Abstract
Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms.
Highlights
Compartments of eukaryotic cells are surrounded by a single- or multiple-layer lipid membrane
C. reinhardtii was fractionated to provide samples enriched for stroma proteins and for pyrenoidassociated proteins according to Mackinder et al (2016), followed by quantification of the abundance of enzymes involved in the Calvin-Benson cycle (CBC) and starch synthesis, using either an enzymatic assay or shotgun proteomics (Figure 2, Supplementary file 1A,B)
Apart from GAPDH (8% in low CO2 (LC) and 11% in high CO2 (HC) grown cells) and PRK (13% in HC grown cells but
Summary
Compartments of eukaryotic cells are surrounded by a single- or multiple-layer lipid membrane. Microcompartments are known in eukaryotes, including: metabolic compartments in liver (Fujiwara and Itoh, 2014) and muscle cells (Saks et al, 2008), and the pyrenoid in chloroplasts of green algae (Gibbs, 1962) Despite these discoveries, it remains challenging to determine the implications of microcompartments for cellular physiology, and to study the function of microcompartments under different conditions that may induce or suppress their formation. The current model of CCM assumes that, apart from Rubisco, the remaining enzymes of the Calvin-Benson cycle (CBC) are situated in the stroma (Jungnick et al, 2014) This implies that the substrate and product of the carboxylation reaction catalyzed by Rubisco, ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglyccerate (3PGA), need to move in and out of the pyrenoid, respectively. Our approach allows us to determine the exchange of fluxes at the boundary of the pyrenoid and to investigate the mode of transport of the exchanged metabolites
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