title: Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.
abstract: BACKGROUND
  Cah3 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.
  
  
  CONCLUSIONS
  In 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.
  
  
  CONCLUSIONS
  This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.
authors.affiliation: Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Amaya
authors.initials: A
authors.lastname: Blanco-Rivero
authors.forename: Tatiana
authors.initials: T
authors.lastname: Shutova
authors.forename: María José
authors.initials: MJ
authors.lastname: Román
authors.forename: Arsenio
authors.initials: A
authors.lastname: Villarejo
authors.forename: Flor
authors.initials: F
authors.lastname: Martinez
mesh_terms: D000064:Acclimatization / Q000187:drug effects; D002245:Carbon Dioxide / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D002457:Cell Extracts; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects; D015151:Immunoblotting; D047468:Immunoprecipitation; D008027:Light; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D045332:Photosystem II Protein Complex / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D020524:Thylakoids / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure
chemical_list: D002457:Cell Extracts; D010455:Peptides; D045332:Photosystem II Protein Complex; D047428:Protein Kinase Inhibitors; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases; D019311:Staurosporine; D010100:Oxygen