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.

Results/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.

Significance: This is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Figure 1. Analysis of thylakoid carbonic anhydrase (Cah3) activity and expression during the acclimation of high-CO₂-grown C. reinhardtii cells to low CO₂ conditions.

(A) 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 (n = 5). (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. (C) Immunoblot analysis of total cell extracts from cells of C. reinhardtii with antibodies raised against over-expressed Cah3 polypeptide. The lanes were loaded with 10 µg protein.

Figure 2. Staurosporine, a protein kinase inhibitor, partially inhibits the activation of Cah3 activity.

High-CO₂-grown C. reinhardtii cells were acclimated to low CO₂ conditions for 2 h in the absence or in the presence of 0.1 µM Staurosporine. CA activity was measured in thylakoid membranes isolated from control and treated cells. As a control, periplasmic CA activity was measured using intact cells of the same cultures. Values are means ± SE (n = 5).

Figure 3. Phosphorylation of LHCIIP and PSII polypeptides during acclimation to low CO₂ conditions.

(A) 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) 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. (C) Changes in the immunoresponse of Thr(P) antibody to a 30-kDa phosphoprotein during the acclimation to low CO₂. The inset 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.

Figure 4. Immunoprecipitation and dephosphorylation experiments of extrinsic thylakoid polypeptides.

Extraction of extrinsic thylakoid proteins was accomplished by washing the thylakoid membranes with a medium containing low concentrations (0.05%) of Triton X-100. (A) The 30-kDa extrinsic phosphoprotein immunoprecipitates with Cah3. Extrinsic thylakoid proteins released from thylakoid membranes of C. reinhardtii cells acclimated to low CO₂ for 2 h (C) were immunoprecipitated with affinity-purified antibodies against Cah3 and protein A-Sepharose CL-4-B beads. The Sepharose beads were washed and the immunoprecipitate (I) and the supernatant (SN) obtained after centrifugation were analysed by SDS-PAGE and immunoblot and probed with antibodies against Cah3 (left) and Thr(P) (right). (B) Effect of Alkaline phosphatase (AP) treatment on extrinsic proteins released from thylakoid membranes isolated from both high-CO₂-grown cells (High) or cells acclimated to low CO₂ for 2 h (Low). All lanes were loaded with 10 µg protein.

Figure 5. Immunogold labelling of C. reinhardtii cells grown on high-CO₂ or acclimated to low CO₂ conditions for 2 and 5 h.

(A) and (B) High-CO₂-grown cells probed with affinity-purified antibodies against Cah3. (C) and (D) Cells acclimated to low CO₂ conditions for 3 h and (E) pyrenoid of a cell acclimated to low CO₂ conditions for 5 h, probed with affinity-purified antibodies against Cah3. Bars indicated 0.5 µm. C, chloroplast; P, pyrenoid; Ssh, starch sheath; and St, stroma chloroplast.

Figure 6. Immunoblot analysis of total cell extracts and isolated pyrenoid fractions from high- (H) and low-CO₂-grown (L) Chlamydomonas cells probed with antibodies against D1 protein of PSII, Cah3 and Rubisco large subunit.

All lanes were loaded with 10 µg protein.

Figure 7. Differential extraction of Cah3 polypeptide from PSII core complexes isolated from C. reinhardtii cells grown under high-CO₂ (A) or acclimated to low CO₂ conditions for 4 h (B).

PSII hydrophobic and hydrophilic proteins were extracted from PSII core complexes using a chloroform/methanol (2∶1, v/v) mixture (see Materials and Methods). Immunoblot analysis of integral (I) and peripheral (P) protein fractions from PSII core complexes were probed with antibodies against Cah3 (Cah3) and PsbO protein (PsbO). The lanes were loaded with 10 µg protein.