The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin–Benson–Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde−3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism.

The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO₂ assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

Carbonic anhydrases (CAs) typically possess a Zn2+ cofactor and are involved in a wide range of adaptive metabolisms involving pH and dissolved inorganic carbon homeostasis. In photosynthetic microorganisms, they are involved in CO2 concentrating mechanisms (CCM) that supply CO2 to ribulose-1,5-bisphosphate carboxylase-oxygenase. Here we show that the model diatom Phaeodactylum tricornutum possesses a CA from the newly identified iota class, P. tricornutum ι-carbonic anhydrase (Pt-ιCA), which exhibited catalytic activity without a metal cofactor, in contrast to most CAs. Metal-free Pt-ιCA activity was 2 orders of magnitude lower than that of the Zn2+-containing bovine erythrocyte CA, yet still in the range of expected values for CA. Zn2+ concentrations in the picomolar range induced the expression of Pt-ιCA in P. tricornutum, together with the Zn2+/Co2+ responsive protein A. Zn2+ scarcity did not prevent the induction of Zn2+-containing CAs involved in biophysical CCM when the cells were transferred from high atmospheric partial pressure of CO2 (pCO2) to very low pCO2 conditions. The induction of CCM at very low pCO2 and low Zn2+ conditions was concomitant with the induction of light harvesting complexes and remodeling of the pentose phosphate pathways. The total cellular CA activity decreased in the Pt-ιCA deletion mutant (ΔιCA) compared with control cells, indicating that Pt-ιCA is active in P. tricornutum under low Zn2+ conditions. P. tricornutum adapted to the absence of Pt-ιCA by a morphometric change, without affecting photosynthesis or growth rate. The newly identified metal-free Pt-ιCA represents an evolutionary advantage for diatoms that live in metal-limited environments.

The small chloroplastic protein CP12 has multiple functions, including the regulation of enzymes in the Calvin–Benson–Bassham cycle. Here, we investigated its role in the acclimation of Chlamydomonas reinhardtii to varying CO2 availability. We show that phosphoribulokinase can interact with CP12 in conditions where the Calvin–Benson–Bassham cycle is active. Compared to the wild type, at high CO2 C. reinhardtii ΔCP12 mutants have less phosphoribulokinase and ribulose-1,5-bisphosphate (RuBP), indicating that the regeneration of RuBP is regulated, in part, by CP12. Chlamydomonas reinhardtii has a CO2-concentrating mechanism that increases the supply of CO2 to Rubisco and involves, among other features, the condensation of Rubisco within the pyrenoid via its interaction with a scaffold protein named Essential Pyrenoid Component 1 (EPYC1). In the ΔCP12 mutants, the expected relocation of Rubisco towards the pyrenoid was not observed upon transition from high to very low CO2, in contrast to wild type cells. The ΔCP12 mutants are therefore a unique example where the induction of the CO2-concentrating mechanism at very low CO2 is not accompanied by Rubisco relocation. Taken together, our results suggest that CP12 contributes to the coordination between RuBP regeneration, Rubisco location, and CO2 acquisition.