title: Visualization and quantification of protein interactions in the biosynthetic pathway of molybdenum cofactor in Arabidopsis thaliana.
abstract: The molybdenum cofactor (Moco) is the active compound at the catalytic site of molybdenum enzymes. Moco is synthesized by a conserved four-step pathway involving six proteins in Arabidopsis thaliana. Bimolecular fluorescence complementation was used to study the subcellular localization and interaction of those proteins catalysing Moco biosynthesis. In addition, the independent split-luciferase approach permitted quantification of the strength of these protein-protein interactions in vivo. Moco biosynthesis starts in mitochondria where two proteins undergo tight interaction. All subsequent steps were found to proceed in the cytosol. Here, the heterotetrameric enzyme molybdopterin synthase (catalysing step two of Moco biosynthesis) and the enzyme molybdenum insertase, which finalizes Moco formation, were found to undergo tight protein interaction as well. This cytosolic multimeric protein complex is dynamic as the small subunits of molybdopterin synthase are known to go on and off in order to become recharged with sulphur. These small subunits undergo a tighter protein contact within the enzyme molybdopterin synthase as compared with their interaction with the sulphurating enzyme. The forces of each of these protein contacts were quantified and provided interaction factors. To confirm the results, in vitro experiments using a technique combining cross-linking and label transfer were conducted. The data presented allowed the outline of the first draft of an interaction matrix for proteins within the pathway of Moco biosynthesis where product-substrate flow is facilitated through micro-compartmentalization in a cytosolic protein complex. The protected sequestering of fragile intermediates and formation of the final product are achieved through a series of direct protein interactions of variable strength.
authors: Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstrasse 1, D-38106 Braunschweig, Germany.
authors: David
authors: D
authors: Kaufholdt
authors: Christian
authors: C
authors: Gehl
authors: Mirco
authors: M
authors: Geisler
authors: Olga
authors: O
authors: Jeske
authors: Sabrina
authors: S
authors: Voedisch
authors: Christine
authors: C
authors: Ratke
authors: Benjamin
authors: B
authors: Bollhöner
authors: Ralf-R
authors: RR
authors: Mendel
authors: Robert
authors: R
authors: Hänsch
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics / Q000502:physiology; D003067:Coenzymes / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D011485:Protein Binding; D011621:Pteridines / Q000378:metabolism*; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D013466:Sulfurtransferases; C022261:molybdopterin; C085585:molybdopterin synthase
references: Annu Rev Biophys. 2008;37:465-87
references: 18573091
references: J Biol Chem. 2004 Apr 16;279(16):15994-9
references: 14761975
references: J Biol Chem. 2010 Feb 26;285(9):6623-35
references: 20040598
references: Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11646-51
references: 12185250
references: Biophys J. 2012 Feb 8;102(3):407-16
references: 22325262
references: Plant Physiol. 2008 Feb;146(2):368-76
references: 18065554
references: BMC Plant Biol. 2007 Jun 06;7:28
references: 17553146
references: Plant J. 1995 Nov;8(5):751-62
references: 8528286
references: EMBO J. 2004 Jul 7;23(13):2510-9
references: 15201864
references: J Biol Chem. 2007 May 4;282(18):13342-50
references: 17350958
references: Biochim Biophys Acta. 2006 Jul;1763(7):621-35
references: 16784786
references: J Biol Chem. 2006 Jul 7;281(27):18343-50
references: 16636046
references: Nature. 2009 Aug 13;460(7257):839-47
references: 19675644
references: Trends Neurosci. 2008 May;31(5):257-64
references: 18403029
references: Plant Cell. 2000 Dec;12(12):2455-2472
references: 11148290
references: Nature. 2004 Aug 12;430(7001):803-6
references: 15306815
references: Plant J. 2011 Aug;67(3):542-53
references: 21481030
references: Anal Biochem. 1976 May 7;72:248-54
references: 942051
references: Biochim Biophys Acta. 2012 Sep;1823(9):1568-79
references: 22370186
references: J Biol Chem. 2006 Nov 17;281(46):34796-802
references: 16973608
references: Nat Struct Biol. 2001 Jan;8(1):42-6
references: 11135669
references: Mol Plant. 2009 Sep;2(5):1051-8
references: 19825679
references: Plant Cell Physiol. 2004 Dec;45(12):1889-94
references: 15653809
references: Annu Rev Plant Biol. 2006;57:623-47
references: 16669776
references: Plant Cell. 2010 Feb;22(2):468-80
references: 20164445
references: EMBO J. 1987 Dec 20;6(13):3901-7
references: 3327686
references: J Biol Chem. 1997 Oct 24;272(43):26811-4
references: 9341109
references: Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5946-51
references: 15073332
references: Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12288-93
references: 15284440
references: J Biol Chem. 2002 May 24;277(21):18303-12
references: 11891227
references: J Biol Chem. 1993 Jun 25;268(18):13493-8
references: 8514781