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First published online November 23, 2005; 10.1104/pp.105.069732 Plant Physiology 139:1946-1958 (2005) © 2005 American Society of Plant Biologists Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii1Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912
Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.
Hemes and chlorophylls are major tetrapyrrole pigments that are essential for energy metabolism in all photosynthetic organisms. Hemes and chlorophylls are synthesized in a multistep pathway that involves nucleus-encoded enzymes (Beale, 2005  ). 5-Aminolevulinic acid (ALA) is the earliest universal biosynthetic precursor of tetrapyrroles. The biosynthetic steps from ALA to protoporphyrin IX are common to the synthesis of both chlorophylls and hemes. The enzyme that catalyzes the last shared step of heme and chlorophyll synthesis is protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4). Ferrochelatase (FeC; EC 4.99.1.1) catalyzes the last step in heme biosynthesis: the insertion of a ferrous ion into protoporphyrin IX to form protoheme.
PPO and FeC have been extensively studied, and the crystal structures of both enzymes have been determined, enabling further determination of their reaction mechanisms (Wu et al., 2001 The unicellular green alga Chlamydomonas reinhardtii is similar to plants in that it contains both a chloroplast and mitochondria. However, unlike plants, C. reinhardtii does not undergo tissue differentiation. We report here that, in contrast to plants, C. reinhardtii contains only one gene each for PPO and FeC, and that the products of these genes are present only in the chloroplast. Our results on the light regulation of PPO and FeC expression and the intracellular location of these proteins lead us to suggest possible roles for the multiple genes encoding these enzymes in plants.
Isolation of C. reinhardtii PPO and FeC cDNAs C. reinhardtii cDNAs coding for PPO and FeC were isolated by complementation of Escherichia coli hemG and hemH mutant strains, which lack PPO and FeC, respectively. There was a noted difference in the yield of colonies of complemented hemG and hemH cells: the C. reinhardtii cDNA library produced high numbers of complemented hemG colonies, whereas the library produced only a single colony after multiple attempts to complement the hemH strain. Possible reasons for this difference in colony yield include a lower abundance of FeC transcripts compared with those for PPO in the mRNA that was used to construct the cDNA library, and differences in the functional state of the two full-length (unprocessed) translation products in the E. coli cells. Nevertheless, the fact that C. reinhardtii PPO and FeC did complement the E. coli mutant strains indicates that the isolated cDNAs code for functional PPO and FeC proteins. Searches of the C. reinhardtii expressed sequence tag (EST) databases at the Joint Genome Institute (JGI; http://genome.jgi-psf.org/cgi-bin/runAlignment?db=chlre2) and Kazusa DNA Research Institute (http://www.kazusa.or.jp/en/plant/chlamy/EST/blast.html) yielded only one short EST clone for PPO and no matches corresponding to FeC.
A search of the C. reinhardtii genome sequence database indicated that there is only one gene each encoding PPO (ppx, JGI C_330078; as previously reported by Lohr et al., 2005
Analysis of C. reinhardtii PPO and FeC cDNA Products
The sequence of the isolated C. reinhardtii PPO cDNA (2,480 bp) was found to be identical to that previously reported (Randolph-Anderson et al., 1998
The C. reinhardtii FeC cDNA sequence was newly obtained and was deposited in the GenBank database under accession number AF332962. The 2,660-bp cDNA contains a 1,479-bp open reading frame (ORF) that encodes a 493-residue 54,433-Mr precursor protein. The TargetP program predicts a 77-residue chloroplast-targeting sequence. The predicted 416-residue 46,100-Mr mature protein has a pI of 5.12. The FeC protein exhibits the invariant His residue that was reported to be required for activity (Wu et al., 2001
The C-terminal portions of FeCs show characteristics specific to certain groups of organisms (Dailey et al., 2000
In photosynthetic organisms, FeCs that are associated with photosynthetic tissues contain a light-harvesting complex (LHC) motif, a conserved hydrophobic stretch that corresponds to the third transmembrane segment found in the LHC family of proteins and that likely plays a role in anchoring the protein in membranes (Jansson, 1999 ). The LHC motif is present in plant FeCs that are expressed in photosynthetic tissues but not in the FeCs that are expressed in nonphotosynthetic tissues (Suzuki et al., 2002; Masuda et al., 2003). This difference could be important for targeting the FeC to specific membranes within the chloroplast. In common with plant FeCs that are associated with chloroplasts, the C. reinhardtii FeC does contain an LHC motif (Fig. 2A).
In plants, PPO and FeC are each encoded by two different genes that have been referred to as Types I and II. PPO-I is located in chloroplasts, whereas PPO-II was variously reported to be located in mitochondria (Lermontova et al., 1997 BLAST searches revealed that C. reinhardtii PPO exhibits an identity of 55% to 62% with Type I plant PPOs, 40% to 43% with cyanobacterial PPOs, 28% to 30% with Type II plant PPOs, and 32% or less with nonplant mitochondrial and bacterial PPOs. C. reinhardtii FeC shows the highest identity to plant Type II FeCs and cyanobacterial FeCs, being 57% to 69% and 52% to 59% identical, respectively, in overlapping regions. The identity with plant Type I FeCs is 47% to 49%, whereas that with nonplant mitochondrial FeCs is 41% or lower. In phylogenetic analyses, both PPO and FeC of C. reinhardtii group most closely with their counterparts in plants that are expressed in photosynthetic tissues, and away from those enzymes that are active in nonphotosynthetic tissues and which, in the case of PPOs, have been reported to reside in mitochondria (Fig. 2B).
To obtain information about the chloroplast-targeting peptides of C. reinhardtii PPO and FeC, in silico analysis was conducted. Although the TargetP program predicted chloroplast targeting of both PPO and FeC, the scores and reliability of the predictions are moderate (Fig. 3B), in contrast to those obtained for plant PPO and FeC precursors. In addition, for the FeC precursor, other targeting prediction programs (PSORT, Predotar, listed under expasy.org) did not predict a clear chloroplast targeting of the FeC precursor. Generally, targeting predictions for C. reinhardtii precursors to the chloroplast are weak; this can be explained by the fact that C. reinhardtii chloroplast presequences generally resemble more closely mitochondrial-targeting signals of yeast and animals than plant chloroplast signals (Franzén et al., 1990
Interestingly, the presequences of the C. reinhardtii PPO and FeC precursors show certain characteristics of chloroplast precursors that are targeted to the thylakoid lumen via the twin-Arg translocase (Tat) pathway. The Tat pathway is capable of translocating proteins across the thylakoid membrane in their folded state. Tat domains consist of two adjacent Arg residues followed by a hydrophobic residue two or three residues afterward (Tat motif), a central hydrophobic region (H-region) directly followed by a consensus thylakoid processing peptidase (TPP) cleavage site (AxA  x; for review, see Robinson and Bolhuis, 2004). The C. reinhardtii PPO and FeC precursors both contain Tat motifs (Fig. 3A) and possible TPP cleavage sites, as found in most lumen-targeted proteins (Mayfield et al., 1989; von Heijne et al., 1989; Merchant et al., 1990). The program LumenP, a neural network predictor of lumen targeting in plants (Westerlund et al., 2003), produced very high scores for known lumenal proteins such as the Rieske iron-sulfur protein (ISP), the PSII oxygen-evolving enhancer proteins OEE2 and OEE3 (Mayfield et al., 1989; Finazzi et al., 2003 and refs. therein), and a putative ascorbate peroxidase (APX2), which is considered very likely to be lumenal because it is found in the lumen in Arabidopsis (NM_116970; Kieselbach et al., 2000; Peltier et al., 2002). Light-harvesting chlorophyll a/b-binding protein (LHCII) receives a low score because it does not contain lumen-targeting sequences (Lamppa, 1988). Although the C. reinhardtii PPO and FeC precursors yielded relatively high LumenP scores (Fig. 3B), the LumenP program returned TPP cleavage site positions well into the mature protein, in the case of the PPO precursor directly after a predicted transmembrane helix, and for the FeC precursor inside a moderately hydrophobic stretch (Fig. 3A). Moreover, the segment that typically comprises the H-region in valid Tat domains, directly following the twin Args, could not be unequivocally identified in the C. reinhardtii precursors. Therefore, although the presence of Tat signals in these proteins is striking, their functional validity is uncertain. However, it should be noted that all of the analyzed C. reinhardtii precursors that are known to be targeted to the lumen exhibit two separate twin Args in their presequences, which is also true for the FeC precursor and potentially also for the PPO precursor, depending on the position of the N terminus of the latter. Another interesting feature of FeC presequences from photosynthetic eukaryotes, including that of C. reinhardtii, is the presence of several Cys residues (Fig. 3C). A survey of plant chloroplast-targeting peptides indicates that the occurrence of multiple Cys residues is uncommon (data not shown).
The predicted mature regions of the PPO and FeC were overexpressed in E. coli using the pQE30 expression vector, which introduces an N-terminal His tag. The recombinant proteins were highly expressed (Fig. 4) and invariably found in inclusion bodies (data not shown). These inclusion bodies were dissolved in 6 M guanidinium hydrochloride and the proteins were purified under denaturing conditions. On SDS-PAGE, purified, overexpressed PPO appeared as a single 59-kD band, whereas purified FeC was seen as a 47-kD monomer but also as a set of higher Mr bands even in the presence of 1% (w/v) SDS and 100 mM dithiothreitol, suggestive of a strong tendency of the protein to aggregate, at least under denaturing conditions. Purified PPO and FeC were used to raise antibodies in rabbits. The obtained antisera cross-reacted with their corresponding overexpressed proteins (data not shown) and recognized bands of the appropriate size in C. reinhardtii cell extracts (Fig. 5).
Subcellular Localization of PPO and FeC To determine whether the products of the single genes encoding PPO and FeC in C. reinhardtii are targeted to specific organelles in the cell, chloroplasts and mitochondria were isolated from mixotrophically grown cells. The subcellular fractions were analyzed on SDS-PAGE, and the proteins were transferred to nitrocellulose for immunoblotting with specific antisera that were raised against the purified, overexpressed PPO and FeC. Coomassie Blue-stained SDS-PAGE gels revealed clearly different polypeptide patterns for the chloroplast and mitochondrial fractions (Fig. 5). In the chloroplast fraction, heme-mediated peroxidase activity staining detected cytochrome (cyt) f, as expected, but also low levels of a 12-kD c-type cyt that was attributed to contamination by mitochondrial cyt c (Fig. 5). In contrast, the mitochondrial fraction contains heme-staining bands at positions corresponding to cyt c and cyt c1, but this fraction contained no evidence for the presence of chloroplast cytochromes. In addition, an anti-Rubisco antibody detected high levels of the large and small Rubisco subunits in the chloroplast fraction. The mitochondrial fraction contained some cross-reacting bands, but none of those bands corresponded to the Rubisco large and small subunits. It can therefore be concluded that the chloroplast fraction might be slightly contaminated by mitochondria whereas the mitochondrial fraction is free of chloroplast contamination. Immunoblot analysis with the anti-PPO antibodies revealed in whole cells a single band of an apparent molecular mass of 59 kD. In the organellar fractions, this protein was detected exclusively in the chloroplast fraction (Fig. 5). Anti-FeC antibodies detected a major band of 47 kD in whole cells and in the chloroplast fraction. Several very faint bands were detected in the mitochondrial fraction, but none were detected at the position of FeC. The apparent molecular mass of the mature PPO was approximately 4 kD higher (59 kD) than predicted; since the overexpressed PPO exhibited the same apparent molecular mass, the observed electrophoretic mobility might be influenced by the high pI value of the PPO protein. The FeC band correlated with the mass calculated for the predicted mature protein. A 36-kD nonspecific band, especially visible in the FeC immunoblot, was caused by a common contaminant in antisera of preimmunized rabbits for C. reinhardtii proteins (data not shown). Altogether, these results indicate that in C. reinhardtii, both PPO and FeC are located exclusively in the chloroplasts.
To determine whether C. reinhardtii PPO and FeC proteins are associated with membranes, intact chloroplasts were fractionated into membrane and soluble fractions. Immunoblot analysis revealed that both PPO and FeC were located only in the membrane fraction (Fig. 6). Membranes were further subjected to high salt or alkaline treatment to examine the nature of the membrane association of the enzymes. PPO was not extracted by 1 M NaCl and was mostly resistant to extraction by alkaline treatments to a similar level as the LHC proteins, indicating that the protein is intrinsic to the membrane. No dissociation of FeC by either 1 M NaCl or alkaline treatment could be detected.
Light and Nutritional Regulation of mRNA and Protein Levels
The regulation of PPO and FeC expression in cells growing in continuous light and dark, and with different acetate levels in the growth medium, was investigated by RNA-blot and immunoblot analysis. It was found that the PPO mRNA level was at least 2-fold lower in cells growing in Tris-acetate phosphate (TAP) medium in the dark, compared to cells growing in the other conditions (Fig. 7A). The PPO protein level was not significantly influenced by either light or acetate concentration. In contrast, the levels of FeC mRNA and protein were both lower in cells growing in the dark than in light-grown cells, regardless of the acetate concentration, and the effect was more pronounced on the mRNA level than on the protein level. It was very important to make sure that the cells were in their exponential growth phase in darkness, especially in TAP medium, due to the limited amount of carbon source. On this medium, dark-adapted cells (cell wall-deficient strain) started dying off within 72 h after the initiation of the exponential growth phase (R. van Lis, unpublished data). As an indication for dark adaptation of the C. reinhardtii cells, the levels of LHC proteins were visualized by immunoblotting, using an antibody that recognizes C. reinhardtii LHCII proteins P11, P16, and P17 (Pineau et al., 2004
In C. reinhardtii cells synchronized to a 12-h-light/12-h-dark cycle, no significant changes in protein levels of either PPO or FeC were observed upon onset of the light phase. This suggests a different type of regulation of these two enzymes in the synchronized cells compared to continuous light and dark conditions. The synchronized cells were previously verified to exhibit the typical synchronization behavior of dividing once during the dark phase and not synthesizing chlorophyll in the dark (Nogaj et al., 2005 ). Experiments to determine the in vivo turnover of PPO and FeC proteins were done by administering 200 µg/mL of the protein synthesis inhibitor cycloheximide to cultures growing in continuous light, continuous dark, and at the beginning of the light phase in light/dark-synchronized cells. Samples were taken at various times during the next 24 h and the levels of PPO and FeC were quantitated by immunoblotting. Under all growth conditions, both PPO and FeC were relatively stable, with at least 75% of the initial levels remaining at 24 h (data not shown).
Using the C. reinhardtii genomic sequence (JGI draft sequence, version 2.0), the regions upstream of the nuclear PPO and FeC genes were searched for transcriptional elements that involve light regulation. In addition to putative TATA boxes, the upstream regions of the two genes exhibit several potential light-responsive elements, which for the FeC gene are all located relatively far from the cDNA start site (Fig. 8). C. reinhardtii-specific light-responsive elements (Hahn and Kück, 1999
The presence of only one gene each for PPO and FeC made the determination of the predominant organellar targeting in C. reinhardtii a less confounding task than in plants. The antibodies raised against the single forms of both overexpressed enzymes are expected to recognize the proteins regardless their intracellular location. Immunoblots using antibodies against the PPO and FeC detected the proteins only in the chloroplast fraction, which makes it likely that in C. reinhardtii, the last two steps of heme synthesis are confined to the chloroplast. C. reinhardtii PPO and FeC proteins were both found to be associated with the membrane fraction of the chloroplast. It was not possible to localize these proteins to a specific chloroplast membrane because a protocol to obtain pure envelope and thylakoids from the green alga is not currently available. Although in silico analysis using the program LumenP suggested that both precursor proteins contained Tat domain characteristics that would direct their localization to the lumen, the program seemed to have based its predictions at least in part on H-regions near the N terminus but within the mature proteins, instead of in the presequence, where true H-regions could not be found. Therefore, conclusions based on this analysis should be interpreted with caution, and in any case it is unlikely in either plants or C. reinhardtii that PPO and FeC are located on the lumenal side of the thylakoid. However, the Tat signals might be involved in directing these proteins and inserting them into the thylakoid membranes. Direct determination of the actual N termini of the mature C. reinhardtii PPO and FeC would help to resolve this issue. An unusually high number of Cys residues is found only in FeC presequences, in C. reinhardtii as well as higher plants. Although the significance of these Cys residues is unknown, it is conceivable that they could be involved in regulation of posttranslational processing or import by the chloroplast redox state, which is strongly influenced by light. It is known that the content of FeCs in plants and in C. reinhardtii is influenced by light (see below).
Several potential transcriptional regulatory elements were found in the genomic regions upstream of C. reinhardtii PPO and FeC. GT-1 transcriptional regulatory elements are ubiquitous (Villain et al., 1996
In C. reinhardtii, only one form each of PPO and FeC is present. The expression profile of the genes for PPO and FeC differed in conditions of continuous darkness or continuous light. The cells grown in continuous darkness were truly dark adapted, as judged by the diminished levels of LHCII proteins. The relative lower amount of LHC protein in high-acetate medium as compared to TAP medium in either light or dark can be viewed as being in accordance with Heifetz et al. (2000)
In cells growing on TAP medium, PPO mRNA levels were lower in continuous darkness than in light. In cells growing on high-acetate medium, this difference is not observed, which suggests that acetate somehow causes the cells to increase transcription of the PPO gene. However, the levels of PPO protein were comparable in dark- and light-grown cells. Since, unlike in angiosperm plants, chlorophyll synthesis in C. reinhardtii does occur in continuous darkness, it might be expected that light modulation of PPO mRNA and protein levels would be less pronounced than in plants; for example, in tobacco leaves, PPO transcript levels were strongly up-regulated in the light (Lermontova et al., 1997 Taken together, the lack of correlation between the PPO level and the rate of chlorophyll synthesis under a variety of growth conditions indicates that the rate of chlorophyll synthesis is not limited by the rate of synthesis of PPO or its cellular abundance. For FeC, it is difficult to interpret the significance of the observed changes in mRNA and protein levels in cells growing under different conditions. If the rate of heme synthesis is reflected by the cellular content of FeC, then it would appear that heme synthesis occurs at a relatively constant rate throughout the light/dark cycle in synchronized cells, but at a lower rate in cells growing in continuous dark than in cells in continuous light. This can be contrasted to the situation in higher plants, where different tissue types can have greatly different needs for heme, which are met by expression of tissue-specific FeC genes. We propose that the existence of multiple genes for PPO and FeC in higher plants fulfils the need for different tissue-specific levels of expression rather than for different organelle-specific targeting of the gene products.
Transcription of many of the genes for pigment biosynthesis in C. reinhardtii is regulated by light (Matters and Beale, 1995
Heme is required in chloroplasts mainly for cofactors of PSII and the cyt b6/f complex. Heme is also needed for components of the mitochondrial electron transport chain and as a cofactor of various nonchloroplast hemoproteins (e.g. catalase, peroxidases). Although all of the steps of heme synthesis leading to protoporphyrinogen IX are generally believed to occur exclusively in the chloroplasts in plants, the site(s) of the last two steps of heme synthesis in plants have not been well understood. Many plants have been found to contain two PPO-encoding genes. In tobacco, the products of two genes, PPO-I and PPO-II, are specifically targeted to chloroplasts and mitochondria, respectively (Lermontova et al., 1997
A hypothetical alternative possibility is that plant mitochondria are devoid of FeC but do contain a PPO that has a nonbiosynthetic role. Tetrapyrroles have been proposed as signaling molecules that convey information about the status of chloroplasts to the rest of the cell, although the mechanisms involved in tetrapyrrole signaling remain unknown (Strand et al., 2003
If all cellular hemes are synthesized exclusively in chloroplasts, export of heme from plastids to mitochondria and the cytoplasm must occur. Heme export from chloroplasts is poorly understood, but in vitro export of heme from isolated pea (Pisum sativum) chloroplasts has been described (Thomas and Weinstein, 1990
Cell Cultures
Wild-type Chlamydomonas reinhardtii strain CC124 was grown with orbital agitation at 25°C in TAP medium (Harris, 1989
A cDNA library, constructed in lZAPII phagemid (Stratagene) with mRNA from C. reinhardtii cells grown in light with 5% (v/v) CO2, was obtained from J. Davies (Iowa State University, Ames, IA). A sample of this library was excised with helper phage VCMS13 in XL1-Blue MRF' Escherichia coli cells, and recovered as pBluescript plasmids. Batches of electro-competent E. coli PPO-deficient hemH (Sasarman et al., 1993
The region of PPO cDNA (corresponding to translated residues I85 to K558) was amplified by PCR using the primers 5'-GACGAGCTCATCGTGGTCGGTGGAGGTC-3' and 5'-CTGAAGCTTCTTGGACACGCTCTTGGC-3'. The FeC cDNA region (corresponding to translated residues K87–L491) was amplified using the PCR primers 5'-GACGAGCTCAAGGTCGGCGTTCTGCTG-3' and 5'-CTGAAGCTTGCCAGGAACAGGTTTTTGAGG-3'. The restriction sites are underlined. The template cDNA was subjected to 30 cycles of PCR amplification (1 min at 94°C, 1 min at 60°C, and 2 min at 72°C) using Taq DNA polymerase (Qiagen). The amplified products were first ligated into pGEM-T-easy (Promega) and then digested with SacI and HindIII and ligated into expression vector pQE30 (Qiagen) that was predigested with the same enzymes.
E. coli strain XL1-Blue MRF' (Stratagene) was transformed with the PPO- and FeC-pQE30 constructs and induced for 4 h at 37°C after adding 1 mM isopropylthio-
C. reinhardtii chloroplasts and mitochondria were isolated from CC400 cells grown on TAP medium in continuous light. Cells were harvested by centrifugation at 2,000g for 10 min, resuspended in nebulizing buffer (50 mM MES, 50 mM Tris, 0.25 M sorbitol, 10 mM MgCl2, 1 mM MnCl2, 3 mM KH2PO4, 2 mM EDTA, pH 7.2) and disrupted using the BioNeb cell disruption system (Glas-Col) with N2 (20 psi; Bollivar and Beale, 1996
Chloroplast membrane and soluble fractions were obtained after centrifugation of sonicated chloroplasts at 100,000g for 30 min. Membranes were resuspended in 10 mM Tricine-NaOH, pH 8.0, in the presence of protease inhibitors and supplemented with 1 M NaCl or 20 mM Na2CO3 (pH 11.0; Pierre et al., 1995
SDS-PAGE gels were stained for heme-associated peroxidase activity by the method of Thomas et al. (1976)
For Southern blotting, genomic DNA (10 µg) was digested with various restriction endonucleases, electrophoresed on 1% (w/v) agarose gels and blotted onto Hybond-N nylon membranes (Amersham). Southern hybridization at 65°C was performed as described (Sambrook and Russell, 2001
Proteins from various extracts were separated on SDS-PAGE gels and then electroblotted onto nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline supplemented with 0.05% (v/w) Tween 20. The blocked membranes were incubated first with the different specific antibodies and then with either horseradish peroxidase-conjugated anti-rabbit IgG (Pierce) and detected using ECL western-blotting detection reagents (Amersham), or alkaline phosphatase-conjugated anti-rabbit IgG (Sigma) and detected with the NBT/BCIP Liquid Substrate system (Sigma). In Figure 5, PPO antibodies were used at a dilution of 1:5,000 and FeC antibodies at 1:2,000, with enhanced chemiluminescence detection. In Figures 6 and 7, PPO antibodies were diluted 1:500 and detection was done using the alkaline phosphatase system. In Figure 6, FeC antibodies were used at a dilution of 1:1,000 and detected using the enhanced chemiluminescence system with freshly prepared solutions to obtain maximum sensitivity to compensate for the deterioration of the signal over time (Durrant, 1990
Multiple sequence alignments were done using the ClustalW 1.82 program (Thompson et al., 1994 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AF332962.
We thank O. Emanuelsson for the use of the LumenP program, J. Davies for providing the C. reinhardtii cDNA library, H.A. Dailey for providing the E. coli hemG and hemH strains, J.V. Morony for the antibody against C. reinhardtii Rubisco, and O. Vallon for the antibody against C. reinhardtii LHCII. The gene sequence data were produced by the U.S. Department of Energy JGI, http://www.jgi.doe.gov/ and are provided for use in this publication/correspondence only. Received August 9, 2005; returned for revision August 9, 2005; accepted September 24, 2005.
1 This work was supported by the National Science Foundation (grant no. MCB–9808578 to S.I.B.) and the U.S. Department of Energy (grant no. DE–FG02-88ER13918 to S.I.B.). 
2 Present address: Physiologie Cellulaire Végétale, Unité Mixte de Recherche 5168, Commissariat à l'Energie Atomique Grenoble, 17 rue des Martyrs, Grenoble cedex 9, 38054, France. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Samuel I. Beale (sib{at}brown.edu). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.069732. * Corresponding author; e-mail sib{at}brown.edu; fax 401–863–1182.
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ABSTRACT
RESULTS
, Experimentally determined cleavage site; 
, cleavage site predicted by TargetP; 
0.47 and cleavage site score (CSscore) 
-galactoside to the medium (Luria-Bertani). The cells were harvested and disrupted by sonication, and the pellet containing PPO or FeC recombinant protein was dissolved in 6 M guanidinium chloride and used for nickel-nitrilotriacetic acid agarose affinity chromatography under denaturing conditions, according to Qiagen product manual. Antibodies were raised against the purified PPO and FeC proteins in rabbits (Animal Pharm Services). 
pH dependence in vivo. EMBO J 22: 807–815