Alternative NADH dehydrogenase (NDH2): intermembrane-space-facing counterpart of mitochondrial complex I in the procyclic Trypanosoma brucei

• Published in: Parasitology 2013-03, Vol.140 (3), p.328-337
• Main Authors: VERNER, ZDENĚK, ŠKODOVÁ, INGRID, POLÁKOVÁ, SIMONA, ĎURIŠOVÁ-BENKOVIČOVÁ, VLADISLAVA, HORVÁTH, ANTON, LUKEŠ, JULIUS
• Format: Article
• Language: English
• Subjects: Animals; Cytosol - enzymology; Dehydrogenase; Electron Transport; Electron Transport Complex I; Enzymatic activity; Intracellular Membranes - metabolism; Membrane Potentials; Mitochondria - enzymology; NAD - metabolism; NADH Dehydrogenase - genetics; NADH Dehydrogenase - metabolism; Oxidoreductases - genetics; Oxidoreductases - metabolism; Oxygen Consumption; Protozoan Proteins - metabolism; Respiration; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology; Trypanosoma brucei brucei - genetics; Trypanosoma brucei brucei - growth & development; Trypanosoma brucei brucei - physiology
• ISSN: 0031-1820; 1469-8161; 1469-8161
• DOI: 10.1017/S003118201200162X

The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.