isolated obtained from Zymed Laboratories (San Francisco, CA). from spinach leaves using discontinuous Percoll 66 For competition experiments with antisera raised gradients and were subsequently osmotically shocked against TCP34 protein with sub-chloroplast fractions the in lysis buffer (10 mM Hepes–KOH (pH 8.0), 5 mM proteins transferred onto nitrocellulose membrane were MgCl2). Thylakoid membranes were separated from the incubated with anti-TCP34 non-treated and pre-treated stroma fraction by centrifugation for 10 min at 5000g. The with denatured overexpressed TCP34 to assess heter- membranes were washed in buffer containing 10 mM ologous specificity. In the latter case, anti-TCP34 diluted Tricine–HCl (pH 8.0), 100 mM sucrose and 5 mM MgCl2. in milk was incubated for 1 h with 1 mg of overexpressed Fractions containing transcriptionally active chromo- 37 in E. coli TCP34 denatured for 10 min at 80 8C. somes were prepared as described.

Import of precursor and C-terminally truncated TCP34 protein into isolated spinach chloroplasts Expression and purification of recombinant TCP34

The plasmids containing the coding region of the The E. coli expression plasmid was constructed by TCP34 precursor or C-terminally deleted protein were cloning of a 1269 bp HincI-fragment, isolated from linearized with KpnI downstream of the inserted cDNA SAMS2B4.1T7, into SmaI-digested pQE30 (Qiagen GmbH,Hilden,Germany).Anovernightcultureof E. coli M15 carrying the His-tagged version of TCP34 a http://www.ncbi.nlm.nih.gov/BLAST/ precursor was used to inoculate 30 ml of LB containing b http://www2.ebi.ac.uk/clustalw/ 100 mg/ml of ampicillin. Four hours after induction of c http://www.tigr.org/tdb/euk/ protein synthesis with 1 mM isopropyl-b-D-thiogalacto- d version 1.1; http://www.cbs.dtu.dk/services/ pyranoside (IPTG) cells were lysed in 300 mM NaCl, TargetP 50 mM NaH2PO4 (pH 8.0), and 10 mM imidazole in the e version 1.03; http://genoplante-info.infobiogen.fr/ presence of lysozyme (1 mg/ml) followed by sonication. predotar/test.seq The pelleted insoluble protein fraction containing recom- f version 3.0; http://www.cbs.dtu.dk/services/ binant TCP34 was resuspended in lysis buffer without SignalP/ lysozyme and used for further analyses. TCP34, a Response Regulator-like TPR Protein 547

Antibody preparation chloroplast DNA clones were isolated for recombinant plasmids. Recombinant TCP34 precursor was expressed in E. coli M15 as described above and the insoluble protein fraction Data Bank accession number was separated by SDS-12% (w/v) PAGE. For further purification the recombinant protein was excised from the The TCP34 sequence data have been submitted to the gel, subjected once more to SDS-PAGE, followed by DDBJ/EMBL/GenBank databank under the accession no. electrotransfer onto nitrocellulose membrane. Prior to Y14198. injection into rabbits a membrane strip (1 cm) containing recombinant TCP34 was dissolved in 200 ml of dimethyl sulfoxide.

Phosphorylation in vitro and immunoprecipitation Acknowledgements of phosphorylated proteins This work was supported by the Deutsche Phosphorylation of chloroplast, thylakoid and stroma Forschungsgemeinschaft (SFB 184 and SFB TR1- 8 proteins was performed for 30 min at 25 C in buffer B3), the Human Frontier Science Program (HFSP) containing 50 mM Tricine–NaOH (pH 7.8), 100 mM and the Fonds der Chemischen Industrie. We thank sorbitol, 5 mM MgCl2, 10 mM NaF and 0.2 mM [g-32P]ATP (0.05 mCi/ml; 1 mCiZ37 kBq). The reaction Dr Ju¨ rgen Soll for providing antisera raised against was stopped by addition of 6 mM ATP. Phosphorylation Cpn60 and TOC34 proteins. experiments with TAC fraction were performed in a 100 ml volume as described.68 TAC proteins were phosphorylated by addition of protein kinase A References (Sigma). Immunoprecipitation of proteins was performed by addition of one volume of buffer containing 100 mM 1. Rochaix, J. D. (1992). Post-transcriptional steps in the Tris–HCl (pH 7.5), 600 mM NaCl, 10 mM EDTA, 2% (v/v) expression of chloroplast genes. Annu. Rev. Cell. Biol. m Triton X-100, 40 l of the TCP34 antibody solution and 8, 1–28. m 40 l of protein A Sepharose. After 60 min of incubation 2. Rochaix, J. D. (1996). Post-transcriptional regulation and centrifugation the pellet was washed four times in of chloroplast gene expression in Chlamydomonas buffer containing 50 mM Tris–HCl (pH 7.5), 300 mM reinhardtii. Plant Mol. Biol. 32, 327–342. NaCl, 2% Triton X-100, 0.1% (w/v) SDS and once in Tris– 3. Mayfield, S. P., Christopher, B. Y., Cohen, A. & HCl (pH 6.8). Proteins were separated by denaturing 6 M Danon, A. (1995). Regulation of chloroplast gene urea SDS-PAGE and transferred onto nitrocellulose expression. Annu. Rev. Physiol. Plant Mol. Biol. 46, membranes. 147–166. 4. Sugita, M. & Sugiura, M. (1996). Regulation of gene Dephosphorylation of phosphorylated chloroplast expression in chloroplasts of higher plants. Plant Mol. polypeptides Biol. 32, 315–326. 5. Goldschmidt-Clermont, M. (1998). Coordination of In order to dephosphorylate thylakoid and stroma nuclear chloroplast expression in plant cells. Int. Rev. proteins the samples were incubated with bovine alkaline Cytol. 177, 115–180. phosphatase (Sigma, Schnelldorf).69 For dephosphoryla- 6. Leon, P., Arroyo, A. & Mackenzie, S. (1998). Nuclear tion assays phosphorylation in vitro was performed as control of plastid and mitochondrial development in described above but without adding phosphatase inhibi- higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. tor (NaF). The phosphorylated proteins were incubated 49, 453–480. with or without (control) alkaline phosphatase in 0.1 M 7. Rochaix, J. D., Kuchka, M., Mayfield, S., Schirmer- Rahire, M., Girard-Bascou, J. & Bennoun, P. (1989). glycine (pH 10.4), 1 mM MgCl2,1mMZnCl2 and proteinase inhibitor cocktail (Sigma, Schnelldorf) for Nuclear and chloroplast mutations affect the syn- 30 min at room temperature. The reaction was stopped thesis or stability of the chloroplast psbC gene by addition of 120 mM EDTA. product in Chlamydomonas reinhardtii. EMBO J. 8, 1013–1021. 8. Danon, A. & Mayfield, S. P. (1991). Light regulated In vitro DNA-binding assay translational activators: identification of chloroplast gene specific mRNA binding proteins. EMBO J. 10, The insoluble protein fraction containing recombinant 3993–4001. TCP34 precursor was separated by SDS-PAGE and 9. Barkan, A. (1993). Nuclear mutants of maize with electrotransferred onto nitrocellulose membrane. The defects in chloroplast polysome assembly have membrane was probed with 100 ng of 32P-labelled DNA altered chloroplast RNA metabolism. Plant Cell, 5, isolated from spinach chloroplasts as described70 in buffer 389–402. containing 10 mM Tris–HCl (pH 7.5), 75 mM NaCl, 0.13% 10. Levy, H., Kindle, K. L. & Stern, D. B. (1997). A nuclear 0 (w/v) BSA, 0.02% (w/v) NaN3 10 mg/ml of salmon sperm mutation that affects the 3 processing of several DNA and 1 mM DTT. For competition experiments mRNAs in Chlamydomonas chloroplasts. Plant Cell, 9, 100 ng, 1 mg and 10 mg of non-labelled plastid DNA or 825–836. 100 ng and 1 mg of genomic E. coli DNA were added 11. Boudreau, E., Nickelsen, J., Lemaire, S. D., during the hybridisation step with radiolabelled DNA Ossenbu¨ hl, F. & Rochaix, J.-D. (2000). The Nac2 gene probe to nitrocellulose membrane with overexpressed of Chlamydomonas encodes a TPR-like protein protein. To determine the binding specificity of TCP34 involved in psbD mRNA stability. EMBO J. 19, for chloroplast DNA fragments, inserts of spinach 3366–3376. 548 TCP34, a Response Regulator-like TPR Protein

12. Vaistij, F. E., Boudreau, E., Lemaire, S. D., 30. Posas, F., Casamayor, A., Morral, N. & Arino, J. (1992). Goldschmidt-Clermont, M. & Rochaix, J.-D. (2000). Molecular cloning and analysis of a yeast protein Characterization of Mbb1, a nucleus-encoded tetra- phosphatase with an unusual amino-terminal region. tricopeptide-like repeat protein required for J. Biol. Chem. 267, 11734–11740. expression of the chloroplast psb/psbT/psbH gene 31. Volz, K. (1993). Structural conservation in the CheY cluster in Chlamydomonas reinhardtii. Proc. Natl Acad. superfamily. Biochemistry, 32, 11741–11753. Sci. USA, 97, 14813–14818. 32. Kyte, J. & Doolittle, R. F. (1982). A simple method for 13. Goebl, M. & Yanagida, M. (1991). The TPR snap helix: displaying the hydrophatic character of a protein. a novel protein repeat motif from mitosis to transcrip- J. Mol. Biol. 23, 337–348. tion. Trends Biochem. Sci. 16, 173–177. 33. von Heijne, G., Steppuhn, J. & Herrmann, R. G. (1989). 14. Lamb, J. R., Tugendreich, S. & Hieter, P. (1995). Domain structure of mitochondrial and chloroplast Tetratricopeptide repeat interactions: to TPR or not targeting peptides. Eur. J. Biochem. 180, 535–545. to TPR? Trends Biochem. Sci. 20, 257–259. 34. Schmitz, G., Schmidt, M. & Feierabend, J. (1996). 15. Allen, J. F. (1992). Protein phosphorylation in regu- Comparison of the expression of a plastidic chaper- lation of photosynthesis. Biochim. Biophys. Acta, 1098, onin 60 in different plant tissues and under photo- 275–335. synthetic and non-photosynthetic conditions. Planta, 16. Parkinson, J. S. & Kofoid, E. C. (1992). Communi- 200, 326–336. cation modules in bacterial signaling proteins. Annu. 35. Itou, H. & Tanaka, I. (2001). The OmpR-family of Rev. Genet. 26, 71–112. proteins: insights into the tertiary structure and 17. Allen, J. F. (1993). Control of gene expression by redox functions of the two-component regulator proteins. potential and the requirement for chloroplast and J. Biochem. 129, 343–350. mitochondrial genomes. J. Theor. Biol. 165, 609–631. 36. Suck, R., Zeltz, P., Falk, J., Acker, J., Ko¨ssel, H. & 18. Kakimoto, T. (1996). CKI1, a histidine kinase homolog Krupinska, K. (1996). Transcriptionally active implicated in cytokinin signal transduction. Science, chromosomes (TACs) of barley chloroplasts contain 274, 982–985. the a-subunit of plastome-encoded RNA polymerase. 19. Hua, J. & Meyerowitz, E. M. (1998). Ethylene Curr. Genet. 30, 515–521. responses are negatively regulated by a receptor 37. Krause, K. & Krupinska, K. (2000). Molecular and gene family in Arabidopsis thaliana. Cell, 94, 261–271. functional properties of highly purified transcription- 20. Imamura, A., Hanaki, N., Umeda, H., Nakamura, A., ally active chromosomes from spinach chloroplasts. Suzuki, T., Ueguchi, C. & Mizuno, T. (1998). Response Physiol. Plantarum, 109, 188–195. regulators implicated in His-to-Asp phosphotransfer 38. Peltier, J. B., Ytterberg, A. J., Sun, Q. & van Wijk, K. J. signaling in Arabidopsis. Proc. Natl Acad. Sci. USA, 95, (2004). New functions of the thylakoid membrane 2691–2696. proteome of Arabidopsis thaliana revealed by a simple, 21. D’Agostino, I. B. & Kieber, J. J. (1999). Phosphorelay fast, and versatile fractionation strategy. J. Biol. Chem. signal transduction: the emerging family of plant 279, 49367–49383. response regulators. Trends Plant Sci. 24, 452–456. 39. Haze, K., Yoshida, H., Yanagi, H., Yura, T. & Mori, K. 22. Sakai, H., Aoyama, T., Bono, H. & Oka, A. (1998). (1999). Mammalian ATF6 is Two-component response regulators from Arabidopsis synthesized as a transmembrane protein and acti- thaliana contain a putative DNA-binding motif. Plant vated by proteolysis to endoplasmic reticulum stress. Cell Physiol. 39, 1232–1239. Mol. Biol. Cell, 10, 3787–3799. 23. Urao, T., Yakubov, B., Yamaguchi-Shinozaki, K. & 40. Rudner, D. Z., Fawcett, P. & Losick, R. (1999). A family Shinozaki, K. (1998). Stress-responsive expression of of membrane-embeded metalloproteases involved in genes for two-component response regulator-like regulated proteolysis of membrane-associated tran- proteins in Arabidopsis thaliana. FEBS Letters, 427, scription factors. Proc. Natl Acad. Sci. USA, 96, 175–178. 14765–14770. 24. Lohrmann, J., Buchholz, G., Keitel, C., Sweere, U., 41. Brown, M. S., Ye, J., Rawson, R. B. & Goldstein, J. L. Kircher, S., Ba¨urle, I. et al. (1999). Differential (2000). Regulated intramembrane proteolysis: a con- expression and nuclear localization of response trol mechanism conserved from to humans. regulator-like proteins from Arabidopsis thaliana. Cell, 100, 391–398. Plant Biol. 1, 495–505. 42. Sokolenko, A., Pojidaeva, E., Zinchenko, V., 25. Sakai, H., Aoyama, T. & Oka, A. (2000). Arabidopsis Panichkin, V., Glaser, V. M., Herrmann, R. G. & ARR1 and ARR2 response regulators operate as Shestakov, S. V. (2002). The gene complement for transcriptional activators. Plant J. 24, 703–711. proteolysis in the cyanobacterium Synechocystis sp 26. Jacobs, M. A., Connel, L. & Cattolico, R. A. (1999). PCC 6803 and Arabidopsis thaliana chloroplasts A conserved His-Asp signal response regulator-like (review). Curr. Genet. 41, 291–310. gene in Heterosigma akashiwo chloroplasts. Plant Mol. 43. Koonin, E. V., Makarova, K. S., Rogozin, I. B., Biol. 41, 645–655. Davidovic, L., Letelier, M.-C. & Pellegrini, L. (2003). 27. Lu¨ tcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., The rhomboids: a nearly uniquitous family of Kern, H. F. & Scheele, G. A. (1987). Selection of AUG intramembrane serine proteases that probably initiation codons differs in plant and . EMBO evolved by multiple ancient horizontal gene transfers. J. 6, 43–48. Genome Biol. 4, R19. 28. Sikorski, R. S., Boguski, N. S., Goebl, M. & Hieter, P. 44. Herrmann, R. G., Westhoff, P. & Link, G. (1992). (1990). A repeating amino acid motif in CDC23 Biogenesis of plastids in higher plants. In Plant Gene defines a family of proteins and a new relationship Research. Cell Organelles (Herrmann, R. G., ed.), pp. among genes required for mitosis and RNA synthesis. 276–332, Springer, Vienna. Cell, 60, 307–317. 45. Small, I. D. & Peeters, N. (2000). The PPR motif—a 29. Hurst, H. C. (1994). Transcription factors 1: bZip TPR-related motiv prevalent in plant organellar proteins. Protein Profile, 1, 123–168. proteins. Trends Biochem. Sci. 25, 46–47. TCP34, a Response Regulator-like TPR Protein 549

46. Herrmann, R. G. & Possingham, J. V. (1980). 60. Hunter, T. & Karin, M. (1992). The regulation of Plastid DNA–the plastome. Results Probl. Cell Differ. transcription by phosphorylation. Cell, 70, 375–387. 10, 45–96. 61. Hunter, T. (1995). Protein kinases: the Yin and Yang of 47. Kuroiwa, T. (1991). The replication, differentiation, an protein phosphorylation and signaling. Cell, 80, inheritance of plastids with emphasis on the concept 225–236. of organelle nuclei. Int. Rev. Cytol. 128, 1–62. 62. Wellmer, F., Scha¨fer, E. & Harter, K. (2001). The DNA 48. Herrmann, R. G. & Kowallik, K. V. (1970). Multiple binding properties of the parsley bZIP transcription amounts of DNA related to the size of chloroplasts. II. factor CPRF4a are regulated by light. J. Biol. Chem. Comparison of electron-microscopic and autoradio- 276, 6274–6279. graphic data. Protoplasma, 69, 365–372. 63. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). 49. Sato, N., Albrieux, C., Joyard, J., Douce, R. & Molecular Cloning: A Laboratory Manual, 2nd edit., Kuroiwa, T. (1993). Detection and characterisation of Cold Spring Harbor Laboratory Press, Cold Spring a plastid envelope DNA-binding protein which may Harbor, NY. anchor plastid nucleids. EMBO J. 12, 555–561. 64. Laemmli, U. (1970). Cleavage of structural proteins 50. Nakano, T., Murakami, S., Shoji, T., Yoshida, S., during the assembly of the head of bacteriophage T4. Yamada, Y. & Sato, F. (1997). A novel protein with Nature, 227, 680–685. DNA binding activity from tobacco chloroplast 65. Towbin, H., Staehelin, T. & Gordon, J. (1979). nucleoids. Plant Cell, 9, 1673–1682. Electrophoretic transfer of proteins from polyacryl- 51. Sato, N., Ohshima, K., Watanabe, A., Ohta, N., amide gels to nitrocellulose sheets: procedure and Nishiyama, Y., Joyard, J. & Douce, R. (1998). some applications. Proc. Natl Acad. Sci. USA, 76, Molecular characterization of the PEND protein, a 4350–4354. novel bZip protein in the envelope membrane that is 66. Clausmeyer, S., Klo¨sgen, R. B. & Herrmann, R. G. the site of nucleoid replication in developing plastids. (1993). Protein import into chloroplasts. The hydro- Plant Cell, 10, 859–872. philic lumenal proteins exhibit unexpected import and sorting specificities inspite of structurally con- 52. Sato, N. & Ohta, N. (2001). DNA-binding specificity served transit peptides. J. Biol. Chem. 268, and dimerization of the DNA-binding domain of the 13869–13876. PEND protein in the chloroplast envelope membrane. 67. Eichacker, L. A., Mu¨ ller, B. & Helfrich, M. (1996). Nucl. Acids Res. 29, 2244–2250. Stabilization of the chlorophyll binding apoproteins, 53. Jeong, S. Y., Rose, A. & Meier, I. (2003). MFP1 is a P700, CP47, CP43, D2, and D1, by synthesis of Zn- thylakoid-associated, nucleoid-binding protein with a pheophytin a in intact etioplasts from barley. FEBS 31 coiled-coil structure. Nucl. Acids Res. , 5175–5185. Letters, 395, 251–256. 54. Pfannschmidt, T., Nilsson, A. & Allen, J. F. (1999). 68. Baginsky, S., Tiller, K. & Link, G. (1997). Transcription Photosynthetic control of chloroplast gene expression. factor phosphorylation by a protein kinase associated Nature, 397, 625–628. with chloroplast RNA polymerase from mustard 55. Mizuno, T., Kaneko, T. & Tabata, S. (1996). Compi- (Sinapis alba). Plant Mol. Biol. 34, 181–189. lation of all genes encoding bacterial two-component 69. de Vitry, C., Diner, B. A. & Popot, J. L. (1991). signal transducers in the genome of the cyanobacter- Photosystem II particles from Chlamydomonas rein- ium Synechocystis sp PCC 6803. DNA Res. 3, 407–414. hardtii. Purification, molecular weight, small subunit 56. Yeh, K. C. & Lagarias, J. C. (1998). Eukaryotic composition, and protein phosphorylation. J. Biol. phytochromes: light-regulated serine/threonine pro- Chem. 266, 16614–16621. tein kinase with histidine ancestry. Proc. Natl Acad. Sci. 70. Polycarpou-Schwarz, M. & Papavassiliou, A. G. USA, 95, 13976–13981. (1993). Distinguishing specific from nonspecific 57. Chang, C. & Stewart, R. C. (1998). The two-component complexes on southwestern blots by rapid DMS system: regulation of diverse signaling pathways in protection assays. Nucl. Acids Res. 21, 2531–2532. prokaryotes and eukaryotes. Plant Physiol. 117, 71. Becker, W., Kentrup, H., Klumpp, S., Schultz, J. E. & 723–731. Joost, H. G. (1994). Molecular cloning of a protein 58. Sassone-Cori, P., Ransone, L. J., Lamph, W. W. & serine/threonine phosphatase containing a putative Verma, I. M. (1988). Direct interaction between Fos regulatory tetratricopeptide repeat domain. J. Biol. and Jun nuclear oncoproteins: role of the ‘leucine Chem. 269, 22586–22592. zipper’ domain. Nature, 336, 692–695. 72. Chen, J., Parsons, S. & Brautigan, D. L. (1994). 59. Yamamoto, K. K., Gonzalez, G. A., Briggs, W. H., III & Tyrosine phosphorylation of protein phosphatase 2A Montminy, M. R. (1988). Phosphorylation-induced in response to growth stimulation and v-src trans- binding and transcriptional efficacy of nuclear factor formation of the fibroblasts. J. Biol. Chem. 269, CREB. Nature, 334, 494–499. 7957–7962.

Edited by J. O. Thomas

(Received 1 September 2005; received in revised form 16 December 2005; accepted 21 December 2005) Available online 11 January 2006