title: Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32.
abstract: 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.
authors.affiliation: Department of Biology I, Ludwig-Maximilians-Universität, Menzinger Strasse 67, D-80638 München, Germany.
authors.forename: Fatima
authors.initials: F
authors.lastname: Chigri
authors.forename: Friederike
authors.initials: F
authors.lastname: Hörmann
authors.forename: Anna
authors.initials: A
authors.lastname: Stamp
authors.forename: David K
authors.initials: DK
authors.lastname: Stammers
authors.forename: Bettina
authors.initials: B
authors.lastname: Bölter
authors.forename: Jürgen
authors.initials: J
authors.lastname: Soll
authors.forename: Ute C
authors.initials: UC
authors.lastname: Vothknecht
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002736:Chloroplasts / Q000187:drug effects* / Q000378:metabolism*; D017124:Conserved Sequence; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D018532:Pisum sativum; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D016415:Sequence Alignment
chemical_list: D002147:Calmodulin; D008565:Membrane Proteins; D010940:Plant Proteins; D009249:NADP; D010088:Oxidoreductases; D002118:Calcium