title: A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei.
abstract: Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.
authors.affiliation: Laboratoire de Génomique Fonctionnelle des Trypanosomatides, UMR-5162 CNRS, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Coustou
authors.forename: Sébastien
authors.initials: S
authors.lastname: Besteiro
authors.forename: Loïc
authors.initials: L
authors.lastname: Rivière
authors.forename: Marc
authors.initials: M
authors.lastname: Biran
authors.forename: Nicolas
authors.initials: N
authors.lastname: Biteau
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Franconi
authors.forename: Michael
authors.initials: M
authors.lastname: Boshart
authors.forename: Théo
authors.initials: T
authors.lastname: Baltz
authors.forename: Frédéric
authors.initials: F
authors.lastname: Bringaud
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008830:Microbodies / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000737:chemistry*; D010084:Oxidation-Reduction; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013385:Succinate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D013386:Succinates / Q000737:chemistry*; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D003470:Culture Media; C501298:FRDm1 protein, Trypanosoma brucei; D020033:Protein Isoforms; D015800:Protozoan Proteins; D013386:Succinates; D009243:NAD; D049452:Green Fluorescent Proteins; D011392:Proline; D013385:Succinate Dehydrogenase; D005947:Glucose; D004072:Digitonin