Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

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. Methodology/Principal Findings: 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/Significance: 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.This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovación (MICINN) to A

Análisis de la mutación en un gen con implicaciones en procesos de adaptación A pHs alcalinos, resistencia al ión Na+ y fijación de N2 en la cianobacteria anabaena sp. pcc7120

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 21-11-200

Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

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 CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions. Results/Conclusions: In the present work we demonstrate that after transfer to low CO2, 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 CO2 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 CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules. Significance: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen

Analysis of thylakoid carbonic anhydrase (Cah3) activity and expression during the acclimation of high-CO₂-grown <i>C. reinhardtii</i> cells to low CO₂ conditions.

<p>(<b>A</b>) CA activity (WA units (mg Chl)⁻¹) was measured in thylakoid membranes isolated from high-CO₂-grown cells or acclimated to low CO₂ for 2, 4 and 8 h. Values are means ± SE (<i>n</i> = 5). (<b>B</b>) Semiquantitative RT-PCR analysis of Cah3 gene expression. Total RNA to be used for RT was isolated by using Trizol™ reagent according to the manufacturers protocol (Life Technologies, US). Aliquots of the reaction mix were loaded and ethidium bromide stained in 1% agarose gels. (<b>C</b>) Immunoblot analysis of total cell extracts from cells of <i>C. reinhardtii</i> with antibodies raised against over-expressed Cah3 polypeptide. The lanes were loaded with 10 µg protein.</p

Calculation of Cah3 fraction in both the pyrenoid and the stroma of the chloroplast of both high-CO₂ cells and cells acclimating to low CO₂ conditions for 3 and 5 h.

<p>Immunogold labelling experiments were carried out using antibodies against Cah3. Evaluation of labelling was made using the computer program UTHSCSA Image Tool version 3.0. The gold particles associated with the pyrenoid and the stroma were counted and the density calculated on the basis of area. To calculate the fraction of Cah3 in the pyrenoid, the particle density of the pyrenoid or stroma was multiplied by the average volume of the compartment (which is 2.4 µm³ and 35.6 µm³, respectively, according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049063#pone.0049063-LacosteRoyal1" target="_blank">[27]</a>, giving the total particles for each compartment. The total number of particles in the pyrenoid was divided by the combined number of particles in the pyrenoid and in the stroma. The data shown are the averages ± SD of 30 samples. Preimmune sera gave immunogold densities of less than 2 particles/µm².</p

Phosphorylation of LHCIIP and PSII polypeptides during acclimation to low CO₂ conditions.

<p>(<b>A</b>) Immunoblot analysis of thylakoid membrane proteins isolated from high-CO₂-grown cells (H), and cells acclimated to low CO₂ for 1 (1 h) and 2 h (2 h) probed with antibodies against phosphothreonine (Thr(P)). (<b>B</b>) Immunoblot analysis of extrinsic thylakoid proteins isolated from thylakoids of high-CO₂-grown cells (H), and cells acclimated to low CO₂ for 1 (1 h) and 2 h (2 h), probed with affinity-purified antibodies against Cah3. (<b>C</b>) Changes in the immunoresponse of Thr(P) antibody to a 30-kDa phosphoprotein during the acclimation to low CO₂. The <i>inset</i> shows immunoblot analysis of extrinsic thylakoid proteins isolated from thylakoids of high-CO₂-grown cells (H), and cells acclimated to low CO₂ for 1 (1 h) and 2 h (2 h), probed with Thr(P) antibodies. The lanes were loaded with 10 µg protein.</p