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. 2009 Sep 18;284(38):25867-78.
doi: 10.1074/jbc.M109.023622. Epub 2009 Jul 7.

"V体育官网" Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii

Aimee M Terauchi  1 , Shu-Fen Lu, Mirko Zaffagnini, Shane Tappa, Masakazu Hirasawa, Jatindra N Tripathy, David B Knaff, Patrick J Farmer, Stéphane D Lemaire, Toshiharu Hase, Sabeeha S Merchant
Affiliations

Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii

Aimee M Terauchi et al. J Biol Chem. .

Abstract

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe2S2] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP+ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (Em = -321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions VSports手机版. .

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FIGURE 1.
FIGURE 1.
The Chlamydomonas genome encodes six highly related ferredoxins. A, multiple alignment of six ferredoxins in C. reinhardtii was created using Multalin (available on the World Wide Web). Conserved cysteine residues that coordinate the [Fe2S2] cluster are shown in red. The red arrow indicates N termini used for generation of recombinant protein. The sequences of peptides used to generate antibodies specific to Fd, Fdx3, and Fdx6 are highlighted in yellow. Transit sequences of Fd (14, 63) and Fdx5 (39) are underlined. Conserved, negatively charged residues implicated in interaction of Fd with Fd:NADP+ oxidoreductase, Fd:thioredoxin reductase, nitrite reductase, and glutamate synthase are indicated by a red dot. B, a phylogenetic tree of ferredoxin proteins from A. thaliana, Z. mays, Physcomitrella patens, Ostreococcus tauri, Micromonas pusilla, Volvox carteri, and C. reinhardtii was generated with Phylogeny.fr (available on the World Wide Web). Only the core sequence was used (indicated by wavy lines in A). Chlamydomonas ferredoxins are indicated in blue. Photosynthetic and non-photosynthetic ferredoxins are indicated by green and red boxes, respectively. Bootstrap values are indicated in red. Bar, 0.4 amino acid substitutions/site. C, FDX transcript abundance was determined by RNA-seq in Chlamydomonas strain 2137 (E. Urzica and S. S. Merchant, unpublished results).
FIGURE 2.
FIGURE 2.
Specificity of ferredoxin antibodies. 20 μg of soluble protein extracts from E. coli cells expressing recombinant ferredoxins was separated by denaturing polyacrylamide gel electrophoresis, and the specificity of each ferredoxin antibody was analyzed by immunoblot.
FIGURE 3.
FIGURE 3.
Localization of Fd, Fdx2, Fdx3, and Fdx6. A, 20 μg of total cellular protein, isolated mitochondria, and chloroplasts was separated on a 15% polyacrylamide gel containing SDS, and the abundance of Fd, Fdx3, and Fdx6 was analyzed by immunoblot. Cox2b and ketoacid reductoisomerase (KARI) abundance were analyzed in order to assess the purity of isolated mitochondria and chloroplasts. B, 40 μg of soluble fraction of total cells, isolated mitochondria, and chloroplasts were separated on a 15% polyacrylamide gel containing SDS and analyzed for the abundance of Fdx2 by immunoblot. C, 20 μg of total cellular protein, isolated mitochondria, and chloroplasts were analyzed as in B to assess the purity of isolated mitochondria and chloroplasts. D, 20 μg of total cellular protein and purified chloroplasts were separated on a 12% polyacrylamide gel containing SDS to assess cytosolic contamination of purified chloroplasts.
FIGURE 4.
FIGURE 4.
FDX2 is induced in nitrate-grown cells. A, RNA was isolated from wild-type Chlamydomonas strain 21gr cells grown in TAP medium containing ammonium or nitrate as a nitrogen source, and FDX expression was analyzed by quantitative real time PCR. Relative expression was normalized to UBI2 expression, and -fold induction was calculated by the 2−ΔΔCT method (64). Amplifications were performed in technical triplicate and averaged. Biological duplicates are shown. Inset, amplification product of NIA1 was visualized on a 1% agarose gel. B, soluble protein extracts of Chlamydomonas strain 21gr grown in TAP medium containing ammonium or nitrate as a nitrogen source were separated on a denaturing polyacrylamide gel containing SDS, and Fdx2 and NiR abundance were analyzed by immunoblot.
FIGURE 5.
FIGURE 5.
Fdx2 is damaged by H2O2in vivo. A, RNA was isolated from Chlamydomonas strain 17D− grown in TAP medium prior to and 2 h after the addition of 1 mm H2O2. Gene expression was measured by quantitative real time PCR. FDX abundance after 2 h of H2O2 treatment was compared with expression prior to the addition of H2O2. RNA abundance was normalized to CBLP. MSD3 was tested as a control for H2O2 treatment. Two independent experiments are shown. B, soluble protein extracts isolated from Chlamydomonas strain 21gr grown in TAP medium containing NO3− prior to and 1 and 2 h after the addition of 1 mm H2O2 were separated on a 15% denaturing polyacrylamide gel containing SDS, and Fdx2 and Fd abundance were determined by immunoblot.
FIGURE 6.
FIGURE 6.
FDX5 is controlled by copper homeostasis factor Crr1. A, RNA was isolated from wild-type Chlamydomonas strain CC124 grown in the dark in order to induce hypoxia and from cells grown aerobically in the light. Relative abundance of FDX RNAs was determined by quantitative real time PCR. Gene expression was normalized to CBLP. Two independent experiments are shown. B, RNA was isolated from wild-type Chlamydomonas cells grown in the presence and absence of copper. FDX gene expression was analyzed as in A. Two independent experiments are shown. Circles, strain 2137; triangles, strain CC125. C, RNA was isolated from wild-type Chlamydomonas cells grown in the presence and absence of nickel. FDX expression was analyzed as in A. Two independent experiments are shown. Circles, strain 2137; triangles, strain CC125. D, soluble protein extracts of Chlamydomonas strain 2137 grown in TAP medium containing 0 (−Cu) or 2 μm copper (+Cu) were separated on a 15% non-denaturing polyacrylamide gel, and Fdx5 abundance was analyzed by immunoblot. Plastocyanin and cytochrome c6 abundance were analyzed as markers for copper-deficient and copper-replete conditions, respectively.
FIGURE 7.
FIGURE 7.
Ferredoxin expression in response to iron nutrition. A, FDX expression was analyzed by quantitative real time PCR in Chlamydomonas strain 17D− cells grown in various concentrations of iron. Relative gene expression was normalized to CBLP. Three independent experiments are shown. B, 20 μg of soluble protein extracts of Chlamydomonas strain 17D− grown in TAP medium containing 0.1, 0.2, 1, 3, 20, or 200 μm iron were separated on a 15% denaturing polyacrylamide gel containing SDS, and Fd, Fdx3, and Fdx6 abundance were analyzed by immunoblot.
FIGURE 8.
FIGURE 8.
The midpoint potentials of Fd and Fdx2. Cyclic voltammograms were obtained at an indium-doped tin oxide electrode from a solution of 100 mm spinach (S.o.) ferredoxin, Chlamydomonas (C.r.) Fd, or Fdx2 in 100 mm MOPS, pH 7.0, at a sweep rate of 50 mV/s and given versus the Ag/AgCl electrode.
FIGURE 9.
FIGURE 9.
Change in FDX transcript abundance in response to copper and iron nutrition. FDX transcript abundance was determined by RNA-seq in Chlamydomonas strain 2137 (M. Castruita and S. S. Merchant, unpublished results; E. Urzica and S. S. Merchant, unpublished results). The sizes of the circles are proportional to the relative abundance of the total ferredoxin transcript pool in each condition.
FIGURE 10.
FIGURE 10.
Surface charge distribution of Fdx proteins. A, the coordinates of the Chlorella fusca ferredoxin (Protein Data Bank code 1AWD) were used to derive coordinates of the Chlamydomonas ferredoxins (31). The N terminus of Fdx2 was estimated based on homology to the transit sequence of Fd. Electrostatic potentials were calculated in PyMOL (available on the World Wide Web) using the Adaptive Poisson-Boltzmann Solver plugin. Red, negatively charged amino acids; blue, positively charged amino acids. B, 0.5 μg of purified Fd from Chlamydomonas and recombinant Fd and Fdx2 were separated on an 18% non-denaturing polyacrylamide gel.
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