Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Mar 2;17(5):1208–1216. doi: 10.1093/emboj/17.5.1208

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

J Karlsson 1, A K Clarke 1, Z Y Chen 1, S Y Hugghins 1, Y I Park 1, H D Husic 1, J V Moroney 1, G Samuelsson 1
PMCID: PMC1170469  PMID: 9482718

Abstract

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.

Full Text

The Full Text of this article is available as a PDF (438.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bogsch E., Brink S., Robinson C. Pathway specificity for a delta pH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein. EMBO J. 1997 Jul 1;16(13):3851–3859. doi: 10.1093/emboj/16.13.3851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brock I. W., Hazell L., Michl D., Nielsen V. S., Møller B. L., Herrmann R. G., Klösgen R. B., Robinson C. Precursors of one integral and five lumenal thylakoid proteins are imported by isolated pea and barley thylakoids: optimisation of in vitro assays. Plant Mol Biol. 1993 Nov;23(4):717–725. doi: 10.1007/BF00021527. [DOI] [PubMed] [Google Scholar]
  3. Burow M. D., Chen Z. Y., Mouton T. M., Moroney J. V. Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2. Plant Mol Biol. 1996 May;31(2):443–448. doi: 10.1007/BF00021807. [DOI] [PubMed] [Google Scholar]
  4. Chaddock A. M., Mant A., Karnauchov I., Brink S., Herrmann R. G., Klösgen R. B., Robinson C. A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase. EMBO J. 1995 Jun 15;14(12):2715–2722. doi: 10.1002/j.1460-2075.1995.tb07272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen Z. Y., Burow M. D., Mason C. B., Moroney J. V. A low-CO2-inducible gene encoding an alanine: alpha-ketoglutarate aminotransferase in Chlamydomonas reinhardtii. Plant Physiol. 1996 Oct;112(2):677–684. doi: 10.1104/pp.112.2.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eriksson M., Karlsson J., Ramazanov Z., Gardeström P., Samuelsson G. Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):12031–12034. doi: 10.1073/pnas.93.21.12031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Franzén L. G., Rochaix J. D., von Heijne G. Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences. FEBS Lett. 1990 Jan 29;260(2):165–168. doi: 10.1016/0014-5793(90)80094-y. [DOI] [PubMed] [Google Scholar]
  8. Fukuzawa H., Fujiwara S., Yamamoto Y., Dionisio-Sese M. L., Miyachi S. cDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii: regulation by environmental CO2 concentration. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4383–4387. doi: 10.1073/pnas.87.11.4383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Funke R. P., Kovar J. L., Weeks D. P. Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1. Plant Physiol. 1997 May;114(1):237–244. doi: 10.1104/pp.114.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hewett-Emmett D., Tashian R. E. Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol Phylogenet Evol. 1996 Feb;5(1):50–77. doi: 10.1006/mpev.1996.0006. [DOI] [PubMed] [Google Scholar]
  11. Husic H. D., Marcus C. A. Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label. Plant Physiol. 1994 May;105(1):133–139. doi: 10.1104/pp.105.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johanningmeier U., Howell S. H. Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors. J Biol Chem. 1984 Nov 10;259(21):13541–13549. [PubMed] [Google Scholar]
  13. Karlsson J., Hiltonen T., Husic H. D., Ramazanov Z., Samuelsson G. Intracellular carbonic anhydrase of Chlamydomonas reinhardtii. Plant Physiol. 1995 Oct;109(2):533–539. doi: 10.1104/pp.109.2.533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Katzman G. L., Carlson S. J., Marcus Y., Moroney J. V., Togasaki R. K. Carbonic Anhydrase Activity in Isolated Chloroplasts of Wild-Type and High-CO2-Dependent Mutants of Chlamydomonas reinhardtii as Studied by a New Assay. Plant Physiol. 1994 Aug;105(4):1197–1202. doi: 10.1104/pp.105.4.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Khalifah R. G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C. J Biol Chem. 1971 Apr 25;246(8):2561–2573. [PubMed] [Google Scholar]
  16. Kindle K. L. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1228–1232. doi: 10.1073/pnas.87.3.1228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Komarova Iu M., Doman N. G., Shaposhnikov G. L. Dve formy karboangidrazy v khloroplastakh bobov. Biokhimiia. 1982 Jun;47(6):1027–1034. [PubMed] [Google Scholar]
  18. Kulkarni R. D., Schaefer M. R., Golden S. S. Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942. J Bacteriol. 1992 Jun;174(11):3775–3781. doi: 10.1128/jb.174.11.3775-3781.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Liljas A., Håkansson K., Jonsson B. H., Xue Y. Inhibition and catalysis of carbonic anhydrase. Recent crystallographic analyses. Eur J Biochem. 1994 Jan 15;219(1-2):1–10. doi: 10.1007/978-3-642-79502-2_1. [DOI] [PubMed] [Google Scholar]
  21. Mason C. B., Matthews S., Bricker T. M., Moroney J. V. Simplified Procedure for the Isolation of Intact Chloroplasts from Chlamydomonas reinhardtii. Plant Physiol. 1991 Dec;97(4):1576–1580. doi: 10.1104/pp.97.4.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moroney J. V., Husic H. D., Tolbert N. E. Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii. Plant Physiol. 1985 Sep;79(1):177–183. doi: 10.1104/pp.79.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moubarak-Milad M., Stemler A. Oxidation-reduction potential dependence of photosystem II carbonic anhydrase in maize thylakoids. Biochemistry. 1994 Apr 12;33(14):4432–4438. doi: 10.1021/bi00180a042. [DOI] [PubMed] [Google Scholar]
  24. Nielsen V. S., Mant A., Knoetzel J., Møller B. L., Robinson C. Import of barley photosystem I subunit N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site. Role of the delta pH in translocation across the thylakoid membrane. J Biol Chem. 1994 Feb 4;269(5):3762–3766. [PubMed] [Google Scholar]
  25. Purton S., Rochaix J. D. Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol. 1994 Feb;24(3):533–537. doi: 10.1007/BF00024121. [DOI] [PubMed] [Google Scholar]
  26. Ramazanov Z., Mason C. B., Geraghty A. M., Spalding M. H., Moroney J. V. The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii. Plant Physiol. 1993 Apr;101(4):1195–1199. doi: 10.1104/pp.101.4.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rawat M., Moroney J. V. Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii. J Biol Chem. 1991 May 25;266(15):9719–9723. [PubMed] [Google Scholar]
  28. Silflow C. D., Chisholm R. L., Conner T. W., Ranum L. P. The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins. Mol Cell Biol. 1985 Sep;5(9):2389–2398. doi: 10.1128/mcb.5.9.2389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Spalding M. H., Spreitzer R. J., Ogren W. L. Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth. Plant Physiol. 1983 Oct;73(2):268–272. doi: 10.1104/pp.73.2.268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stemler A., Jursinic P. The effects of carbonic anhydrase inhibitors formate, bicarbonate, acetazolamide, and imidazole on photosystem II in maize chloroplasts. Arch Biochem Biophys. 1983 Feb 15;221(1):227–237. doi: 10.1016/0003-9861(83)90139-x. [DOI] [PubMed] [Google Scholar]
  31. Sueoka N. MITOTIC REPLICATION OF DEOXYRIBONUCLEIC ACID IN CHLAMYDOMONAS REINHARDI. Proc Natl Acad Sci U S A. 1960 Jan;46(1):83–91. doi: 10.1073/pnas.46.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tashian R. E. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays. 1989 Jun;10(6):186–192. doi: 10.1002/bies.950100603. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES