title: Identification of a protein-protein interaction network downstream of molybdenum cofactor biosynthesis in Arabidopsis thaliana.
abstract: The molybdenum cofactor (Moco) is ubiquitously present in all kingdoms of life and vitally important for survival. Among animals, loss of the Moco-containing enzyme (Mo-enzyme) sulphite oxidase is lethal, while for plants the loss of nitrate reductase prohibits nitrogen assimilation. Moco is highly oxygen-sensitive, which obviates a freely diffusible pool and necessitates protein-mediated distribution. During the highly conserved Moco biosynthesis pathway, intermediates are channelled through a multi-protein complex facilitating protected transport. However, the mechanism by which Moco is subsequently transferred to apo-enzymes is still unclear. Moco user enzymes can be divided into two families: the sulphite oxidase (SO) and the xanthine oxidoreductase (XOR) family. The latter requires a final sulphurisation of Moco catalysed via ABA3. To examine Moco transfer towards apo-Mo-enzymes, two different and independent protein-protein interaction assays were performed in vivo: bimolecular fluorescence complementation and split luciferase. The results revealed a direct contact between Moco producer molybdenum insertase CNX1, which represents the last biosynthesis step, and members of the SO family. However, no protein contact was observed between Moco producer CNX1 and apo-enzymes of the XOR family or between CNX1 and the Moco sulphurase ABA3. Instead, the Moco-binding protein MOBP2 was identified as a mediator between CNX1 and ABA3. This interaction was followed by contact between ABA3 and enzymes of the XOR family. These results allow to describe an interaction matrix of proteins beyond Moco biosynthesis and to demonstrate the complexity of transferring a prosthetic group after biosynthesis.
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: d.kaufholdt@tu-bs.de.
authors: David
authors: D
authors: Kaufholdt
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: c.baillie@tu-bs.de.
authors: Christin-Kirsty
authors: CK
authors: Baillie
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: martin.meyer@tu-bs.de.
authors: Martin H
authors: MH
authors: Meyer
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: Schwich@bio.uni-frankfurt.de.
authors: Oliver D
authors: OD
authors: Schwich
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: u.timmerer@tu-bs.de.
authors: Ulrike L
authors: UL
authors: Timmerer
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: l.tobias@tu-bs.de.
authors: Lydia
authors: L
authors: Tobias
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: d.van-thiel@tu-bs.de.
authors: Daniela
authors: D
authors: van Thiel
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: r.haensch@tu-bs.de.
authors: Robert
authors: R
authors: Hänsch
authors: Department of Plant Biology, Technical University of Braunschweig, Humboldtstrasse 1, 38106 Braunschweig, Germany. Electronic address: r.mendel@tu-bs.de.
authors: Ralf R
authors: RR
authors: Mendel
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D053898:Biosynthetic Pathways; D003067:Coenzymes / Q000096:biosynthesis*; D005453:Fluorescence; D008667:Metalloproteins / Q000096:biosynthesis*; D000090006:Molybdenum Cofactors; D030821:Plants, Genetically Modified; D011485:Protein Binding; D060066:Protein Interaction Maps; D011621:Pteridines; D050876:Sulfite Oxidase / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D050876:Sulfite Oxidase; C022261:molybdopterin
keywords: Arabidopsis thaliana; Bimolecular fluorescence complementation assay (BiFC); Floated leaf luciferase complementation imaging assay (FLuCI); Moco biosynthesis; Molybdenum cofactor (Moco); Protein-protein interaction