Proceedings of the National Academy of Sciences · 2006 · 114 citations · 14 references
Life SciencesMolecular BiologyPlant BiologyPlant BiochemistryPlant Cell BiologyPlant Molecular BiologyCalcium SignalingCalcium-binding ProteinsChloroplast BiochemistryChloroplast BiologyEndomembrane SystemThylakoid Membrane StructureCalmodulin-mediated RegulationChloroplast Envelope MembranesChloroplast Membrane Protein ComplexesChloroplast Protein ImportChloroplast Redox RegulationNuclear Envelope ProteinsProtein TranslocationProtein Translocation Across The Chloroplast Envelope
The import of nuclear-encoded proteins into chloroplasts is tightly controlled on both sides of the envelope membranes. Regulatory circuits include redox-control as well as calcium-regulation, with calmodulin being the likely mediator of the latter. Using affinity-chromatography on calmodulin-agarose, we could identify the inner envelope translocon component Tic32 as the predominant calmodulin-binding protein of this membrane. Calmodulin-binding assays corroborate the interaction for heterologously expressed as well as native Tic32. The interaction is calcium-dependent and is mediated by a calmodulin-binding domain between Leu-296 and Leu-314 close to the C-proximal end of the pea Tic32. We furthermore could establish Tic32 as a bona fide NADPH-dependent dehydrogenase. NADPH but not NADH or NADP(+) affects the interaction of Tic110 with Tic32 as well as Tic62. At the same time, dehydrogenase activity of Tic32 is affected by calmodulin. In particular, binding of NADPH and calmodulin to Tic32 appear to be mutually exclusive. These results suggest that redox modulation and calcium regulation of chloroplast protein import convene at the Tic translocon and that both could be mediated by Tic32.
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