Abstract: | |
L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (L-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.
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License of this version: | CC BY 4.0 Unported - https://creativecommons.org/licenses/by/4.0/ |
Publication type: | Article |
Publishing status: | publishedVersion |
Publication date: | 2012 |
Keywords english: | Activity assays, Arabidopsis thaliana, Gel electrophoresis, Immunoblotting, L-Ascorbate, Membrane domain, Mitochondrial complex, NADH dehydrogenase, Plant mitochondria, Protein complexes, Protein identification, Subcomplexes, Vitamin C, Biochemistry, Electrophoresis, Mass spectrometry, Plants (botany), Mitochondria, levo galactono 1,4 lactone dehydrogenase, oxidoreductase, reduced nicotinamide adenine dinucleotide dehydrogenase (ubiquinone), unclassified drug, Arabidopsis, article, complex formation, enzyme assay, enzyme binding, enzyme conformation, gel electrophoresis, immunoblotting, mass spectrometry, nonhuman, priority journal, protein domain, Arabidopsis, Arabidopsis Proteins, Ascorbic Acid, Electron Transport Complex I, Mitochondria, Mitochondrial Proteins, Oxidoreductases Acting on CH-CH Group Donors, Protein Structure, Tertiary, Arabidopsis thaliana |
DDC: | 570 | Biowissenschaften, Biologie, 540 | Chemie |
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