RuBP availability contributes to the regulation of RUBISCO activity in Synechocystis

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) and the Calvin-Benson-Bassham (CBB) cycle assimilate most of the inorganic carbon on earth. Conserved CP12 proteins control diurnal C-assimilation of the CBB cycle. Their function originated in cyanobacteria that deactivate GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) and PHOSPHORIBULOKINASE (PRK) during darkness by redox-regulated complex-formation with CP12. This mechanism indicates metabolic regulation of C-assimilation. We measured C-assimilation in vivo at the day/night transition by dynamic ¹³CO₂ labeling and 1-C-positional monitoring of ¹³C-incorporation into 3-phosphoglycerate (3PGA) and aspartate of Synechocystis sp. PCC 6803 grown with high (5%) CO₂ supply. Unlike wild type, a Δcp12 mutant accumulated ribulose-1,5-bisphosphate (RuBP) and assimilated ¹³CO₂ in darkness. We validated these observations by proteome measurements and orthogonal quantifications of RUBISCO abundances and specific activities. Complementation of Δcp12 with native CP12 and point-mutants of its GAPDH- and PRK-binding domains demonstrated that defective complex formation is causal for aberrant C-assimilation during darkness and that RuBP accumulation correlates significantly with the time-shifted ¹³C-incorporation into 3PGA. RUBISCO carboxylase activity is present throughout the diurnal cycle and depends on CP12-controlled substrate availability. ¹³C-positional analyses suggest dysregulated RuBP regeneration in Δcp12 during darkness via futile metabolic cycling through gluconeogenesis and the oxidative pentose phosphate path. We propose that blocked regeneration and rapid depletion of RuBP contributes to the regulation of RUBISCO activity at day-night transitions.