Abstract
BackgroundThe 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.Methodology/Principal FindingsTransient 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/SignificanceWe 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.
Highlights
Carbonic anhydrase (CA) is a ubiquitous zinc metalloenzyme that catalyzes the reversible interconversion of carbon dioxide (CO2) and bicarbonate (HCO32) at very high turnover rates [1]
The HA-tagged CAH1 (HC) was stably transformed into an Arabidopsis thaliana cell suspension culture and sub-cellular localization of the expressed protein was analyzed using immunogold (IG) labelling followed by electron microscopy
The wild type HC was mainly localized to the chloroplast of cells, where the highest immunogold labelling density was observed (Table 2, Figure S1)
Summary
Carbonic anhydrase (CA) is a ubiquitous zinc metalloenzyme that catalyzes the reversible interconversion of carbon dioxide (CO2) and bicarbonate (HCO32) at very high turnover rates [1]. In order to study the importance of endomembrane-specific post-translational modifications for correct trafficking and functioning of a chloroplast-localized protein, an epitope-tagged version of CAH1 was constructed that enabled both stable and transient expression in Arabidopsis suspension culture cells and protoplasts.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
