tailieunhanh - Báo cáo khoa học: Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

he kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase⁄glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex fromChlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. | iFEBS Journal Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii Functional consequences for glyceraldehyde-3-phosphate dehydrogenase Sandrine Lebreton Simona Andreescu Emmanuelle Graciet and Brigitte Gontero Institut Jacques Monod CNRS-Universites Paris VI et Paris VII France Keywords CP12 GAPDH interaction site intrinsically unstructured protein protein-protein interactions Correspondence B. Gontero Institut Jacques Monod UMR 7592 CNRS-Universites Paris VI et Paris VII 2 place Jussieu 75251 Paris cedex 5 France Fax 33 1 44 27 59 94 Tel 33 1 44 27 47 19 E-mail meunier@ The authors contributed equally to this work. Present address California Institute of Technology Pasadena CA USA Received 29 March 2006 revised 16 May 2006 accepted 25 May 2006 doi The kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase glyceraldehyde-3-phosphate dehydrogenase GAPDH complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1 3-bisphosphoglycerate leads to dissociation of the GAPDH CP12 complex. Consequently a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1 3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central a-helix of CP12 which includes residues at position 36 D and 39 E . Mutants of