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• W2015454276 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. 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. Ascorbate (vitamin C) is of central importance for several biological processes. In plants, it was shown to be essential for growth (1Pignocchi C. Foyer C.H. Apoplastic ascorbate metabolism and its role in the regulation of cell signaling.Curr. Opin. Plant Biol. 2003; 6: 379-389Crossref PubMed Scopus (382) Google Scholar), programmed cell death (2de Pinto M.C. Paradiso A. Leonetti P. De Gara L. Hydrogen peroxide, nitric oxide, and cytosolic ascorbate peroxidase at the cross-road between defense and cell death.Plant J. 2006; 48: 784-795Crossref PubMed Scopus (174) Google Scholar), pathogen response (3Barth C. Moeder W. Klessig D.F. Conklin P.L. The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin C-1.Plant Physiol. 2004; 134: 1784-1792Crossref PubMed Scopus (219) Google Scholar), signal transduction (4Barth C. De Tullio M. Conklin P.L. The role of ascorbic acid in the control of flowering time and the onset of senescence.J. Exp. Bot. 2006; 57: 1657-1665Crossref PubMed Scopus (226) Google Scholar), and the stress response with respect to ozone (5Conklin P.L. Barth C. Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senesence.Plant Cell Environ. 2004; 27: 959-970Crossref Scopus (342) Google Scholar), UV radiation (6Gao Q. Zhang L. Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana.J. Plant Physiol. 2008; 165: 138-148Crossref PubMed Scopus (123) Google Scholar), high temperature (7Larkindale J. Hall J.D. Knight M.R. Vierling E. Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance.Plant Physiol. 2005; 138: 882-897Crossref PubMed Scopus (597) Google Scholar), and high light (8Müller-Moulé P. Golan T. Niyogi K.K. Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress.Plant Physiol. 2004; 134: 1163-1172Crossref PubMed Scopus (165) Google Scholar). Ascorbate is the cofactor of several enzymes and one of the major components adjusting the redox state of cells. In plant tissue, it can reach millimolar concentrations and form up to 10% of the soluble carbohydrate content. Biosynthesis of ascorbate in plants mainly takes place via the “l-galactose” also known as “Smirnoff-Wheeler” pathway (9Wheeler G.L. Jones M.A. Smirnoff N. The biosynthetic pathway of vitamin C in higher plants.Nature. 1998; 393: 365-369Crossref PubMed Scopus (857) Google Scholar). The terminal step of this pathway, the conversion of l-galactono-1,4-lactone (GL) 2The abbreviations used are: GLl-galactono-1,4-lactoneGLDHl-galactono-1,4-lactone dehydrogenaseCAcarbonic anhydraseBNblue native. into ascorbate, is catalyzed by l-galactono-1,4-lactone dehydrogenase (GLDH). GLDH is localized in mitochondria. During ascorbate formation, GLDH needs oxidized cytochrome c as the electron acceptor (10Mapson L.W. Isherwood F.A. Chen Y.T. Biological synthesis of l-ascorbic acid. The conversion of l-galactono-γ-lactone into l-ascorbic acid by plant mitochondria.Biochem. J. 1954; 56: 21-28Crossref PubMed Scopus (38) Google Scholar, 11Mapson L.W. Breslow E. Biological synthesis of ascorbic acid. l-Galactono-γ-lactone dehydrogenase.Biochem. J. 1958; 68: 395-406Crossref PubMed Scopus (75) Google Scholar, 12Bartoli C.G. Pastori G.M. Foyer C.H. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.Plant Physiol. 2000; 123: 335-344Crossref PubMed Scopus (321) Google Scholar). Indeed, GL represents a respiratory substrate for oxidative phosphorylation in plants (12Bartoli C.G. Pastori G.M. Foyer C.H. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.Plant Physiol. 2000; 123: 335-344Crossref PubMed Scopus (321) Google Scholar, 13Millar A.H. Mittova V. Kiddle G. Heazlewood J.L. Bartoli C.G. Theodoulou F.L. Foyer C.H. Control of ascorbate synthesis by respiration and its implications for stress responses.Plant Physiol. 2003; 133: 443-447Crossref PubMed Scopus (273) Google Scholar). l-galactono-1,4-lactone l-galactono-1,4-lactone dehydrogenase carbonic anhydrase blue native. GLDH has been purified and characterized for several plant species (11Mapson L.W. Breslow E. Biological synthesis of ascorbic acid. l-Galactono-γ-lactone dehydrogenase.Biochem. J. 1958; 68: 395-406Crossref PubMed Scopus (75) Google Scholar, 14Oba K. Ishikawa S. Nishikawa M. Mizuno H. Yamamoto T. Purification and properties of l-galactono-γ-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots.J. Biochem. 1995; 117: 120-124Crossref PubMed Scopus (132) Google Scholar, 15Ostergaard J. Persiau G. Davey M.W. Bauw G. Van Montagu M. Isolation of a cDNA coding for l-galactono-γ-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants. Purification, characterization, cDNA cloning, and expression in yeast.J. Biol. Chem. 1997; 272: 30009-30016Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The primary GLDH translation product has a molecular mass of about 68 kDa but is processed to a mature protein of 56–58 kDa. Processing is based on removal of an N-terminal peptide of about 100 amino acids and probably takes place during transport of GLDH into mitochondria (15Ostergaard J. Persiau G. Davey M.W. Bauw G. Van Montagu M. Isolation of a cDNA coding for l-galactono-γ-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants. Purification, characterization, cDNA cloning, and expression in yeast.J. Biol. Chem. 1997; 272: 30009-30016Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar). GLDH is most active with l-galactono-1,4-lactone but also has some low l-gulono-1,4-lactone activity (14Oba K. Ishikawa S. Nishikawa M. Mizuno H. Yamamoto T. Purification and properties of l-galactono-γ-lactone dehydrogenase, a key enzyme for ascorbic acid biosynthesis, from sweet potato roots.J. Biochem. 1995; 117: 120-124Crossref PubMed Scopus (132) Google Scholar, 16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar). The enzyme needs noncovalently bound FAD as a co-factor. GLDH so far has not been crystallized but amino acid positions essential for regulation and activity were identified by the investigation of recombinant forms of the enzyme (16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar, 17Leferink N.G. Jose M.D. van den Berg W.A. van Berkel W.J. Functional assignment of Glu-386 and Arg-388 in the active site of l-galactono-γ-lactone dehydrogenase.FEBS Lett. 2009; 583: 3199-3203Crossref PubMed Scopus (17) Google Scholar, 18Leferink N.G. van Duijn E. Barendregt A. Heck A.J. van Berkel W.J. Galactonolactone dehydrogenase requires a redox-sensitive thiol for optimal production of vitamin C.Plant Physiol. 2009; 150: 596-605Crossref PubMed Scopus (35) Google Scholar, 19Leferink N.G. Fraaije M.W. Joosten H.J. Schaap P.J. Mattevi A. van Berkel W.J. Identification of a gatekeeper residue that prevents dehydrogenases from acting as oxidases.J. Biol. Chem. 2009; 284: 4392-4397Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). GLDH is localized in the inner mitochondrial membrane (12Bartoli C.G. Pastori G.M. Foyer C.H. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV.Plant Physiol. 2000; 123: 335-344Crossref PubMed Scopus (321) Google Scholar, 20Imai T. Karita S. Shiratori G. Hattori M. Nunome T. Oba K. Hirai M. l-Galactono-γ-lactone dehydrogenase from sweet potato. Purification and cDNA sequence analysis.Plant Cell Physiol. 1998; 39: 1350-1358Crossref PubMed Scopus (87) Google Scholar, 21Siendones E. Gonzalez-Reyes J.A. Santos-Ocana C. Navas P. Cordoba F. Biosynthesis of ascorbic acid in kidney bean. l-Galactono-γ-lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane.Plant Physiol. 1999; 120: 907-912Crossref PubMed Scopus (105) Google Scholar). Because the mature protein lacks membrane spanning segments (16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar) it most likely is peripherally attached to the inner mitochondrial membrane. If overexpressed in Escherichia coli, GLDH forms part of the soluble fraction of this bacterium (16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar). About a decade ago, it surprisingly was discovered that GLDH is attached to the mitochondrial NADH dehydrogenase complex (complex I) of the respiratory chain (22Heazlewood J.L. Howell K.A. Millar A.H. Mitochondrial complex I from Arabidopsis and rice. Orthologs of mammalian and fungal components coupled with plant-specific subunits.Biochim. Biophys. Acta. 2003; 1604: 159-169Crossref PubMed Scopus (160) Google Scholar). Complex I has a molecular mass of 1000 kDa and in plants includes at least 48 different subunits, several of which represent proteins specific for this enzyme complex in plants (23Klodmann J. Sunderhaus S. Nimtz M. Jänsch L. Braun H.P. Internal architecture of mitochondrial complex I from Arabidopsis thaliana.Plant Cell. 2010; 22: 797-810Crossref PubMed Scopus (153) Google Scholar, 24Klodmann J. Braun H.P. Proteomic approach to characterize mitochondrial complex I from plants.Phytochemistry. 2011; 72: 1071-1080Crossref PubMed Scopus (37) Google Scholar). Some of these extra subunits integrate side activities into this respiratory complex, e.g. carboanhydrase (CA) subunits, which were proposed to support CO2 transfer from mitochondria to chloroplasts in plant cells (25Braun H.P. Zabaleta E. Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants.Physiol. Plant. 2007; 129: 114-122Crossref Scopus (58) Google Scholar). However, GLDH was not found to be attached to the 1000-kDa holoenzyme, but only to a slightly smaller version of complex I, which is of comparatively low abundance (13Millar A.H. Mittova V. Kiddle G. Heazlewood J.L. Bartoli C.G. Theodoulou F.L. Foyer C.H. Control of ascorbate synthesis by respiration and its implications for stress responses.Plant Physiol. 2003; 133: 443-447Crossref PubMed Scopus (273) Google Scholar, 22Heazlewood J.L. Howell K.A. Millar A.H. Mitochondrial complex I from Arabidopsis and rice. Orthologs of mammalian and fungal components coupled with plant-specific subunits.Biochim. Biophys. Acta. 2003; 1604: 159-169Crossref PubMed Scopus (160) Google Scholar, 26Pineau B. Layoune O. Danon A. De Paepe R. l-Galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I.J. Biol. Chem. 2008; 283: 32500-32505Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This complex has a molecular mass of about 850 kDa and obviously lacks some of the subunits present in the main form of complex I. The identity of these subunits is not known so far. GLDH activity is inhibited in the presence of rotenone, an inhibitor of electron transfer within complex I, if pyruvate and malate are used as respiratory substrates (13Millar A.H. Mittova V. Kiddle G. Heazlewood J.L. Bartoli C.G. Theodoulou F.L. Foyer C.H. Control of ascorbate synthesis by respiration and its implications for stress responses.Plant Physiol. 2003; 133: 443-447Crossref PubMed Scopus (273) Google Scholar). It therefore was speculated that a subpopulation of complex I particles are important for GLDH regulation by monitoring the rate of NADH-driven electron flow through complex I (13Millar A.H. Mittova V. Kiddle G. Heazlewood J.L. Bartoli C.G. Theodoulou F.L. Foyer C.H. Control of ascorbate synthesis by respiration and its implications for stress responses.Plant Physiol. 2003; 133: 443-447Crossref PubMed Scopus (273) Google Scholar). Silencing of the gene encoding GLDH in tomato does not much affect ascorbate concentration, indicating that GLDH activity is not rate-limiting for ascorbate formation (27Alhagdow M. Mounet F. Gilbert L. Nunes-Nesi A. Garcia V. Just D. Petit J. Beauvoit B. Fernie A.R. Rothan C. Baldet P. Silencing of the mitochondrial ascorbate synthesizing enzyme l-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato.Plant Physiol. 2007; 145: 1408-1422Crossref PubMed Scopus (157) Google Scholar). However, silenced plants have a clearly retarded growth and produce smaller fruits. At the same time, the central metabolism of plant mitochondria is significantly changed. It is concluded that GLDH is important for other processes besides ascorbate formation. Recently, characterization of an Arabidopsis knock-out mutant lacking the gene encoding GLDH was found to have drastically reduced amounts of complex I (26Pineau B. Layoune O. Danon A. De Paepe R. l-Galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I.J. Biol. Chem. 2008; 283: 32500-32505Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In contrast, the amounts of the other protein complexes of the respiratory chain were not changed. It therefore is speculated that GLDH, besides its role in ascorbate formation, represents an assembly factor for complex I. Here we present a biochemical investigation on GLDH localization within plant mitochondria. Protein complexes of the inner mitochondrial membrane were carefully solubilized by the use of nonionic detergents and resolved protein complexes were separated by blue native PAGE. Using a newly developed in gel GLDH activity assay and immunoblotting, three distinct GLDH containing protein complexes of 850, 470, and 420 kDa were discovered. The 850-kDa complex represents the known smaller version of mitochondrial complex I. GLDH is shown to be attached to the membrane arm of the 850-kDa complex I. Subunits of the novel 470 and 420 kDa complexes were identified by mass spectrometry. Like the 850-kDa complex, they also include complex I subunits. We propose that GLDH has a more extended function in complex I assembly by specifically binding to several of its assembly intermediates. A. thaliana cells (var. Columbia-0) were cultivated as previously described (28Sunderhaus S. Dudkina N.V. Jänsch L. Klodmann J. Heinemeyer J. Perales M. Zabaleta E. Boekema E.J. Braun H.P. Carbonic anhydrase subunits form a matrix-exposed domain attached to the membrane arm of mitochondrial complex I in plants.J. Biol. Chem. 2006; 281: 6482-6488Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Isolation of mitochondria was performed according to Werhahn et al. (29Werhahn W. Niemeyer A. Jänsch L. Kruft V. Schmitz U.K. Braun H. Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20.Plant Physiol. 2001; 125: 943-954Crossref PubMed Scopus (156) Google Scholar). One-dimensional BN-PAGE and two-dimensional BN/SDS-PAGE was performed as previously described (30Heinemeyer J. Lewejohann D. Braun H.P. Blue native gel electrophoresis for the characterization of protein complexes in plants.Methods Mol. Biol. 2007; 355: 343-352PubMed Google Scholar). Two-dimensional BN/BN-PAGE was carried out as outlined in Sunderhaus et al. (31Sunderhaus S. Eubel H. Braun H.P. Two-dimensional blue native/blue native polyacrylamide gel electrophoresis for the characterization of mitochondrial protein complexes and supercomplexes.Methods Mol. Biol. 2007; 372: 315-324Crossref PubMed Scopus (24) Google Scholar). For the experiments of the current investigation, first dimension BN-PAGE was carried out in the presence of digitonin, second dimension BN-PAGE in the presence of Triton X-100. Proteins were visualized by Coomassie colloidal staining (32Neuhoff V. Arold N. Taube D. Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.Electrophoresis. 1988; 9: 255-262Crossref PubMed Scopus (2350) Google Scholar, 33Neuhoff V. Stamm R. Pardowitz I. Arold N. Ehrhardt W. Taube D. Essential problems in quantification of proteins following colloidal staining with Coomassie Brilliant Blue dyes in polyacrylamide gels, and their solution.Electrophoresis. 1990; 11: 101-117Crossref PubMed Scopus (199) Google Scholar). After separation on polyacrylamide gels proteins were blotted onto a nitrocellulose membrane using the Trans Blot Cell from Bio-Rad. The transfer of proteins was performed as described in Kruft et al. (34Kruft V. Eubel H. Jänsch L. Werhahn W. Braun H.P. Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.Plant Physiol. 2001; 127: 1694-1710Crossref PubMed Scopus (313) Google Scholar). Immunostainings were carried out using the VectaStain ABC Kit (Vector Laboratories, Burlingame, CA). The carbonic anhydrase antibody was provided by Eduardo Zabaleta (Mar del Plata University, Argentina). The GLDH antibody was purchased from Agrisera Antibodies (Vännäs, Sweden). In gel staining for NADH-ubiquinone-oxidoreductase was carried out as previously described (35Zerbetto E. Vergani L. Dabbeni-Sala F. Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.Electrophoresis. 1997; 18: 2059-2064Crossref PubMed Scopus (254) Google Scholar). In gel activity staining of GLDH was performed as follows. After half-completion of the electrophoretic run of a BN-PAGE the Coomassie-containing cathode buffer was replaced by a cathode buffer without Coomassie for dye reduction within the gel. The gel was incubated in 100 ml of GLDH staining solution (40 mm Tris(hydroxymethyl)aminomethane, 2 mm l-galactono-1,4-lactone, 1 mg/ml of nitro blue tetrazolium chloride, 200 μm phenazine methosulfate) in the dark. The pH of the solution was adjusted to 8.8 (HCl). GLDH activity becomes visible as purple bands or spots after 15–30 min. The activity staining was stopped by rinsing the gel with water. To improve visualization and destain the background the gel was transferred into destaining solution (40% methanol and 10% acetic acid) overnight. The resulting gels were finally scanned on a transmission scanner (PowerLook III, UMAX). Tryptic digestion of proteins and MS were performed as described previously (23Klodmann J. Sunderhaus S. Nimtz M. Jänsch L. Braun H.P. Internal architecture of mitochondrial complex I from Arabidopsis thaliana.Plant Cell. 2010; 22: 797-810Crossref PubMed Scopus (153) Google Scholar). Protein identifications were based on the MASCOT search algorithm using the A. thaliana protein data base, release TAIR10 (www.arabidopsis.org). Immunoblotting experiments were carried out to first get information on GLDH localization in plant mitochondria. For this approach, mitochondria were isolated from a suspension cell culture of A. thaliana. Isolated organelles were solubilized by 5% digitonin and protein complexes were subsequently separated by two-dimensional blue native (BN)/SDS-PAGE (Fig. 1). Upon Coomassie staining, subunits of the mitochondrial protein complexes are visible as reported before (36Klodmann J. Senkler M. Rode C. Braun H.P. Defining the protein complex proteome of plant mitochondria.Plant Physiol. 2011; 157: 587-598Crossref PubMed Scopus (145) Google Scholar). The main form of complex I runs at about 1000 kDa. In addition, a slightly smaller version of complex I is visible in accordance with previous investigations (13Millar A.H. Mittova V. Kiddle G. Heazlewood J.L. Bartoli C.G. Theodoulou F.L. Foyer C.H. Control of ascorbate synthesis by respiration and its implications for stress responses.Plant Physiol. 2003; 133: 443-447Crossref PubMed Scopus (273) Google Scholar, 22Heazlewood J.L. Howell K.A. Millar A.H. Mitochondrial complex I from Arabidopsis and rice. Orthologs of mammalian and fungal components coupled with plant-specific subunits.Biochim. Biophys. Acta. 2003; 1604: 159-169Crossref PubMed Scopus (160) Google Scholar, 26Pineau B. Layoune O. Danon A. De Paepe R. l-Galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I.J. Biol. Chem. 2008; 283: 32500-32505Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). On our gels, it runs at 850 kDa on the first gel dimension and is designated complex I*. It also includes an additional 58-kDa subunit not present in the main form of complex I. This protein represents GLDH as shown by a parallel immunoblotting experiment. Furthermore, the 58-kDa immune signal is detectable at two further regions on the two-dimensional gels, which correspond to 470 and 420 kDa on the native gel dimension. Finally, the 58-kDa GLDH signal is visible in the <100 kDa region of the native gel dimension. This signal represents the monomeric form of GLDH, which was reported previously (36Klodmann J. Senkler M. Rode C. Braun H.P. Defining the protein complex proteome of plant mitochondria.Plant Physiol. 2011; 157: 587-598Crossref PubMed Scopus (145) Google Scholar); see the two-dimensional BN/SDS-PAGE GelMap of Arabidopsis mitochondria (www.gelmap.de/47, spot 116). We conclude that GLDH not only forms part of the 850-kDa complex I*, but additionally of two unknown protein complexes of 470 and 420 kDa. An in gel activity assay was developed to investigate if the 850-, 470-, and 420-kDa complexes have GLDH activity. The assay includes 2 mm l-galactono-1,4-lactone to avoid substrate inhibition, which was reported to take place at higher concentrations (16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar). Oxidized cytochrome c was substituted by the electron acceptor phenazine methosulfate, which increases sensitivity of the assay. The pH of the assay solution was adjusted to 8.8 in accordance with the pH optimum of GLDH reported previously (16Leferink N.G. van den Berg W.A. van Berkel W.J. l-Galactono-γ-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.FEBS J. 2008; 275: 713-726Crossref PubMed Scopus (65) Google Scholar). Finally, nitro blue tetrazolium was used for GLDH activity visualization. This redox dye is yellow in its oxidized form and purple upon reduction into formazan (37Eadie M.J. Tyrer J.H. Kukums J.R. Hooper W.D. Aspects of tetrazolium salt reduction relevant to quantitative histochemistry.Histochemie. 1970; 21: 170-180Crossref PubMed Scopus (50) Google Scholar). Reduction takes place in the presence of ascorbate. Additionally, reduced phenazine methosulfate can directly reduce nitro blue tetrazolium, which may enhance the ascorbate-mediated color reaction. Nitro blue tetrazolium itself cannot be reduced directly by the GLDH. Using this assay, GLDH activity becomes visible on native protein gels as purple bands or spots. The principle of the assay is summarized in Fig. 2A and the details are given under “Experimental Procedures.” For performing the GLDH in gel activity assay, mitochondria were solubilized by digitonin and protein complexes were subsequently separated by one-dimensional BN PAGE (Fig. 2B). The 850-, 470-, and 420-kDa complexes exhibit strong GLDH activity. No activity is detectable in the absence of GL. The activity-stained bands exactly correspond to the signals obtained by immunoblotting (Fig. 2B). The bands at 850, 470, and 420 kDa are not visible on a parallel Coomassie-stained gel indicating that the in gel GLDH activity assay has very high sensitivity. For further investigation of the 470- and 420-kDa complexes, a mitochondrial protein fraction was resolved by two-dimensional BN/BN PAGE (Fig. 3). First dimension BN PAGE was carried out in the presence of digitonin, second dimension BN PAGE in the presence of Triton X-100. On the resulting two-dimensional gels, most protein complexes are positioned on a diagonal line, but resolution is increased in comparison to one-dimensional BN PAGE due to differential effects of the two detergents on the individual protein complexes. Visualization of 850-, 470-, and 420-kDa complexes was carried out by in gel GLDH activity staining (Fig. 3). Both, the 470- and 420-kDa complexes again were not visible on a parallel Coomassie-stained BN/BN gel due to their low concentration (supplemental Fig. S3). The 470-kDa complex runs close to dimeric complex III (500 kDa). Although present at extremely low concentrations, gel spots representing the 470- and 420-kDa complexes were further analyzed by mass spectrometry. As expected, both protein complexes include GLDH (Table 1). Furthermore, and as expected, MS analysis of the 470-kDa complex revealed identification of complex III subunits (supplemental Table S2). This complex is of very high abundance and migrates in very close proximity to the 470-kDa complex on the BN/BN gel. However, the GLDH activity stain clearly is not at the position of complex III (Fig. 3). Strikingly, the 470-kDa complex additionally includes the CA2, CAL2, and Grim-19 subunits, which form part of the membrane arm of complex I in plants. Similarly, MS analysis of the 420-kDa complex revealed, besides GLDH, several subunits of the membrane arm of complex I: CA2, CA3, CAL2, and NAD2 (Table 1). Further complex I subunits were not identified, probably due to the low abundance of the 470- and 420-kDa complexes and due to the fact that the membrane arm of complex I mainly includes very hydrophobic subunits, which are difficult to detect by MS. Besides, some proteins of the HSP60 and malic enzyme complexes and the F1-part of ATP synthase were identified in the spot representing the 420-kDa complex (supplemental Table S2). However, as in the case of the 470-kDa complex, identification of these subunits rather reflects spot overlappings on our BN/BN gel than physical association of these proteins with GLDH, because these complexes run in very close proximity to the 420-kDa complex and are of very high abundance.FIGURE 1Immunological detection of l-galactono-1,4-lactone dehydrogenase in a mitochondrial protein fraction of A. thaliana. Proteins were separated by BN/SDS-PAGE and either stained by Coomas" @default.
• W2015454276 created "2016-06-24" @default.
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• W2015454276 date "2012-04-01" @default.
• W2015454276 modified "2023-10-15" @default.
• W2015454276 title "l-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana" @default.
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