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Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii.

ABSTRACT: Two copies of structurally related genes (CAH1 and CAH2) for carbonic anhydrase (EC 4.2.1.1) were found to be tandemly clustered on the Chlamydomonas reinhardtii genome. The previously isolated cDNA clones for carbonic anhydrase polypeptides were derived from the upstream gene, CAH1, which has 10 introns in its coding region. The downstream gene, CAH2, also has 10 introns, at positions identical to those of CAH1. Although amino acid sequences deduced from the two genes showed 91.8% identity, partial sequences of the authentic enzyme isolated from air-induced cells were identical only to those of the CAH1 product. Northern hybridization using gene-specific probes showed that the level of 2.0-kilobase CAH1 mRNA increased in response to a decrease in CO2 concentration in the presence of light. The CAH1 mRNA did not accumulate when CO2 was lowered in the dark. In contrast, the level of 2.0-kilobase CAH2 mRNA decreased in response to lowering of CO2 and increased upon transfer to the high-CO2 condition in light. The decrease of CAH2 mRNA under the low-CO2 condition was not observed in the dark. The fully induced mRNA level was much higher for CAH1 than for CAH2. These results indicate that CAH1 is a gene coding for the major periplasmic carbonic anhydrase whose level of transcript is rapidly induced under the low-CO2 condition in the presence of light, and that CAH2 may encode another periplasmic isozyme, which is made under the high-CO2 condition.

SUBMITTER: Fujiwara S

PROVIDER: S-EPMC55257 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii.

Fujiwara S S Fukuzawa H H Tachiki A A Miyachi S S

Proceedings of the National Academy of Sciences of the United States of America 19901201 24

Two copies of structurally related genes (CAH1 and CAH2) for carbonic anhydrase (EC 4.2.1.1) were found to be tandemly clustered on the Chlamydomonas reinhardtii genome. The previously isolated cDNA clones for carbonic anhydrase polypeptides were derived from the upstream gene, CAH1, which has 10 introns in its coding region. The downstream gene, CAH2, also has 10 introns, at positions identical to those of CAH1. Although amino acid sequences deduced from the two genes showed 91.8% identity, par ...[more]

PMID: 2124702

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Project description:BackgroundCah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2) fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2) conditions.Results/conclusionsIn the present work we demonstrate that after transfer to low CO(2), Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO(2) conditions, the Cah3 activity increased about 5-6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO(2) conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO(2) conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO(2) the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.SignificanceThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Dataset Information

Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii.

Publications

Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii.

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