Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants

Abstract

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.

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12

“…This suggests that CP12 is involved in the post-translational regulation and at the transcriptional level. A recent study showed that reduced C. reinhardtii CP12 stabilizes PRK in vitro and in vivo, but the mechanism of this protection needs further investigation [ 76 , 77 ]. In the mutant strain of C. reinhardtii , where the CP12 protein is absent, while the abundance of numerous proteins increases (see below), the abundance of others, including PRK, involved in photosynthesis, decreases.…”

“…In C. reinhardtii , the deletion of the protein induced a re-routing of the metabolism under the light. In particular, metabolic pathways involving malate shuttles increased in the mutant such as the tricarboxylic acid cycle (TCA) and the glyoxylate pathway [ 77 ]. Malate shuttles, combined with other signaling factors, play a putative role in algal CO 2 -concentrating mechanisms (CCM) [ 82 , 83 ].…”

“…In both A. thaliana and N. tabacum , the antisense suppression of CP12 increased the expression of proteins related to oxidative stress [ 87 ]. Recently, it has been shown in C. reinhardtii that the suppression of CP12 leads also to an increase in the proteins involved in stress [ 77 ]. In addition, in cyanobacteria, CP12 might be involved in oxidative stress by controlling the electrons flux from Photosystem I.…”

“…In C. reinhardtii , CP12 protected GAPDH against heat inactivation and aggregation and therefore plays the role of a specific chaperone [ 88 ]. As mentioned above, CP12 also protects PRK against irreversible inactivation in vitro [ 77 ]. Besides these roles as a specific chaperone, CP12 from other organisms is more abundant in stress conditions, and this is the case for the T. pseudonana CP12-2.…”

First in vivo analysis of the regulatory protein CP12 of the model cyanobacterium Synechocystis PCC 6803: Biotechnological implications

“…Then, the N-terminal region of CP12 containing one cysteine pair followed by the consensus sequence binds PRK. In the eukaryotic alga Chlamydomonas reinhardtii , the core sequence of CP12 was found to act in the formation of the GAPDH–CP12–PRK complex and the protection of PRK from irreversible inactivation in vitro ( Gérard et al, 2022 ). In the light, complex dissociation mediated by CP12 reduction via a thioredoxin allows GAPDH and PRK reactivation, and consequently CO 2 fixation ( Gurrieri et al, 2022 ).…”