Redox Regulation of Chloroplast Enzymes in ... - Oxford Journals

Plant Cell Physiol. 48(9): 1359–1373 (2007) doi:10.1093/pcp/pcm108, available online at www.pcp.oxfordjournals.org ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

Redox Regulation of Chloroplast Enzymes in Galdieria sulphuraria in View of Eukaryotic Evolution Christine Oesterhelt 1, Susanne Klocke 2, Simone Holtgrefe 2, Vera Linke 2, Andreas P. M. Weber Renate Scheibe 2, *

3

and

1

Department of Plant Physiology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24–25, D-14476 Potsdam-Golm, Germany 2 Department of Plant Physiology, Faculty of Biology and Chemistry, University of Osnabru¨ck, D-49069 Osnabru¨ck, Germany 3 Department of Plant Biochemistry, Heinrich-Heine-University, D-40225 Du¨sseldorf, Germany

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found. Keywords: Chloroplast enzymes — Complex formation — CP12 — Galdieria sulphuraria — Light/dark modulation — Molecular evolution. Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; EST, expressed sequence tag; FBPase, fructose 1,6-bisphosphatase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; G6PDH, glucose 6-phosphate dehydrogenase;

MDH, malate dehydrogenase; PRK, RT–PCR, reverse transcription–PCR.

phosphoribulokinase;

Introduction In higher plants, redox regulation of chloroplast enzymes is an efficient mechanism for light/dark modulation (Buchanan 1980) and the basis for a fine-tuning of enzyme activities by metabolites (Scheibe 1991). Photoautotrophic algae from the families of chlorophytes and rhodophytes also use the ferredoxin–thioredoxin system for redox modulation of Calvin cycle enzymes. However, the molecular mechanism and the redox-active structures of the target enzymes are often distinct from those of higher plants (Martin et al. 1999, Dietz et al. 2002). These enzymes and their regulatory properties are rather well understood in higher plants and also in green algae. However, much less is known about light/dark modulation and the target enzymes in red algae, although these organisms contribute a large share to the global CO2 assimilation. In our study, we focus on the redox regulation of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37), comparing their sequences and their regulatory properties. In chloroplasts of unicellular green algae, NADPGAPDH and PRK have been described to form inactive complexes of high molecular weight in darkness. Upon illumination, these complexes are reversibly dissociated and the enzymes are thereby activated (Lazaro et al. 1986, Nicholson et al. 1987, Clasper et al. 1994, Lebreton et al. 1997). Complex formation is achieved by interaction of GAPDH and PRK with the small chloroplast protein CP12 (Graciet et al. 2003). This protein is present in all

*Corresponding author: E-mail, [email protected]; Fax, þ49-541-969-2265. 1359

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Redox regulation of red algal chloroplast enzymes

photoautotrophic organisms, including cyanobacteria (Pohlmeyer et al. 1996, Wedel and Soll 1998), and regulation of enzyme activity via CP12 appears to be of general importance. In higher plants, the presence of two NADP-GAPDH isoforms, GapA and GapB, led to a new achievement: a hexadecameric A8B8 complex as the dark-inactivated form of NADP-GAPDH (Baalmann et al. 1994, Baalmann et al. 1995). GapB has never been found in a unicellular organism. It appears first in the multicellular charophyte Chlorokybus atmophyticus (R. Cerff and J. Petersen, personal communication). In addition to this A8B8 complex, land plants retained the mixed complex of GAPDH, PRK and CP12, present in algae, and are therefore able to modulate the activities of NADP-GAPDH and PRK in an advanced manner (Scheibe et al. 2002). Another Calvin cycle enzyme, FBPase, is activated upon reduction in higher plants, but alternatively also by high pH, or by high Mg2þ and substrate (FBP) concentrations (Schu¨rmann and Wolosiuk 1978). This redox regulation involves two of the three cysteines present on an amino acid insert of the enzyme (Chiadmi et al. 1999, Reichert et al. 2003). In red algae, these two cysteines are also present, but there is only a small activity change of FBPase upon oxidation/reduction (Reichert et al. 2003). In cyanobacteria, PRK, GAPDH and FBPase were found to be insensitive towards redox changes (Tamoi et al. 1998). On the other hand, the key enzyme of the oxidative pentose phosphate cycle, G6PDH, proved responsive towards redox changes, in both higher plants and cyanobacteria, although the conserved cysteines are different in these organisms (Wenderoth et al. 1997). Another chloroplast enzyme, NADP-MDH as part of the malate valve, is strictly redox regulated in higher plants. This is due to two regulatory cysteine/cystine pairs located on both N- and C-terminal extensions of the enzyme (Miginiac-Maslow et al. 2000). In unicellular green algae, NADP-MDH lacks the N-terminal cysteine pair, although the sequence extension is already present. Still, the enzyme is inactive if export of reducing equivalents from the chloroplast is not required. However, this redox regulation is less strict than in higher plants (Ocheretina et al. 2000). In contrast, no such enzyme is present in cyanobacteria, which comprise only a single compartment. In this study, we focus on the chloroplast enzymes from the red alga Galdieria sulphuraria (Cyanidiaceae). Recent data identified the Cyanidiaceae as a highly conserved sister group of the rhodophytes and place them at the basis of secondary endosymbiosis (Yoon et al. 2002, Yoon et al. 2004). It is therefore interesting to analyze how enzyme regulation in G. sulphuraria fits into the scheme of eukaryotic evolution. Recently the Galdieria genome has

been completely sequenced (http://genomics.msu.edu/galdieria/index.html) and molecular data and tools are now available to analyze redox regulation in the red alga on a molecular level.

Results Redox-dependent complex formation and activities of PRK and GAPDH from G. sulphuraria In order to determine whether there is redox-dependent complex formation between PRK and NADP-GAPDH in G. sulphuraria, cell extracts were subjected to gel filtration under reducing [dithiothreitol (DTT)] and oxidizing [oxidized glutathione (GSSG)] conditions, and elution profiles of PRK and GAPDH were compared. Depending on the pre-incubation of the extract, we obtained the complexed form of GAPDH and PRK with an apparent molecular mass of 550 kDa, or the tetrameric GAPDH of 160 kDa and the dimeric PRK of 80 kDa (Fig. 1). Incubation of extracts with GSSG prior to gel filtration led to formation of the high molecular weight complex (Fig. 1A), while incubation with DTT yielded exclusively tetrameric GAPDH and dimeric PRK (Fig. 1B). Extracts from darkened cells contained only the complexed form of PRK and GAPDH, while untreated extracts, harvested and extracted in dim light, yielded both the aggregated and the dissociated forms of the enzymes (Fig. 1C). Enzymatic activities of PRK and GAPDH in the high molecular weight complex were determined after incubation of the respective fractions with GSSG and with DTT (Fig. 1A). PRK activity was only slightly decreased upon incubation with GSSG, suggesting that the complexed enzyme was almost fully active. For GAPDH, the difference was more pronounced. In the presence of GSSG, GAPDH in the complex exhibited only about 20% of its full activity obtained after incubation with DTT (Fig. 1A). Enzymes in the DTT-treated profile eluted in their active state and already possessed their maximal activities which could not be stimulated further (Fig. 1B). Involvement of CP12 in complex formation To verify the presence of CP12 in the PRK–GAPDH complex, fractions from the gel filtration were separated by SDS–PAGE, blotted onto a polyvinylidene fluoride (PVDF) membrane and incubated with the antiserum against recombinant CP12 from G. sulphuraria. In the oxidized extract, immunostaining revealed a band at about 15 kDa (Fig. 2A). It was most intense in the fractions of the PRK–GAPDH complex and weaker or absent in fractions eluting later in the profile. When the cell extract was treated with DTT prior to gel filtration, PRK and GAPDH eluted as a dimer (PRK) and a tetramer (GAPDH) in a noncomplexed form (Fig. 1B). Immunolocalization of CP12 revealed the protein in fractions eluting later than dimeric

Redox regulation of red algal chloroplast enzymes 550

160 80 kDa 0.8

0.4

0.6

0.2

0.4

0.1

0.2

0.0

0.0

B 0.6

0.2

0.4 0.1 0.2 0.0

PRK activity (U/ml)

GAPDH activity (U/ml)

A 0.3

0.0

C 0.4

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Fraction #

Fig. 1 GAPDH and PRK activities after gel filtration of Galdieria sulphuraria crude cell extracts. The extract was incubated with 10 mM GSSG (A), or with 20 mM DTTred (B) prior to gel filtration. PRK (open triangles) and NADP-GAPDH (open circles) activities were determined after full activation of enzymes with 50 mM DTT. Both enzyme activities were also determined after incubation of the fractions with GSSG (PRK, filled triangles; GAPDH, filled circles). (C) The cell extract was prepared in dim light and without pre-incubation. Enzyme activities after gel filtration were determined after incubation of the fractions with DTT.

PRK, i.e. probably as a free dimer (Fig. 2B). Some signal in the first fractions results from highly complexed forms of a few thousand kiloDaltons containing membrane fragments and phycobilins, as could be concluded from the colored fractions. When recombinant CP12 from Galdieria was linked to an Ni2þ-NTA column and crude algal cell extract was applied to the column, the CP12–matrix exhibited a strong affinity for binding of NADP-GAPDH, as opposed to the empty matrix material (Fig. 3).

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Redox-dependent activation of other chloroplast enzymes from G. sulphuraria The above-described complex formation between GAPDH and PRK could not be detected for any other chloroplast enzyme of G. sulphuraria tested so far. Chloroplast FBPase eluted as a tetramer under all conditions, and the oxidized enzyme still exhibited significant activity (Fig. 4). In contrast, G6PDH, the key enzyme of the oxidative pentose phosphate pathway, is clearly subject to redox modulation in G. sulphuraria. The tetrameric enzyme, obtained in its oxidized form when the crude cell extracts was pre-incubated with GSSG prior to gel filtration, was inactivated by incubation with 10 mM DTT (Table 1, Fig. 4)—as would be expected from the key enzyme of a metabolic pathway that is only active in the dark. A complete enzyme inactivation, however, could not be achieved due to the presence of a cytosolic isoform. In order to determine whether NADP-MDH, the key enzyme of the malate valve in higher plants, was also present in red algae, the elution profiles from gel filtration were assayed for NAD- and NADP-dependent MDH activities, both before and after enzyme activation by DTT. No specific NADP-MDH was detected, while residual NADP-dependent activity (Table 1) was due to some unspecificity of NAD-MDH, as also described for higher plants (Scheibe and Stitt 1988). This finding is in agreement with an earlier analysis of NADPMDH from various algae, where no DTT-sensitive NADP-MDH activity was found in the cyanobacateria, Odontella sinensis (Bacillariophyta), Cyanophora paradoxa (Glaucocystophyta) and G. sulphuraria (Ocheretina et al. 2000). Comparison of PRK and GAPDH sequences from G. sulphuraria with the enzymes from higher plants We have isolated the cDNA clones for PRK and GAPDH from a cDNA library prepared from autotrophic cells (W. Gross, Berlin). The GAPDH (AJ012286) clone from Galdieria was sequenced and translated, leading to 77 amino acids for the transit peptide (predicted by analogy with the enzyme from other organisms) and 337 residues for the mature enzyme. BLAST analyses identified GAPDH from Galdieria as GapA, and phylogenetic analyses place Galdieria GapA in the vicinity of other GapA and GapB sequences and not next to GapC and GapCp (Fig. 5). Two additional GAPDH proteins of G. sulphuraria fall into the cluster of GapC, and a closely related red alga, Cyanidioschyzon merolae, also possesses only a single sequence of the plastid-localized GapA, but two sequences for GapC. Mature Galdieria GapA exhibits between 70 and 73% identity to mature GapA from higher plants and green algae, and 77–79% identity (95% similarity) to the GapA

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A MW

(kDa) GSSG GSSG

72 55 43 34 26

17

11

B

72 55

DTT

43 34 26

17

11 5

10

15 20 25 28

31 34 37 40 43

46 49 52 55

Fraction #

GAPDH activity (U/ml)

0.5

0.4

0.3

0.2

0.1

0 100 mM NAD+ 100 mM NADP+ 500 mM NaCl 1 M Imidazol

+ − + −

+ − + −

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Fig. 3 Affinity chromatography of GAPDH from Galdieria sulphuraria crude extract on a CP12 column. Recombinant CP12 was immobilized on an Ni2þ-NTA column. After incubation with the algal extract for 3 h, elution was done stepwise using NADþ, NADPþ, NaCl and imidazole as indicated. GAPDH activity was determined in the fractions (hatched bars). A column without bound CP12 was used as a control (black bars).

58 61 65

Fig. 2 Immunodetection of CP12 after gel filtration of cell extracts. Extracts from Galdieria sulphuraria, treated with 20 mM GSSG (A) or with 10 mM DTT (B) prior to gel filtration, were separated on a Superdex 200 column. Aliquots (20 ml) from both gel filtrations were separated by SDS–PAGE (15%) and blotted onto a PVDF membrane. Detection of CP12 protein was achieved with anti-G. sulphuraria CP12 serum from rabbit (1 : 10,000), anti-rabbit IgG-horseradish peroxidase (1 : 20,000) and ECL.

sequences from the red algae Chondrus crispus and Gracilaria gracilis (synonymous with G. verrucosa). The cysteine (C149; numbering derived from the enzyme of Bacillus stearothermophilus), involved in catalysis, is present in all of these GapA forms. Since no GapB sequence has been found in Galdieria, inactivation of GAPDH is likely to depend only on complex formation with CP12 (and PRK). The PRK (AJ012719) clone from Galdieria was also sequenced and translated, comprising 106 amino acid residues coding for a transit peptide (predicted by analogy) and 342 amino acids representing the mature enzyme sequence. Comparison of the Galdieria PRK with PRK from other organisms revealed an identity of 61–66% to the enzyme from plants, green algae and cyanobacteria. Most parts of the mature enzyme are conserved in all organisms, e.g. all four cysteines (Cys16, Cys55, Cys244 and Cys250; numbering derived from the spinach enzyme), as well as Trp155 and Asp160 as part of the active site (Charlier et al. 1994, Brandes et al. 1998). The major difference between the various PRK proteins is the size of the insert between Cys16 and Cys55. Galdieria PRK contains seven amino acids less than the enzyme from higher plants (Fig. 6), while the insert of the cyanobacterial enzyme is even 17–18 amino acids


shorter than in higher plants. It has been argued that a reduction of the insert size leads to a less efficient light/dark modulation in cyanobacteria (Wadano et al. 1995, Tamoi et al. 1998, Kobayashi et al. 2003, Tamoi et al. 2005). However, heterokonts appear to possess inserts that are five amino acids longer than in higher plants, yet inactivation of their PRK is also incomplete (Michels et al. 2005). Finally, the PRK from C. merolae even lacks one of the regulatory cysteines, namely Cys16 (Fig. 6).

0.05 FBPase 0.04 0.03

Enzyme activity (U/ml)

0.02 0.01 0.00 G6PDH 0.025 0.020 0.015 0.010 0.005 0.000 0

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Fig. 4 FBPase and G6PDH activities after gel filtration of Galdieria sulphuraria crude cell extracts under oxidizing conditions. Experimental conditions were as described in the legend of Fig. 1. Activities of FBPase and G6PDH were determined in the fractions directly after elution (oxidized sample, filled symbols) and after incubation with DTT (open symbols).

Table 1 extracts

a

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Comparison of the G. sulphuraria CP12 sequence with CP12 proteins from other sources Attempts to amplify a Galdieria CP12 sequence from the autotrophic cDNA library were not successfull. Therefore, we used the recently established expressed sequence tag (EST) collection of G. sulphuraria (Weber et al. 2004a) to identify a sequence encoding a protein partially homologous to CP12 (A4_22D02). From this EST sequence, primers were generated to amplify the full-length cDNA sequence from a cDNA library of heterotrophic cells. The genomic sequence of CP12 was identified within the contig_746_Nov18_2004 from the Galdieria genome project (http://genomics.msu.edu/Galdieria/index.html). The CP12-encoding gDNA (AJ870345) has a length of 551 bp and contains three introns. The cDNA (AJ870344) has a size of 390 bp and translates into a protein of 129 amino acids. There are two putative start codons present in the cDNA sequence (at þ1 and þ151). An alignment of the Galdieria protein with CP12 from other organisms shows that the C-terminal half of the protein, which corresponds to the GAPDH-binding loop and the linker region, exhibits high similarity to other CP12 sequences, while the N-terminal part appears to be less conserved (Fig. 7). According to Wedel and Soll (1998), CP12 proteins contain four conserved cysteine residues. Two of these are also present in the Galdieria protein (Cys115 and Cys124); the two cysteines in the N-terminal region are absent (Fig. 7). Phylogenic analyses of CP12 proteins from Galdieria and various other organisms place the Galdieria protein next to prokaryotic homologs and not on the branch of higher plants or green algae (Fig. 8). Interestingly, there are several examples among cyanobacteria, Glaucophytae and red microalgae for CP12 sequences lacking one pair of conserved cysteine residues, either the N- or the C-terminal one. The cyanobacterial CP12 from Synechococcus PCC 7942 (Tamoi et al. 2005), and that from C. paradoxa and C. merolae all lack the N-terminal cysteine loop, as does the Galdieria protein. This lack of N-terminal cysteine residues has an effect on the secondary structure of CP12. We subjected the recombinant protein as well as crude cell

Redox-dependent activities of chloroplast enzymes from Galdieria sulphuraria after gel filtration of crude cell

Incubation

NADP-GAPDH (U ml1)

PRK (U ml1)

FBPase (U mg1 protein)

G6PDH (U ml1)

NADP-MDH (U ml1)

NAD-MDH (U ml1)

20 mM GSSG 2.5 mM Diamide 10 mM DTT

0.12 n.d. 0.36

0.6 n.d. 0.75

14a n.d. 34a

0.075 0.055 0.025

n.d. 0.017 0.014

0.50 n.d. 0.45

From Reichert et al. (2003) for the recombinant protein. n.d., not determined.

1364


A. thaliana (At3g26650)

GapC


GapA

P. sativum N. tabacum

798

C. merolae C. merolae (CMJ250C) (CMM167C) Synechocystis G. sulphuraria (PCC6803, Gap1) (GapC-1) G. sulphuraria 1000 (GapC-2) 1000 1000

470

S. oleracea

513 1000

912

C. reinhardtii

P. sylvestris 996 963

479

925 992

N. tabacum


468

S. oleracea Synechocystis (PCC6803,Gap2)

588 748

632

GapCp

A. thaliana (At3g04120) A. thaliana (At1g13440)

GapB

1000

896

1000 1000

1000 C. annuum

G. sulphuraria C. merolae G. gracilis (CMJ042C)

C. crispus

A. thaliana A. thaliana (At1g16300) (At1g79530)

GapC Fig. 5 Phylogenic tree of GAPDH proteins from higher plants, green algae, rhodophytes, glaucophytes and cyanobacteria. The unrooted tree was generated using PHYLIP (Felsenstein 1993), and 1,000 bootstraps were performed. Numbers on the branches indicate the distance matrix (Dayhoff PAM). Closely related orthologs are circled.

Spinacia oleracea Oryza sativa Triticum aestivum Pisum sativum Arabidopsis thaliana Chlamydomonas reinhardtii Odontella sinensis Synechocystis PCC6803 Galdieria sulphuraria Cyanidioschyzon merolae

1 16 55 SQQQTIVIGLAADSGCGKSTFMRRLTSVFGGAAEPPKGGNPD-----SNTLISDTTTVICLDDFHSLDRNGRKV SVDKPVVIGLAADSGCGKSTFMRRLTSVFGGAAEPPKGGNPD-----SNTLISDTTTVICLDDYHSLDRTGRKE AVEQPIVIGLAADSGCGKSTFMRRLTSVFGGAAEPPKGGNPD-----SNTLISDTTTVICLDDYHSLDRTGRKE GDSQTIVIGLAADSGCGKSTFMRRLTSVFGGAAEPPKGGNPD-----SNTLISDTTTVICLDDYHSLDRTGRKE CAQETIVIGLAADSGCGKSTFMRRLTSVFGGAAKPPKGGNPD-----SNTLISDTTTVICLDDYHSLDRYGRKE DKDKTVVIGLAADSGCGKSTFMRRMTSIFGGVPKPPAGGNPD-----SNTLISDMTTVICLDDYHCLDRNGRKV EGEKPIVIGVAADSGCGKSTFMRRLTSIFGGEGVGPLGGGFDNGGWETNSLVSDLTTVLCLDDYHLNDRNGRKV QLDRVVLIGVAGDSGCGKSTFLRRLTDLFG----------------------EEFMTVICLDDYHSLDRQGRKA GIERPVIIGVAADSGCGKSTFLRRVNEIFGTKVSQ------------SHTPQGELVTVICLDDFHTLDRKGRAE NIQRPVMVGVAADSGAGKSTFLRRVMRMFGSDIPK------------GHTPQGELITVICLDDWHNRDRQGRKE * * *** ***** ** ** ** **** * ** **

Fig. 6 Sequence alignment of the N-terminal part of PRK proteins from cyanobacteria, glaucophytes, higher plants, green and red algae. Conserved cysteines (Cys16 and Cys55 of the mature spinach sequence) are shaded. Identical amino acids in all sequences are labeled with asterisks () To facilitate comparison between enzymes, putative plastid targeting signals have been omitted, and numbering of sequence residues has been adjusted to that of the mature spinach enzyme.

extracts from G. sulphuraria and spinach chloroplast stroma to non-reducing SDS–PAGE (Fig. 9). In spinach, both recombinant and native CP12 migrated significantly faster in the oxidized than in the reduced form. This can be ascribed to

the formation of intramolecular disulfide bonds which results in a shortening of the unfolded chain. The increased size of the recombinant protein (Fig. 9A) as compared with the native protein (Fig. 9B) is due to the His tag. When the

Redox regulation of red algal chloroplast enzymes C.merolae C.paradoxa At1g76560 G.sulphuraria Anabaena As12850 Synechococcus S.elongatus PCC7942 Prochlorothrix Synechocystis Anabaena Alr0765 Gloeobacter At2g47400 At3g62410 P.sativum N.tabacum S.oleracea O.sativa C.reinhardtii

C.merolae C.paradoxa At1g76560 G.sulphuraria Anabaena As12850 Synechococcus S.elongatus PCC7942 Prochlorothrix Synechocystis Anabaena Alr0765 Gloeobacter At2g47400 At3g62410 P.sativum N.tabacum S.oleracea O.sativa C.reinhardtii

C.merolae C.paradoxa At1g76560 G.sulphuraria Anabaena As12850 Synechococcus S.elongatus PCC7942 Prochlorothrix Synechocystis Anabaena Alr0765 Gloeobacter At2g47400 At3g62410 P.sativum N.tabacum S.oleracea O.sativa C.reinhardtii

1365

-------------MKAAFLIVPSN----ATHFSSVSVSKTRATFAVRRTFLRTRKTTRVA ----------------------AP----KIQFG---------VFAS-------------------MISGSATASHGRVLLPSQRERRPVSTGSNILRFRETVPRQFSLMMVTKATAKYM MLGFVQTFPIRVTRCKRDISFCSNTVKHREWKPRQSRFCQQPIQRVD---------LLTM -----------------------------------------------------------M ----------------------------------------------------------------------------------------------------------------------MTKPCITVNPDLEVEYVARLFAMTGIRRAPVIQGQLLGMISTTDILV---------KSNF -------------------------------------------------------------------------------------------------MTMTFNVAP---------SGAN ---------------------------------------------------------------MTTIAAAGLNVATPRVVVRP--VAR---VLGPVRLNYPWKFGS-------MKRMVV ----MATIATG-LNIATQRVFVTS--ENRPVCLAGPVHLNNSWNLGSRT--TNRMMKLQP ----MATISGL--SLSNPRLLFNSPGFPQTIKISSASPLSTRQTLTG----SGRMKIVQP ----MASIAGV--SITTPRILSKTSDSPK-VQTLKFQSLNKPWKNSSLVQFGHGKLYLKA ----MASMSLS----MTPKILLPN--NPTTNFDAPKLANMVTYKLHG----GRRSHGLVA ----MASTLTN-VGLSTPAAAASS--LVR--PVAGAGRVVFPRVGRG------GFAAVRA -------------MMLTKSVVISR---------PAVRPVSTRR------------AVVVR

ALRMANEKLNTQIQEAIKNAEDAAKKYGKASKEAKVAWDFVEELEAERSHQAANKQSE-----ADLSIQERIQKAISQARAVAEEKGATSKEARVAWDEVEELEAELSHQKAQPKA--GTKMREEKLSEMIEEKVKEATEVCEA-EEMSEECRVAWDEVEEVSQARADLRIKLKLLNSSSDSSDGWKQQIQESIKKAEEATKKFGRDSKEAAAAWDAVEELDAEASHQRVRQ----TDTQSKD-IQDQIQEEVEQARAVCDISGSNSAECAAAWDAVEELQAEASHQRQDK---------MSNLEKQIEQAREEAHKICDTEGATSGQCAAAWDALEELQAEAAHQRAEQQD-------MSDIQEKIEQARQEAHAISEEKGATSPDAAAAWDAVEELQAEAAHQRQQKSE--VEEPKAQRLEQLIQEAIAAARQICADEGTTSPGCAAAWDVVEELQAEAAHQEAKG-L-------MSNIQEKIEQELANARQVCSTDEASPAECAAAWDAVEELEAEAAHQRQQH-P--SDNNGTTNLEKAILAAIAEARTTCEQNGDGSPNCAVAWDIVEELQAEKSHQQQAQ-K------MTTNYDKLIQQEKAEAKEICSINGDGSAQCAAAWDAVEEVQAAASHAGDKD-K--VKATSEGEISEKVEKSIQEAKETC-ADDPVSGECVAAWDEVEELSAAASHARDKKKAGGIKAAPEGGISDVVEKSIKEAQETC-AGDPVSGECVAAWDEVEELSAAASHARDKKKADGVRAAPE-QISKKVEESIKSAQETC-ADDPVSGECVAAWDEVEELSAAASHARDRKKE--ISATPDNKLSDLVAESVKEAEEAC-AENPVSGECAAAWDVVEEASAAASHARDKKKES-VRAAPDNRISENVEKSIKEAQETC-SDDPVSGECVAAWDVVEELSAAASHARDKAKD--SGPATPPDISDKMSESIDKAKEAC-AEDTASGECAAAWDEVEELSAAASHARDKLKET-ASGQPAVDLNKKVQDAVKEAEDAC-AKG-TSADCAVAWDTVEELSAAVSHKKDAVKADVT

--DPLEKYCNEVPEADECRVYED --DPLQEFCKENPETDECRLYED -QDPLESFCQENPETDECRIYED KTDPLETFCDESPEAEECRVYDN KKNSLEQYCDDNPEAAECRVYDE HKTSFQQYCDDNPDAAECRIYDD TEPFFGDYCSENPDAAECLIYDD IKTAFEEYLEENPEALEARVYDV TQTTLEKFCDENPDAAECRIYDD RKSSLESFCDLHPEALECLIYDV KMNSLQSYCADNPDAAECRIYED -SDPLEEYCNDNPETDECRTYDN -SDPLEEYCKDNPETNECRTYDN -SDPLEDYCKDNPETDECKTYDN -SDPLENYCKDNPETDECRTYDN -VEPLEEYCKDNPETDECRTYDN -SDPLEAYCKDNPETDECRTYDN LTDPLEAFCKDAPDADECRVYED

Fig. 7 Comparison of CP12 protein sequences. Alignment of CP12 from prokaryotic and eukaryotic organisms. The position of the conserved cysteines is indicated by arrows. Transit peptides and putative transit peptides are included in the alignment.

same method (Allore and Barber 1984) was applied to recombinant CP12 from G. sulphuraria and to algal cell extracts, the oxidized CP12 showed hardly any difference in migration as compared with the reduced protein. Significant changes in CP12 secondary structure upon oxidation are therefore unlikely for the Galdieria protein.

Expression of CP12 in G. sulphuraria In order to compare the expression of CP12 in autotrophically and heterotrophically grown Galdieria, we isolated mRNA and total protein from both cell types and used Northern blot analysis, reverse transcription–PCR (RT–PCR) and Western blot analysis to follow CP12 levels.

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DN S. elongatus (PCC7942) Synechocystis sp. (PCC 6803)

Anabaena (Alr0765)

353 502

Synechococcus

Prochlorothrix

DC

313 357


460

G. violaceus

828 Anabaena sp. (As12850)

O. sativa

534

358 284

N. tabacum

987

844

C. paradoxa DN

751 S. oleracea

C. reinhardtii 370


G. sulphuraria DN

833

605 P. sativum A. thaliana (At2g47400)

C. merolae

DN

Fig. 8 Phylogenic tree of CP12 proteins from cyanobacteria, rhodophytes, chlorophytes and higher plants. The unrooted tree was generated using PHYLIP (Felsenstein 1993), and 1,000 bootstraps were performed. Numbers on the branches indicate the distance matrix (Dayhoff PAM). The lack of N- or C-terminal cysteine pairs is indicated by N and C, respectively.

CP12 transcripts were present in both autotrophic and heterotrophic cells—as indicated by Northern blot analysis and RT–PCR (Fig. 10A, B). CP12 protein could also be detected in both cell types (Fig. 10C). Immunoblots of extracts from autotrophic cells gave a single band at 15 kDa, while two bands were present in extracts from heterotrophic cells—one at 15 kDa and the other at 20 kDa. The identity of the larger protein remains to be determined. Comparison of redox-active cysteine motifs in FBPase and G6PDH from various organisms A comprehensive search of the Galdieria genome resulted in the identification of two isoforms of plastidic FBPases. This was surprising, as the protein is established as a single-copy gene in other organisms. The protein encoded by Contig 06901.g19.t1 is predicted to be plastid localized by TargetP, while FBPase-06101.g25.t1, which is almost identical to the EMBL GenBank entry AJ 302644

(CAC82800) (Reichert et al. 2003), is not addressed as a plastidic enzyme by this prediction tool for intracellular localization. Both sequences contain the two conserved cysteines involved in redox modulation in the corresponding enzymes of higher plants. Apparently, a gene duplication event in Galdieria gave rise to the two paralogous genes encoding putative plastidial FBPases. In the genome of the closely related red alga C. merolae, only a single copy of the gene encoding plastidial FBPase is found. Since C. merolae is an obligate photoautotroph whereas G. sulphuraria is also able to grow heterotrophically, it is tempting to speculate that this gene duplication might be related to the metabolic versatility of the latter alga. Some support for this assumption comes from the fact that an EST (EST A4_17H07), mapping to FBPase-06101.g25.t1, could be isolated from a cDNA library generated from autotrophically grown cells (Weber et al. 2004a), whereas no autotrophic ESTs could be mapped to gene 06901.g19t1.

Redox regulation of red algal chloroplast enzymes Spinach

A

red

ox

Galdieria red

1367

A

ic

ph

ic

ro ot

ox

r ete

MW (kDa) 40 -

h

ph

ro

t to

au

33 CP12 24 -

17 -

rRNA

11 -

B Spinach

B

red

Galdieria ox

red

CP12

ox

MW (kDa) 55 40 33 -

C

MW (kDa) 24

24 17

CP12

17 11 11 -

Fig. 9 SDS–PAGE (non-reducing) of reduced and oxidized CP12 from Galdieria sulphuraria and spinach. (A) The recombinant proteins with a His tag were used. Samples were either reduced and alkylated (red) or oxidized (ox) as described. The gel was stained with Coomassie brilliant blue. (B) Crude cell extracts from G. sulphuraria and spinach chloroplast stroma, either reduced and alkylated or oxidized, were subjected to non-reducing SDS–PAGE and blotted onto a PVDF membrane. Immunodetection was achieved with polyclonal antisera against G. sulphuraria or spinach CP12 (1 : 10,000), respectively, anti-rabbit IgG–horseradish peroxidase as the second antibody (1 : 20,000) and ECL.

Both genes are expressed in G. sulphuraria since multiple sequence tags, generated from a mixed cDNA library (i.e. prepared from autotrophic, mixotrophic and heterotrophically grown cells), could be mapped to both genes (not shown). While there are four plastidic isoforms of G6PDH in A. thaliana (Wakao and Benning 2005), we could identify only two isoforms of this enzyme in the G. sulphuraria genome. One of them appears to be identical to the previously isolated clone CAB52681 and to the EMBL entry AJ006246. Both enzymes of G. sulphuraria are also

Fig. 10 CP12 mRNA and protein in crude extracts of heterotrophically and autotrophically grown Galdieria sulphuraria. (a) Northern-blot and (b) RT–PCR analysis of mRNA preparations using a homologous full-length probe. (c) For immunodetection, 25 mg of total protein of the crude extracts were subjected to SDS–PAGE and blotted onto a PVDF membrane. Immunodetection was achieved with anti-G. sulphuraria CP12 serum from rabbit (1 : 10,000), antirabbit IgG–horseradish peroxidase (1 : 20,000) and ECL.

very similar to the two isoforms of C. merolae. These plastidic G6PDH isoforms of the Cyanidiaceae all contain two conserved cysteines in the N-terminal part of the sequence. These cysteines have previously been identified as redox active (Cys149 and Cys157) in potato (Wenderoth et al. 1997). In contrast, the cyanobacterial G6PDH does not possess these cysteines, although its activity is also redox-modulated (Udvardy et al. 1984). Instead, it contains two conserved bacterial-type cysteine residues in the C-teminal half of the sequence (Wendt et al. 1999). NADP-dependent malate dehydrogenase in algae and higher plants When the genome of G. sulphuraria was analyzed for the presence of MDH sequences that could be localized in

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the plastid, no hit was found. This is in agreement with previous studies indicating the absence of redox-modulated NADP-dependent MDH activity in extracts of various algae, including G. sulphuraria (Ocheretina et al. 2000). Only in green algae such as Chlamydomonas, is a plastidtargeted NADP-dependent MDH activity evident. This enzyme is redox-regulated, but less strictly than in higher plants (Ocheretina et al. 2000, Lemaire et al. 2005), possessing the C-terminal and N-terminal sequence extensions, but with no cysteine on the N-terminal extension.

Discussion Regulation of Calvin cycle enzymes is necessary in all photosynthetic organisms, prokaryotes as well as eukaryotes. In darkness, this pathway is down-regulated, and anapleurotic carbon as well as NADPH are generated by the oxidative pentose phosphate pathway. Simultaneous activity of the oxidative and reductive pathways would result in futile cycling of organic carbon and a loss of ATP. Tight regulation of both pathways is therefore essential. We have focused on the plastidic enzymes PRK, NADPGAPDH and FBPase as key enzymes of the Calvin cycle, on G6PDH as a key enzyme of the oxidative pentose phosphate pathway, and on NADP-MDH as part of the malate valve of the chloroplast. PRK of Rhodobacter sphaeroides, a facultative photoheterotrophic bacterium, has been studied in great detail (Harrison et al. 1998). This enzyme is not redox modulated and does not contain regulatory cysteines in its active site, as is the case for the orthologous enzyme in higher plants (Gibson et al. 1990). Instead, it is rather sensitive to NADH as allosteric effector (Harrison et al. 1998). Cyanobacteria contain PRK with a redox-active cysteine pair; however, the domain between the two cysteines is 17–18 amino acids shorter than in higher plants (Wadano et al. 1995), and light/dark modulation of enzyme activity appears to be less efficient (Tamoi et al. 1998, Tamoi et al. 2005). PRK from the chromophytes Heterosigma carterae (Hariharan et al. 1998) and O. sinensis both contain the functional active site with the same regulatory cysteines as in higher plants. However, the insert between the two cysteines in PRK from these diatoms is even longer than in the higher plant enzymes. Yet, the enzyme does not appear to be subjected to redox modulation (Michels et al. 2005). The diatom PRK was suggested to originate from green algae, mediated by lateral gene transfer, not from red algae (Petersen et al. 2006). PRK from the unicellular red alga G. sulphuraria, isolated in this study, contains seven amino acids less in the insert between the two cysteines as compared with sequences of higher plants (Fig. 6). Gel filtration experiments indicated that PRK activity is not redox modulated in the red alga, since the aggregated form of the enzyme

yielded nearly as much activity when oxidized as after incubation with DTT. This lack of redox modulation can most probaby be attributed to the reduced size of the insert, as has also been suggested for the cyanobacterial enzyme (Tamoi et al. 1998). In a mutant strain of Synechococcus PCC 7942, a lack of PRK down-regulation by CP12 leads to lower respiration rates and a higher ribulose 1,5-bisphosphate content. Growth rates of this strain are compromised under light/dark cycles, but normal in continuous light (Tamoi et al. 2005). It is still unclear how Galdieria compensates for the apparent lack of PRK regulation. GapA from Galdieria is characterized by a high similarity to Gap genes from other organisms. In none of our attempts to isolate a GAPDH clone from the unicellular red alga could a sequence for GapB be identified. Regulation of GAPDH activity by GapA/GapB complex formation (A8B8) is therefore unlikely in Galdieria. The GapA sequence from the marine red alga G. verrucosa (synonymous with G. gracilis) displays similarity to GapA/B from higher plants, suggesting a common origin of the plastids of rhodophytes and higher plants (Zhou and Ragan 1993, Zhou and Ragan 1994). Also, GapA from C. crispus (Rhodophytae) is positioned between cyanobacteria as ancestor and the chloroplast enzyme from higher plants (Liaud et al. 1993). A GapB sequence, on the other hand, has so far not been found in any unicellular alga. It appears for the first time in the multicellular Characea Chlorokybus atmophyticus, possibly the ancestor of land plants (J. Petersen and R. Cerff, personal communcation). Interestingly, this GapB sequence consists of a core part with high similarity to GapA, and a C-terminal extension, similar to the C-terminal part of CP12. Regulation of GAPDH activity in Galdieria appears to take place via complex formation with CP12 (and PRK). However, inactivation of GAPDH in the high molecular weight complex was not complete, as the complexed enzyme still exhibited about 20% of its maximal activity (Fig. 1A). Alternatively, the complex might be unstable due to dilution in the assay, leading to increased activities even without activation by DTT. Wedel and Soll (1998) have suggested that CP12 arose from fusions of cyanobacterial genes. Its two different protein-binding loops as well as the extra C-terminus of GapB from higher plants are a good indication for such a scenario. From sequence comparison of all currently available genes encoding CP12-like proteins, it is obvious that, apart from ‘classical’ ones with two 2-Cys-loops, there are various other forms lacking one or two of the four cysteines (Figs. 7, 8). The CP12 sequence from Prochlorotrix hollandica lacks both cysteines of the second loop, while CP12 from Synechococcus elongatus (PCC 7942), C. paradoxa and C. merolae lacks the two cysteines of the


first loop. In this respect, the new sequence from Galdieria is similar to those of the latter organisms. The N-terminal part of Galdieria CP12 contains three unique cysteine residues which are absent from the other homologs. However, a function for these additional cysteines is unlikely, since they are located within the putative targeting sequence of the protein. Also, they do not seem to affect the secondary structure of the recombinant protein upon oxidation (Fig. 9A). Similarly, native CP12 migrates without a significant difference between the oxidized and reduced form (Fig. 9B). In higher plants, two intramolecular loops are formed upon oxidation of CP12, causing a shift to smaller molecular mass in the SDS gel (Allore and Barber 1984). In Galdieria it is even questionable whether a single loop (at the C-terminus) is formed, since no major shift could be observed between oxidized and reduced CP12 on SDS–PAGE (Fig. 9). Galdieria is able to grow autotrophically as well as heterotrophically. Under autotrophic conditions in a day– night cycle, light–dark modulation of Calvin cycle activity is essential to avoid futile cycling of organic carbon. Growth in continuous darkness, e.g. on glucose, would not require such a regulation if the Calvin cycle would be otherwise inactivated, e.g. at the transcriptional level. However, the transcript and protein abundance of CP12 in auto- and heterotrophic cells (Fig. 10) indicates that, in a dark environment, constant inactivation of the two Calvin cycle enzymes PRK and NADP-GAPDH is necessary on the post-translational level. This observation is in accordance with a constitutive expression of the vast majority of metabolic enzymes in the alga. Galdieria strain 074G retains full pigmentation even under heterotrophic conditions (Gross and Schnarrenberger 1995) and also still contains the enzymatic equipment for CO2 assimilation (e.g. ribulose 1,5-bisphosphate carboxylase/oxygenase), yet at a reduced level compared with autotrophic cell cultivation (Oesterhelt et al. 2007). For complete inactivation of the Calvin cycle, complex formation of PRK and GAPDH with CP12 would therefore be required. However, as inactivation of PRK by complex formation was only marginal, other mechanisms for regulation of enzyme activity have to be postulated. While the gene encoding plastidic FBPase is present only as a single copy in most photosynthetic organisms, red algae, as the ancestors of complex plastids derived from secondary endosymbiosis, appear to contain two FBPase isoforms that are closely related and both contain the two cysteines that are responsible for S–S bridge formation in the higher plant enzyme (Chiadmi et al. 1999, Reichert et al. 2003). However, in the red algal sequences, a shorter, negatively charged insertion and no arginine residue in front of the second cysteine are the features thought to be responsible for the lack of redox modulation previously

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observed for the recombinant protein (Reichert et al. 2003). Nevertheless, the two FBPase sequences of G. sulphuraria, and also those of C. merolae, cluster with the plastidic FBPases of photoautotrophic organisms. The regulatory properties of the algal enzyme have been characterized as different from those of the higher plant FBPase, the former exhibiting only 50% decrease of activity upon oxidation, but no shift in the S0.5 values for Mg2þ and FBP or in the pH optimum (Reichert et al. 2003). In contrast, the cyanobacterial enzymes as well as those from purple bacteria (form II) are more closely related to other bacterial sequences and do not contain the conserved motif of the plastidic forms (Gibson et al. 1990). In addition, FBPase from cyanobacteria has been described as H2O2 insensitive and not subjected to light/dark modulation (Tamoi et al. 1998), indicating that there is no such strict requirement for regulation of the Calvin cycle in cyanobacteria. It is tempting to hypothesize that, in red algae, plastidic FBPase activity is required for the biosynthesis of hexose phosphates from triose phosphates in the dark. Red algae do not appear to have plastidic pentose phosphate or hexose phosphate, but only triose phosphate translocators (Weber et al. 2004a; M. Linka and A.P.M.W. unpublished results). While in green plants, these pentose and hexose phosphate translocators provide the plastidial starch biosynthesis and the oxidative pentose phosphate pathway with carbon precursors from the cytosol (Weber et al. 2004b, Weber et al. 2005), starch biosynthesis in red algae is a cytosolic pathway (Viola et al. 2001). Support for the oxidative pentose phosphate pathway in the dark can only be sustained if imported triose phosphates are converted into hexose phosphates by a plastidic FBPase. Complete inactivation of FBPase in the dark would block this flux of triose phosphates into the plastidial hexose phosphate pool. G6PDH isoforms are present as multiple copies in the cytosol and plastids of higher plants (Wakao and Benning 2005), but only the plastidic isoforms are redox regulated, being inactivated by reduction and in the light (Scheibe et al. 1989). This property apparently depends on two conserved cysteines (Wenderoth et al. 1997). Although the cyanobacterial G6PDH is also redox modulated, it does not contain this cysteine motif, but instead contains two other conserved cysteines, thus giving an example of functional convergence in these G6PDH isoforms (Wendt et al. 1999). From this study and from the present analysis of the G. sulphuraria and C. merolae genomes, it is obvious that the red algal plastidic isoforms are more closely related to plastidic than to cytosolic G6PDH of higher plants and can also be clearly distinguished from bacterial G6PDH sequences. Since all forms are present in the same compartment as the Calvin cycle, the strict inactivation in the light prevents futile cycling due to simultaneous activity

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of both pathways. Apparently, two alternative mechanisms for inactivation of G6PDH have evolved in cyanobacteria and eukaryotes. The apparent absence of a plastid-targeted NADPdependent MDH from the genomes of red algae, as reported here, coincides with the absence of plastidic dicarboxylate translocators from this group of photosynthetic eukaryotes (Weber et al. 2004a, Barbier et al. 2005). Plastidic dicarboxylate translocators (2-oxoglutarate/ malate translocator, DiT1; glutamate/malate translocator, DiT2) are required for a functional photorespiratory carbon cycle in land plants (Renne´ et al. 2003, Schneidereit et al. 2006, Reumann and Weber 2006). In addition, DiT1 was shown in in vitro assays to act as an oxaloacetate/malate exchanger (Taniguchi et al. 2002, Renne´ et al. 2003, Taniguchi et al. 2004). Based on the latter observation, it was proposed that DiT1 is a component of the plastidic malate valve, namely the oxaloacetate/malate shuttle that is required for the transfer of redox equivalents across the chloroplast envelope membrane in green plants (Heineke et al. 1991, Scheibe 2004). Apparently, the plastidic malate valve, as it is known from land plants, is not present in red algae. In conclusion, a complex picture arises, showing different variations of redox regulation in photosynthetic organisms. It appears that for each case specific adaptations have evolved, driven by the needs for more sophisticated regulation and fine-tuning. At the various stages, the degree of functional redox regulation is different depending on the enzyme. With the stepwise evolution of cell compartments, multicellular organisms and land plants, the molecular properties of redox-regulated enzymes became more efficient in coping with the rapid changes of environmental conditions encountered by these organisms.

Materials and Methods Growth and extraction of the algal cells Galdieria sulphuraria (strain 074G) was cultivated in minimal medium at pH 2.0 (Gross and Schnarrenberger 1995). Heterotrophic cultures were supplemented with 25 mM glucose and grown in 2 liter Erlenmeyer flasks on a rotary table (130 r.p.m.). Autotrophic cultures were supplied with sterile air (enriched with 2% CO2) from the bottom of the culture flask and irradiated with white incandescent light (80 mE m2 s1). Light–dark cycles of 12 h were used for illumination. Cells were harvested during exponential growth. After centrifugation (5 min; 3,000g), cells were washed twice with 10 mM Bicine-KOH, pH 8.0. The pellet was stored at 808C or immediately used for extraction. Cells were homogenized with mortar, pestle and quartz sand, adding repeatedly small portions of liquid nitrogen. Extraction was then achieved with two cell volumes of buffer (50 mM Bicine-KOH, pH 8.0, 0.14 mM NAD). Insoluble material was removed by repeated centrifugation for 5 min at 20,000g and 48C. The crude extract was supplemented with protease inhibitor (100 mM Pefabloc).

Gel filtration of algal extracts, enzyme assays and protein determination For determination of the molecular mass of the native enzymes, the soluble protein fraction was subjected to gel filtration on a Superdex-200 column (Pharmacia, Freiburg, Germany) using an FPLC system. To simulate oxidizing conditions, the sample was incubated with 20 mM GSSG and 0.14 mM NAD for 30 min prior to gel filtration. For complete reduction, the sample was treated with 10 mM DTT for 30 min. For elution from the column, 10 mM Bicine-KOH, pH 7.9, 0.14 mM NAD, 150 mM NaCl was used as buffer (flow rate, 1 ml min1; fractions, 1 ml). Activities were measured after incubation of an aliquot sample with either DTT or oxidant at 258C as indicated. PRK activity was measured according to Porter et al. (1986). NADP-GAPDH activity was determined as described by Baalmann et al. (1995). G6PDH activity was determined as in Graeve et al. (1994). FBPase activity was determined as in Reichert et al. (2000). NAD- and NADPdependent MDH activities were assayed as in Scheibe and Stitt (1988). Protein concentration was measured according to Bradford (1976).

cDNA library screening A ZAP II cDNA library, prepared from autotrophic G. sulphuraria, was kindly provided by C. Schnarrenberger and W. Gross, FU Berlin. For the preparation of a ZAP II cDNA library of heterotrophic cells, G. sulphuraria was cultivated on glucose in the dark. Partial sequences for PRK and GAPDH were obtained by screening the library with heterologous probes derived from higher plants. A subsequent screening with homologous probes generated both full-length clones. The cDNA for CP12 was identified from an EST collection of G. sulphuraria (http://genomics.msu.edu/Galdieria/index.html) (Weber et al. 2004a). The full-length sequence was generated by PCR with cDNA of heterotrophic cells (CP12-specific primers: 50 -CCA GAA GCA GAA GAG TGC AGA GTA TAT GAC-30 as forward and the reverse complementary sequence as reverse primer; T3 and T7 primers were used as plasmid-specific primers). The gDNA sequence of CP12 originates from the genome project of G. sulphuraria, available at http://genomics.msu.edu/Galdieria/ index.html (Weber et al. 2004a, Barbier et al. 2005). Sequence data for comparisons of G6PDH and FBPase were also taken from this source. Sequences from C. merolae are available at http://merolae. biol.s.u-tokyo.ac.jp/(Matsuzaki et al. 2004).

Overexpression in Escherichia coli and purification of CP12 The full-length sequence of CP12 from G. sulphuraria was cloned into the vector pET16b (Novagen) containing in-frame the sequence of an N-terminal His tag with NdeI–BamHI as restriction sites. This construct was transformed into E. coli strain BL21 DE3 pLysS (Novagen). Cells were grown at 378C to a density of OD600 ¼ 0.6, and induction was started with 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 308C. After 4 h, cells were harvested by centrifugation at 2,000g. A nickel-NTA column (Novagen) was used for purification according to the manufacturer’s protocol. From 800 ml of culture, 40 mg of CP12 protein were obtained routinely. Recombinant spinach CP12 with an N-terminal His tag was cloned into pET14b and obtained as described by Pohlmeyer et al. (1996).

Redox regulation of red algal chloroplast enzymes Affinity chromatography on Ni2þ-NTA with immobilized G. sulphuraria CP12 Recombinant Galdieria CP12 was bound to a 1 ml nickelNTA column (Novagen) and incubated with clarified algal cell extract (1.7 mg protein) for 3 h at 48C. As a control, the gel matrix without bound CP12 was used. Washing and elution were performed stepwise (in 1 ml portions) with 100 mM NADþ plus 500 mM NaCl, 100 mM NADPþ plus 500 mM NaCl, and finally 1 M imidazole plus 500 mM NaCl, all in 20 mM Tris–HCl, pH 7.9. Aliquot samples of all fractions were assayed for GAPDH activity. Preparation of an antiserum against G. sulphuraria CP12 For immunization of a rabbit, purified CP12 (portions of 200–300 mg in polyacrylamide gel pieces) was used to immunize rabbits three times (BioScience, Goettingen, Germany). The total serum of the final blood was used for the experiments, diluted as indicated in Tris-buffered saline containing 0.3% skimmed milk powder and 0.03% bovine serum albumin (BSA) as blocking proteins. SDS–PAGE, Western blotting and immunodetection of CP12 For reduction of purified recombinant CP12 from spinach and G. sulphuraria, samples were treated with 20 mM DTT and, after 1 h, with 60 mM iodoacetamide. For oxidation, samples were left untreated as this presumably generates their oxidized forms (Pohlmeyer et al. 1996). For SDS–PAGE (15%), proteins (8 mg) were separated under non-reducing conditions and stained with Coomassie brilliant blue R250. Protein samples with crude cell extracts were separated on SDS–polyacrylamide (15%) gels and blotted onto PVDF membranes. After blocking, the antisera against G. sulphuraria CP12 or spinach CP12, respectively, were added overnight in a 1 : 20,000 dilution. The second antiserum against rabbit IgG conjugated with horseradish peroxidase was used at a 1 : 10,000 dilution. The signal was developed using the chemiluminescent substrate ECL (GE Healthcare Bio-Sciences, Freiburg, Germany). RT–PCR and Northern blot analysis For RT–PCR, total RNA was prepared from frozen cells using the RNAeasy Plant Mini Kit (Qiagen, Hilden, Germany). For removal of DNA, a DNase digestion was performed with 2.5 mg of total RNA. Afterwards total RNA was used for the synthesis of first-strand cDNA (RevertAidTM H Minus First Strand cDNA Synthesis Kit, Fermentas, St. Leon-Rot, Germany). For PCR amplification of CP12, the following primers were used: 50 -GGG TTT TGT ACA AAC TTT CCC-30 and 50 -GTC ATA TAC TCT GCA CTC TTC-30 . The expected PCR product comprises 378 bp. The CP12 transcript was amplified in 40 PCR cycles. For Northern blot analysis, total RNA was isolated from frozen cells using the Purescript RNA Extraction Kit (Gentra Systems, Minneapolis, MN., USA). For RNA gel-blot hybridization analysis, 10 mg of total RNA was denatured and separated on a 1.2% (w/v) agarose–2.5% (v/v) formaldehyde gel. Homidium bromide was included in the loading buffer to assure equal sample loading. RNA was blotted onto a positively charged nylon membrane (Nytran-Plus, Schleicher and Schuell, Germany) by downstream capillary transfer. RNA was cross-linked to the membrane by UV irradiation. Pre-hybridization and hybridization were performed at 658C in Church buffer medium [0.25 M sodium phosphate buffer, pH 7.2, 1 mM EDTA, 7% (w/v) SDS and 1% BSA]. Hybridization was performed with an a-32PdCTP-labeled

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CP12-cDNA-specific probe (Ready-To-Go DNA labeling beads, Amersham Biosciences). Membranes were washed twice for 30 min at 658C in washing buffer (40 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA), containing 5% (w/v) SDS and 5% (w/v) BSA, then for 10 min at room temperature in washing buffer containing 1% (w/v) SDS. Finally, membranes were exposed to Kodak MS X-ray film at 808C. Bioinformatic analysis Alignments and constructions of phylogenetic trees were performed using the CLUSTAL W and PHYLIP tools available at http://www.pasteur.fr/english.html.

Acknowledgments The authors wish to thank the following people for help with some experiments: Martina Herrig, Roland Krauss, Carola Kuhn, Alexandra Hackmann, Dr. Stanislav Vishnyakov, Dr. Achim Tegeler, Dr. Carsten Sanders and Dr. Andre´ Dennes. Dr. Wolfgang Gross, FU Berlin, was initially involved with much enthusiasm; his early death leaves a gap. This work was supported by NSF Award EF-0332882 (to A.P.M.W.) and the Deutsche Forschungsgemeinschaft (Emmy Noether Fellowship to C.O.).

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(Received May 31, 2007; Accepted August 10, 2007)