Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in Chlamydomonas reinhardtii

Abstract

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.

Figure 1.

Southern-blot hybridization with C. reinhardtii genomic DNA, digested with the indicated restriction endonucleases and hybridized as described in the text. PvuI does not cut, whereas StuI and NcoI cut once and twice, respectively, in the PPO probe sequence; in the FeC probe sequence, PvuII does not cut, whereas SacI and AvaI both cut once.

Figure 2.

Analysis of the amino acid sequences of PPO and FeC. A, The C-terminal portion of C. reinhardtii FeC contains an LHC motif that is found in Type II plant FeCs and also in cyanobacterial FeCs. The approximate region of a transmembrane segment (TM segment) as predicted by TMPred is indicated for the sequences of Type II plant FeCs and the FeCs of cyanobacteria and C. reinhardtii. Chlre, C. reinhardtii; Arath, Arabidopsis; Cucsa, cucumber (Cucumis sativus); Glyma, pea; Horvu, barley (Hordeum vulgare); Nicta, tobacco; Orysa, rice (Oryza sativa); Nosto, Nostoc; Synec, Synechococcus; Theel, Thermosynechococcus elongatus; Bacsu, Bacillus subtilus. B, Bootstrapped neighbor-joined rootless trees showing the relatedness of PPO and FeC enzymes from various sources. For the eukaryotic enzymes, full-length preproteins were used in the analyses. Bootstrap values above 50 are shown at the nodes. The bar labeled “0.1” is the branch length representing a mean difference of 0.1 per residue along each branch. C. reinhardtii PPO groups most closely with the Type I chloroplast counterparts in plants, which are found only in photosynthetic tissues. Mitochondrial and nonphotosynthetic refer, respectively, to PPO enzymes reported to be present in mitochondria and to FeCs that are up-regulated in nonphotosynthetic conditions (see text). PPO GenBank accession numbers (clockwise, starting with Chlre): AF068635, BAA96808, Y13465, BAB39760, AAM26644, Y13466, BAB60710, P32397, P40012, NP_484159, BAA18074, NP_000300, BAC07926; FeC GenBank accession numbers (clockwise, starting with Chlre): AF332962, S75788, NP_487791, BAC09768, O04921, BAB20760, CAC50871, XP_475111, P32396, P22830, P16622, P42043, BAA05102, P42045, BAD33213.

Figure 3.

Sequence analysis of transit peptides. A, Twin Arg-containing presequences of C. reinhardtii chloroplast-targeted precursors and their in silico analysis using TargetP v1.1 and LumenP v1.3. OEE2/3, PSII oxygen-evolving enhancer proteins; APX2, putative ascorbate peroxidase; LHCII, chlorophyll a/b-binding protein. Twin Args and the hydrophobic residue (if present) two or three positions downstream are highlighted in black; note that in ISP, KR was found to be important for luminal targeting instead of RR. Charged residues are in bold, and hydrophobic regions are underlined. The invariant GxGxxG motif, which occurs well into the mature protein sequence of PPO, is boxed. ♦, Experimentally determined cleavage site; ▾, cleavage site predicted by TargetP; ↓, TPP cleavage site predicted by LumenP. B, In silico analysis of C. reinhardtii presequences. TargetP cTP, Chloroplast-targeting peptide; RC, reliability coefficient (1 is high and 5 is low); LumenP prediction cutoff, LumenP score ≥ 0.47 and cleavage site score (CSscore) ≥ 6.8; lTPlen, the predicted luminal-targeting peptide length. Predicted lTPlen for ISP and LHCII are incorrect, as these precursors do not contain TPP cleavage sites. C, Cys residues in the precursor sequences of plant and C. reinhardtii FeCs. The I and II suffixes refer to the isoforms present in photosynthetic and nonphotosynthetic tissues, respectively, as described in the text. ▾, Cleavage sites predicted by TargetP.

Figure 4.

Expression and purification of cloned C. reinhardtii PPO and FeC. Coomassie Blue-stained SDS-PAGE of His-tagged C. reinhardtii PPO and FeC proteins in extracts of E. coli cells before (0) and after 4 h of induction (4), and after purification of the expressed proteins by N-affinity chromatography (Ni-NTA). Positions of molecular mass marker proteins are indicated.

Figure 5.

Cell fractionation and immunoblot hybridization with antibodies raised against the overexpressed PPO and FeC proteins. In FeC immunoblots, a 36-kD contaminant of chloroplast origin was usually observed. TMBZ detected heme-containing proteins indicative of mitochondria (cyt c and cyt c₁) and chloroplasts (cyt f). Antibody against Rubisco detected the two subunits of 50 and 15 kD only in cells and the chloroplast fraction. For each sample, 100 μg of total proteins were loaded onto the gel. W, Whole cells; C, chloroplasts; M, mitochondria; RBC, Rubisco.

Figure 6.

Distribution of PPO and FeC between chloroplast membrane and soluble fractions. Chloroplast membranes were treated either with high pH (20 mm Na₂CO₃, pH 11.0) or high salt (1 m NaCl) to determine the nature of membrane associations. An anti-LHC antibody was used as control for a set of proteins known to be confined to the membranes. C, Whole chloroplasts; S, soluble fraction; Mb, membrane fraction; MbA and SA, alkaline-treated membranes and supernatant; MbN and SN, high salt-treated membranes and supernatant; M, M_r marker. Blots were stained with Ponceau Red to detect proteins and then treated with the indicated specific antibodies.

Figure 7.

Expression of PPO and FeC in cells grown in continuous light and dark and in light/dark-synchronized cells. A, RNA blots and concomitant immunoblots of C. reinhardtii cells grown in continuous light and dark, in either TAP or high-acetate medium. For RNA blots, 10 μg of total RNA was transferred; for immunoblots, 50 μg of protein. TL and TD, Cells grown on TAP medium in continuous light and dark, respectively; HL and HD, cells grown on high-acetate medium in light and dark, respectively. B, Left, Ponceau Red-stained blot after transfer of an SDS-polyacrylamide gel (12% acrylamide) loaded with identical samples as in A; right, immunoblot using an anti-LHC antibody. C, PPO and FeC protein levels in 12-h-light/12-h-dark-synchronized cells grown on TAP medium. The samples were from cells harvested 1 h before the light went on (L11) and then at every hour during the light period (L0–L12); 40 μg protein per lane for the anti-PPO immunoblot and 120 μg per lane for the anti-FeC immunoblot was electrophoresed on 8% polyacrylamide SDS gels. Blots were stained with Ponceau Red to detect proteins and with the indicated specific antibodies.

Figure 8.

Schematic representation of potential transcription factor-binding and/or cis-acting elements present upstream of the C. reinhardtii PPO and FeC ORFs that are potentially implicated in the light responsiveness of the genes. The elements were identified using the PLACE database, and the C. reinhardtii-specific light-responsive elements from Hahn and Kück (1999). S-numbers refer to transcription factor elements in the PLACE database. CSS, cDNA start site; TATA box, putative RNA polymerase-binding site; CrLRE, C. reinhardtii-specific light-responsive element; CircLeLhc, element necessary for circadian expression of tomato (Lycopersicon esculentum) Lhc (S000252); EnvStr, environmental stress including high light (S000402), involves phytochrome in plants (S000153); GT-Box, GT-1-binding sites involved in light-activated transcriptional activation (S000198); I-Box (S000199, S000424), conserved sequence upstream of light-regulated genes; T-box (S000383), found involved in Arabidopsis light-induced gene activation. Also shown is the end of a putative ABC transporter gene (JGI C_420004) upstream of the C. reinhardtii FeC gene.