title: Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana.
abstract: BACKGROUND
  The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.
  
  
  RESULTS
  Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.
  
  
  CONCLUSIONS
  We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.
authors.affiliation: Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Burén
authors.forename: Cristina
authors.initials: C
authors.lastname: Ortega-Villasante
authors.forename: Amaya
authors.initials: A
authors.lastname: Blanco-Rivero
authors.forename: Andrea
authors.initials: A
authors.lastname: Martínez-Bernardini
authors.forename: Tatiana
authors.initials: T
authors.lastname: Shutova
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Shevela
authors.forename: Johannes
authors.initials: J
authors.lastname: Messinger
authors.forename: Laszlo
authors.initials: L
authors.lastname: Bako
authors.forename: Arsenio
authors.initials: A
authors.lastname: Villarejo
authors.forename: Göran
authors.initials: G
authors.lastname: Samuelsson
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport
chemical_list: D029681:Arabidopsis Proteins; D004220:Disulfides; D011134:Polysaccharides; C517158:CAH1 protein, Arabidopsis; D002256:Carbonic Anhydrases