Figure 2. Experimental data for protein distributions (outer yellow circle) and metabolite concentrations (inner blue circle) in CCM-supressed (white bars, HC) and CCM-induced (grey bars, LC for proteins and LC* for metabolites) conditions.

Chlamydomonas reinhardtii CC1690 cells were grown under high CO₂ (HC for proteins and metabolites; white bars), ambient CO₂ (LC for proteins; grey bars) and ambient CO₂ bubbled for 15 min with high CO₂ (LC* for metabolites; grey bars). Enzyme distribution between a pyrenoid-enriched fraction (P) and a stroma-enriched fraction (S) was determined by enzyme activity measurements (Rubisco; n = 4 ± SE) and shotgun proteomics (all other proteins; n = 4 ± SE). Metabolites of the Calvin-Benson cycle (CBC) in total cells were measured by HPLC-MS/MS. The metabolite concentrations were normalized to the chloroplast volume as described in the text and Supplementary file 1D, and given as absolute concentrations (µM) in the chloroplast, which includes both microcompartments, the stroma and the pyrenoid (S + P) (n = 4 ± SE). Student´s t-test (alpha = 0.05), significantly changed metabolites are marked with one asterisk.

Figure 2—figure supplement 1. Induction of carbon concentrating mechanism (CCM).

Chlamydomonas reinhardtii CC1690 were grown at 46 μmol photons*m⁻²*s⁻¹, 24°C and bubbled with 5% CO₂ (HC) for two days at constant turbidity in a bioreactor. CO₂ in the outlet air of the bioreactor was measured continuously during a 48 hr run (A). From time point zero onwards the culture was aerated with ambient air (0.039% CO₂). The inserted graph shows the same CO2 data at lower CO₂ concentrations. Cultures were harvested before (HC) and 25 and 34 hr (LC) after low-CO₂ exposure for Western blot analysis (B). Protein amounts equivalent to 1 μg chlorophyll were loaded per lane and separated by 12% SDS-PAGE before transferred to a nitrocellulose membrane for detection via chemilumminescence by an antiserum recognizing mtCA (AgriSera Cat# AS11 1737, RRID:AB_10752086). Loading control: CF₁β, β-subunit of the CF₁-component of CF₁F_O-ATP synthase AgriSera Cat# AS10 1590, RRID:AB_10754669). Transmission electron microscopy (TEM) of cells exposed for 30 hr to low CO₂ and 15 min to high CO₂ (LC*; C). Cells were then quenched in the light for metabolite analysis by LC-MS/MS. Measure bar = 2 μm.