|Do Carmo Silva, Anaelisabete|
Submitted to: Protein and Peptide Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2011
Publication Date: 6/1/2011
Citation: Do Carmo Silva, A., Marri, L., Sparla, F., Salvucci, M.E., 2011. Isolation and compositional analysis of a CP12-associated complex of Calvin cycle enzymes from nicotiana tabacum. Protein and Peptide Letters. 18:618-624. Interpretive Summary: In the process of photosynthesis, plants convert light into chemical energy. The energy produced by photosynthesis is then used to synthesize sugars and other foodstuffs. Heat stress inhibits photosynthesis, reducing the overall yield of the plant. Previous research identified an enzyme called Rubisco activase as the component of photosynthesis that is most sensitive to inhibition by heat. Rubisco activase is a regulatory enzyme that controls the activity of the major carbon dioxide-fixing enzyme in plants. In this manuscript, we examine the involvement of a regulatory mechanism involving a small protein called CP12 in controlling Rubisco activase activity. The research isolated a complex containing CP12 from tobacco and Arabidopsis and identified the enzymes that CP12 regulates. That Rubisco activase was not among these proteins indicates that regulation of the important Rubisco activase enzyme does not involve CP12. The results provide new insights into our understanding of how photosynthesis is regulated and eliminates a possible mechanism that could confer sensitivity to heat stress. This information eventually can be used to make changes that improve the Rubisco activase in order to improve the efficiency of photosynthesis under heat stress.
Technical Abstract: CP12 is a small intrinsically unstructured protein that forms a multiprotein complex with two Calvin Cycle enzymes, phosphoribulokinase (PRK) and NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex can be reconstituted in vitro from recombinant proteins under conditions that resemble those prevailing in darkened chloroplasts. Light/dark reversible dissociation/association of CP12 with PRK and GAPDH has been demonstrated immunologically in leaves and algal cells, but an intact, native GAPDH/CP12/PRK complex has not been isolated for characterization. In the present study, a ~500 kD multiprotein complex containing CP12 was isolated from darkened chloroplasts of tobacco and darkened leaves of Arabidopsis and tobacco. The complex was analyzed by gel electrophoresis, immunoblotting and nano-liquid chromatography coupled with mass spectrometry to determine its composition. The analyses verified that the native tobacco complex contained GAPDH, CP12 and PRK, but no additional partners that could be regulated by the redox-modulated scaffolding activity of CP12. Despite the similarities between Rubisco activase ' and ' isoforms and GAPDH subunits A and B, the results specifically eliminated activase as a component of high molecular mass complexes with CP12. The ~500 kD GAPDH/CP12/PRK complex was present in dark- but not light-adapted leaves and dissociated upon reduction with dithiothreitol. Taken together, the results suggest that CP12 plays a specific role in the dark/light regulation of GAPDH and PRK, through aggregation in supramolecular complexes involving no other stromal enzymes. Of the three CP12 isoforms in Arabidopsis, both CP12-1 and -2 were present in the native complex isolated from darkened leaves, but CP12-1 was predominant.