Abstract
Calcium plays an important role in the regulation of several chloroplast processes. However, very little is still understood about the calcium fluxes or calcium-binding proteins present in plastids. Indeed, classical EF-hand containing calcium-binding proteins appears to be mostly absent from plastids. In the present study we analyzed the stroma fraction of Arabidopsis chloroplasts for the presence of novel calcium-binding proteins using 2D-PAGE separation followed by calcium overlay assay. A small acidic protein was identified by mass spectrometry analyses as the chloroplast protein CP12 and the ability of CP12 to bind calcium was confirmed with recombinant proteins. CP12 plays an important role in the regulation of the Calvin-Benson-Bassham Cycle participating in the assembly of a supramolecular complex between phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, indicating that calcium signaling could play a role in regulating carbon fixation.
Highlights
Chloroplasts are key organelles for plant autotrophism, where many essential metabolic processes take place
Chloroplasts were disrupted by treatment with hypertonic buffer and soluble and membrane proteins were separated by centrifugation
Were able to compete for binding of 45Ca. All together these results indicate that CP12 is a novel chloroplast calcium-binding protein
Summary
Chloroplasts are key organelles for plant autotrophism, where many essential metabolic processes take place. Calcium has been shown to be involved in the regulation of processes such as photosynthesis, CO2 fixation, protein transport and protein phosphorylation. The thylakoid localized calcium sensing protein (CAS) is one of the most extensively studied calcium-binding proteins in plastids It is involved in stomatal closure [8,9,10,11] and photoacclimation [12] and was one of three thylakoid targets of calcium-dependent phosphorylation identified in a recent study [13]. Calmodulins (CaMs) are one of the best studied families of calcium-binding proteins They are comprised of four EF-hand structural motifs that are able to bind calcium with high affinity [14] and bind to their targets in a calcium-dependent manner. Our data suggest that CP12 represents a novel calcium-binding protein in chloroplasts
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