Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii

Abstract

Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated alpha-, beta-, and gamma-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Here we identify a sixth gene encoding a beta-type CA. This new beta-CA, designated Cah6, is distinct from the two mitochondrial beta-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.

Figure 1.

The genomic map of Cah6. Cah6 is 2,886 bp in length. The arrows represent the four exons while the horizontal line represents the three introns. The start and stop codons are labeled by black vertical lines.

Figure 2.

Alignment of Cah6 protein sequence with those of other well-characterized β-CAs. Ca1 represents the C. reinhardtii mitochondrial β-CA. Mitochondrial β-Ca1 and β-Ca2 (sequence not shown in the alignment) are almost identical in amino acid sequence and have only one amino acid difference in their sequences. Coccomyxa represents the cytosolic β-CA from the symbiotic alga Coccomyxa. Active site residues are in bold and highlighted. Asterisks represent a completely conserved amino acid; colons represent conserved amino acid substitutions; and periods represent semiconserved amino acid substitutions.

Figure 3.

Generation of the recombinant pMal-Cah6 expression construct. A, A schematic figure showing the alignment of primers used to amplify Cah6 cDNA. PCR primers are denoted by black arrows. SP2 and R4H primers were used to amplify a cDNA PCR product that codes for the full-length protein while MP and R4H primers were used to amplify a cDNA product that codes for the putative mature protein. R4H primer has a HindIII site incorporated at the 5′ end. The numbers on the top of the primers denote the primer position in bp. B, A part of the pMal vector showing the polylinker cloning site (PCS),β-galactosidase (LacZα), and β-lactamase (Amp^r) genes, XmnI and HindIII recognition sites. Xa denotes Factor Xa cleavage site. MalE codes for MBP. C, A schematic figure of the pMal-Cah6 recombinant construct.

Figure 4.

A 12% SDS-polyacrylamide gel showing overexpression of recombinant MBP-Cah6. Lane 1 represents prestained low molecular mass markers. Lanes 2 and 3 represent 30 μg of proteins from uninduced and induced E. coli cells, respectively.

Figure 5.

A 12% SDS-polyacrylamide gel showing undigested and Factor Xa digested purified MBP-Cah6 protein. Lane 1, 55 μg of undigested purified recombinant protein; lane 2, 55 μg of purified recombinant protein digested by 1 μg of Factor Xa; lane 3, 1 μg of Factor Xa; lane 4, prestained low molecular mass markers.

Figure 6.

A western blot probed by the Cah6 antibody using purified overexpressed MBP-Cah6 protein. Lane 1, 20 μg of purified undigested fusion protein; lane 2, 20 μg of fusion protein cleaved by 1 μg of Factor Xa; lane 3, 1 μg of Factor Xa. ST represents prestained low molecular mass markers.

Figure 7.

Western blots probed by the Cah6 and mitochondrial β-CA antibodies using wild-type Chlamydomonas cells. A, A western blot probed by Cah6 antibody using high (5% CO₂ in air [v/v]) and air (0.035% CO₂ [v/v]) acclimated wild-type Chlamydomonas cells grown in minimal medium. B, A western blot probed by mitochondrial β-CA antibody using high and air acclimated wild-type Chlamydomonas cells grown in minimal medium.

Figure 8.

Northern-blot analyses of Cah6 expression. A, A schematic figure showing the alignment of primers used for making the probe for northern-blot analyses of Cah6 expression. B, A stained RNA gel showing RNA extracted from high and low CO₂ acclimated wild-type cells grown in minimal medium. Each lane contains 20 μg of total RNA. C, Northern-blot result using 826-bp PCR product as a probe.

Figure 9.

A, Transmission electron micrograph showing the immunogold labeling of C. reinhardtii CC-124 cells probed with the Cah6 antibody. Cells grown under low CO₂ (0.035%) conditions in minimal medium were probed with the Cah6 antibody. B, Transmission electron micrograph showing the immunogold density around the pyrenoid in C. reinhardtii cells probed with the Cah6 antibody. Cells grown under low CO₂ (0.035%) conditions in minimal medium were probed with the Cah6 antibody. C, Transmission electron micrograph showing the immunogold labeling of C. reinhardtii cells probed with the preimmune serum. Low CO₂ (0.035%) acclimated CC-124 cells grown in minimal medium were used. For all micrographs, Ss, Py, and C denote starch sheath, pyrenoid, and chloroplast, respectively. Immunogold labelings are shown by small black arrows.

Figure 10.

A model showing the potential role of Cah6 and other known CAs in the operation of CCM in C. reinhardtii. The font sizes of CO₂ and HCO₃⁻ indicate the relative concentrations of these Ci species. Cyt CA? represents a putative cytoplasmic carbonic anhydrase. P, Cy, Ch, and Py represent periplasm, cytoplasm, chloroplast, and pyrenoid, respectively. Cah1 and Cah3 represent the periplasmic and thylakoid CAs, respectively. Putative HCO₃⁻ transporters are denoted by small black circles.