d-Lactate Dehydrogenase Links Methylglyoxal Degradation and Electron Transport through Cytochrome c1

Mitochondrial d-lactate dehydrogenase links the last step of methylglyoxal detoxification to the mitochondrial electron transport chain through cytochrome c. Glycolysis generates methylglyoxal (MGO) as an unavoidable, cytotoxic by-product in plant cells. MGO scavenging is performed by the glyoxalase system, which produces d-lactate as an end product. d-Lactate dehydrogenase (d-LDH) is encoded by a single gene in Arabidopsis (Arabidopsis thaliana; At5g06580). It catalyzes in vitro the oxidation of d-lactate to pyruvate using flavin adenine dinucleotide as a cofactor; knowledge of its function in the context of the plant cell remains sketchy. Blue native-polyacrylamide gel electrophoresis of mitochondrial extracts combined with in gel activity assays using different substrates and tandem mass spectrometry allowed us to definitely show that d-LDH acts specifically on d-lactate, is active as a dimer, and does not associate with respiratory supercomplexes of the inner mitochondrial membrane. The combined use of cytochrome c (CYTc) loss-of-function mutants and respiratory complex III inhibitors showed that CYTc acts as the in vivo electron acceptor of d-LDH. CYTc loss-of-function mutants, as well as the d-LDH mutants, were more sensitive to d-lactate and MGO, indicating that they function in the same pathway. In addition, overexpression of d-LDH and CYTc increased tolerance to d-lactate and MGO. Together with fine-localization of d-LDH, the functional interaction with CYTc in vivo strongly suggests that d-lactate oxidation takes place in the mitochondrial intermembrane space, delivering electrons to the respiratory chain through CYTc. These results provide a comprehensive picture of the organization and function of d-LDH in the plant cell and exemplify how the plant mitochondrial respiratory chain can act as a multifunctional electron sink for reductant from cytosolic pathways.

[1]  D. Gonzalez,et al.  Cytochrome c, a hub linking energy, redox, stress and signaling pathways in mitochondria and other cell compartments. , 2016, Physiologia plantarum.

[2]  M. Fricker,et al.  The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis[OPEN] , 2015, Plant Cell.

[3]  M. Engqvist,et al.  2-Hydroxy Acids in Plant Metabolism , 2015, The arabidopsis book.

[4]  K. Tomii,et al.  MitoFates: Improved Prediction of Mitochondrial Targeting Sequences and Their Cleavage Sites , 2015, Molecular & Cellular Proteomics.

[5]  M. Fricker,et al.  The EF-Hand Ca 2 + Binding Protein MICU Choreographs Mitochondrial Ca 2 + Dynamics in Arabidopsis , 2015 .

[6]  H. Braun,et al.  Respiratory electron transfer pathways in plant mitochondria , 2014, Front. Plant Sci..

[7]  K. Gabriel,et al.  The ins and outs of the intermembrane space: diverse mechanisms and evolutionary rewiring of mitochondrial protein import routes. , 2014, Biochimica et biophysica acta.

[8]  Chankyu Park,et al.  Novel glyoxalases from Arabidopsis thaliana , 2013, The FEBS journal.

[9]  P. Westhoff,et al.  pAUL: A Gateway-Based Vector System for Adaptive Expression and Flexible Tagging of Proteins in Arabidopsis , 2013, PloS one.

[10]  J. Martinou,et al.  Intermembrane Space Proteome of Yeast Mitochondria* , 2012, Molecular & Cellular Proteomics.

[11]  Tatjana M. Hildebrandt,et al.  Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III. , 2012, Biochimica et biophysica acta.

[12]  Yoshimasa Nakamura,et al.  Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis. , 2012, Journal of plant physiology.

[13]  Hans-Peter Braun,et al.  Functional Annotation of 2D Protein Maps: The GelMap Portal , 2012, Front. Plant Sci..

[14]  U. Flügge,et al.  d‐Lactate dehydrogenase as a marker gene allows positive selection of transgenic plants , 2012, FEBS letters.

[15]  GelMap-a novel software tool for building and presenting proteome reference maps. , 2011, Journal of proteomics.

[16]  C. Miyake,et al.  Methylglyoxal functions as Hill oxidant and stimulates the photoreduction of O(2) at photosystem I: a symptom of plant diabetes. , 2011, Plant, cell & environment.

[17]  H. Braun,et al.  Defining the Protein Complex Proteome of Plant Mitochondria1[W] , 2011, Plant Physiology.

[18]  M. Fricker,et al.  The circularly permuted yellow fluorescent protein cpYFP that has been used as a superoxide probe is highly responsive to pH but not superoxide in mitochondria: implications for the existence of superoxide 'flashes'. , 2011, The Biochemical journal.

[19]  Chankyu Park,et al.  Hsp31 of Escherichia coli K‐12 is glyoxalase III , 2011, Molecular microbiology.

[20]  H. Braun,et al.  Internal Architecture of Mitochondrial Complex I from Arabidopsis thaliana , 2010, Plant Cell.

[21]  J. Thelen,et al.  The Plastid Isoform of Triose Phosphate Isomerase Is Required for the Postgerminative Transition from Heterotrophic to Autotrophic Growth in Arabidopsis[W] , 2010, Plant Cell.

[22]  M. Cristescu,et al.  Evolutionary History of d-Lactate Dehydrogenases: A Phylogenomic Perspective on Functional Diversity in the FAD Binding Oxidoreductase/Transferase Type 4 Family , 2009, Journal of Molecular Evolution.

[23]  U. Flügge,et al.  Two d-2-Hydroxy-acid Dehydrogenases in Arabidopsis thaliana with Catalytic Capacities to Participate in the Last Reactions of the Methylglyoxal and β-Oxidation Pathways* , 2009, The Journal of Biological Chemistry.

[24]  Federica Brandizzi,et al.  Non-invasive topology analysis of membrane proteins in the secretory pathway. , 2009, The Plant journal : for cell and molecular biology.

[25]  E. Boekema,et al.  Carbonic Anhydrase Subunits Form a Matrix-exposed Domain Attached to the Membrane Arm of Mitochondrial Complex I in Plants* , 2006, Journal of Biological Chemistry.

[26]  H. Schägger,et al.  Blue native PAGE , 2006, Nature Protocols.

[27]  F. Fiorani,et al.  Characterization of Transformed Arabidopsis with Altered Alternative Oxidase Levels and Analysis of Effects on Reactive Oxygen Species in Tissue1[W] , 2005, Plant Physiology.

[28]  M. Ray,et al.  Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione. , 2005, Biochemical and biophysical research communications.

[29]  P. Gardeström,et al.  Preparation of leaf mitochondria from Arabidopsis thaliana , 2005 .

[30]  D. Valenti,et al.  Transport and metabolism of D-lactate in Jerusalem artichoke mitochondria. , 2005, Biochimica et biophysica acta.

[31]  M. Iacovino,et al.  Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement. , 2004, Biochimica et biophysica acta.

[32]  T. Fujii,et al.  Electron Acquisition System Constructed from an NAD-Independent D-Lactate Dehydrogenase and Cytochrome c 2 in Rhodopseudomonas palustris No. 7 , 2004, Bioscience, biotechnology, and biochemistry.

[33]  P. Goffrini,et al.  Carbon catabolite repression in Kluyveromyces lactis: isolation and characterization of the KINLD gene encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase , 1994, Molecular and General Genetics MGG.

[34]  I. Ferrero,et al.  Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase , 1993, Molecular and General Genetics MGG.

[35]  Paul J Thornalley.  Glyoxalase I--structure, function and a critical role in the enzymatic defence against glycation. , 2003, Biochemical Society transactions.

[36]  D. Logan,et al.  Mitochondrial and Cytosolic Calcium Dynamics Are Differentially Regulated in Plants1 , 2003, Plant Physiology.

[37]  G. Principato,et al.  D-Lactate transport and metabolism in rat liver mitochondria. , 2002, The Biochemical journal.

[38]  H. Braun,et al.  Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20. , 2001, Plant physiology.

[39]  D. Logan,et al.  Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. , 2000, Journal of experimental botany.

[40]  M. Kalapos,et al.  Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. , 1999, Toxicology letters.

[41]  S. Ray,et al.  Glyoxalase III from Escherichia coli: a single novel enzyme for the conversion of methylglyoxal into D-lactate without reduced glutathione. , 1995, The Biochemical journal.

[42]  Paul J Thornalley,et al.  The formation of methylglyoxal from triose phosphates , 1993 .

[43]  Kay Hofmann,et al.  Tmbase-A database of membrane spanning protein segments , 1993 .

[44]  Paul J Thornalley,et al.  The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. , 1993, European journal of biochemistry.

[45]  H. Braun,et al.  Affinity purification of cytochrome c reductase from potato mitochondria. , 1992, European journal of biochemistry.

[46]  K. Kinnally,et al.  The effect of antimycin A on mouse liver inner mitochondrial membrane channel activity. , 1992, The Journal of biological chemistry.

[47]  B. Trumpower,et al.  An inhibitor of mitochondrial respiration which binds to cytochrome b and displaces quinone from the iron-sulfur protein of the cytochrome bc1 complex. , 1984, The Journal of biological chemistry.