FER1 and FER2 encoding two ferritin complexes in Chlamydomonas reinhardtii chloroplasts are regulated by iron

Abstract

Two unlinked genes FER1 and FER2 encoding ferritin subunits were identified in the Chlamydomonas genome. An improved FER2 gene model, built on the basis of manual sequencing and incorporation of unplaced reads, indicated 49% identity between the ferritin subunits. Both FER1 and FER2 transcripts are increased in abundance as iron nutrition is decreased but the pattern for each gene is distinct. Using subunit-specific antibodies, we monitored expression at the protein level. In response to low iron, ferritin1 subunits and the ferritin1 complex are increased in parallel to the increase in FER1 mRNA. Nevertheless, the iron content of the ferritin1 complex is decreased. This suggests that increased expression results in increased capacity for iron binding in the chloroplast of iron-limited cells, which supports a role for ferritin1 as an iron buffer. On the other hand, ferritin2 abundance is decreased in iron-deprived cells, indicative of the operation of iron-nutrition-responsive regulation at the translational or post-translational level for FER2. Both ferritin subunits are plastid localized but ferritin1 is quantitatively recovered in soluble extracts of cells while ferritin2 is found in the particulate fraction. Partial purification of the ferritin1 complex indicates that the two ferritins are associated in distinct complexes and do not coassemble. The ratio of ferritin1 to ferritin2 is 70:1 in iron-replete cells, suggestive of a more dominant role of ferritin1 in iron homeostasis. The Volvox genome contains orthologs of each FER gene, indicating that the duplication of FER genes and potential diversification of function occurred prior to the divergence of species in the Volvocales.

Figure 1.—

Comparison of algal ferritins. (A) Chlamydomonas FER1 (accession no. AAM27205) and FER2 (accession no. EU223296), Volvox FER2 (protein ID 58531) and FEN1 (protein ID 81290), Ostreococcus lucimarinus PFE1 (protein ID 27552) and PFE2 (protein ID 29953), and O. tauri FER1 (protein ID 5942) were aligned with Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.html). Lowercase sequence indicates transit peptides for plastid targeting for Chlamydomonas FER1 based on the N terminus determined in this study. The sequences of peptides used to generate antibodies specific to Chlamydomonas FER1 and FER2 (anti-FER1 and anti-FER2, respectively) are highlighted in yellow. Peptides used to generate antibodies that recognize both ferritins (anti-FER_core, experimentally verified) are underlined and double underlined (Busch et al. 2008). Residues corresponding to the ferroxidase center are shown in red. (B) A phylogenetic tree was generated with MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/phylogeny.html).

Figure 1.—

Comparison of algal ferritins. (A) Chlamydomonas FER1 (accession no. AAM27205) and FER2 (accession no. EU223296), Volvox FER2 (protein ID 58531) and FEN1 (protein ID 81290), Ostreococcus lucimarinus PFE1 (protein ID 27552) and PFE2 (protein ID 29953), and O. tauri FER1 (protein ID 5942) were aligned with Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.html). Lowercase sequence indicates transit peptides for plastid targeting for Chlamydomonas FER1 based on the N terminus determined in this study. The sequences of peptides used to generate antibodies specific to Chlamydomonas FER1 and FER2 (anti-FER1 and anti-FER2, respectively) are highlighted in yellow. Peptides used to generate antibodies that recognize both ferritins (anti-FER_core, experimentally verified) are underlined and double underlined (Busch et al. 2008). Residues corresponding to the ferroxidase center are shown in red. (B) A phylogenetic tree was generated with MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/phylogeny.html).

Figure 2.—

Transcripts encoding both ferritin subunits are increased in iron-limited cells. The abundance of FER1 and FER2 mRNAs was analyzed by quantitative real-time PCR. Strains 17D⁻ and 2137 were cultured in TAP medium supplemented with the indicated amounts of iron. cDNA corresponding to RNA isolated from Chlamydomonas strains (A) 2137 (top) or (B) 17D⁻ (bottom) was used as a template with primers specific to FER1 or FER2. The fold change in abundance of FER1 (left) or FER2 (right) after normalization to CβLP and relative to the abundance in a 20-μm grown culture was calculated according to the 2^−ΔΔCT method (Livak and Schmittgen 2001). Each data point is the average of technical triplicates and represents a separate experiment.

Figure 3.—

Specificity of anti-ferritin1 and anti-ferritin2. (A) Recombinant protein. Extracts from E. coli cells expressing recombinant ferritin 1, rFER1_1–249 (corresponding to residues 1–249 of the preprotein), or recombinant ferritin 2, rFER2_78–298 (corresponding to residues 78–298 of the preprotein), were separated by denaturing polyacrylamide gel electrophoresis (15% monomer). The lane on the left, containing 3 μg of extract, was stained with Coomassie Blue. The specificity of the antibodies was analyzed by immunoblotting after transfer to PVDF membranes. (B) Endogenous protein. Total protein extracts from Chlamydomonas strain CC400, corresponding to 35 μg protein were analyzed by immunoblotting as in A. Ferritin antibodies were incubated for 60 min with 0.01 μg of E. coli extract from cells expressing recombinant ferritin 1 (+rFER1), ferritin 2 (+rFER2), or no protein (none) prior to immunoblotting (see materials and methods).

Figure 4.—

Ferritin1 and ferritin2 are located in the chloroplast. Proteins (20 μg) from total cellular protein extracts of Chlamydomonas strain CC425 (T), isolated mitochondria (M), and chloroplasts (C) were separated on a denaturing polyacrylamide gel containing SDS and transferred to PVDF membranes for immunoblot analysis with anti-FER1, anti-FER2, anti-KARI (marker for chloroplast), and COX2b (marker for mitochondria).

Figure 5.—

Differential accumulation of ferritin1 as a function of iron nutrition. (A) Chlamydomonas strain 17D⁻ was grown in TAP medium containing the indicated iron concentration to midlog cell density. Soluble (S) and insoluble (P) fractions were prepared from the cells (see materials and methods). Five micrograms of protein from each growth condition were separated on a denaturing SDS–PAGE gel (15% monomer) and transferred to PVDF for immunoblot analysis with anti-FER1 and anti-FER2. Five micrograms of soluble protein (17D⁻) from each growth condition were separated on a nondenaturing gel (6% monomer) and analyzed with subunit-specific anti-FER1 and anti-FER2 (B) or stained for iron content with ferricyanide (C). Horse spleen ferritin (5 μg) was used as a marker (M).

Figure 6.—

Modification of ferritin1 in iron-limited cells. Protein (5 μg) from iron-replete (18 μm) vs. iron-limited (0.5 μm) cells was separated on a denaturing gel and analyzed for the abundance of ferritin by immunoblotting with anti-FER_core, which recognizes the internal segment of both ferritins on the basis of reactivity with the recombinant proteins (see Figure 1 legend).

Figure 7.—

Ferritin2 is not associated with ferritin1. The ferritin1 complex was purified on an affinity column. Equivalent volumes of material from various steps in the immunopurification procedure were analyzed for the presence of ferritin1 (top) or ferritin2 (bottom) by immunodetection after separation on SDS-containing gels. Lane 1, total soluble cell extract; lane 2, flow through; lane 3, final wash; lane 4, eluate.