NADH oxidation drives respiratory Na + transport in mitochondria from Yarrowia lipolytica
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ORIGINAL PAPER
NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica Po-Chi Lin Æ Andrea Puhar Æ Julia Steuber
Received: 25 September 2007 / Revised: 29 April 2008 / Accepted: 26 May 2008 / Published online: 13 June 2008 Ó Springer-Verlag 2008
Abstract It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophoreresistant Na+ uptake, indicating that a redox-driven, primary Na+ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 lmol min-1 mg-1) to Na+ translocation (2.0 lmol min-1 mg-1). NADH-driven Na+ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na+ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na+ gradient established by complex I.
Communicated by Ercko Stackebrandt.
Electronic supplementary material The online version of this article (doi:10.1007/s00203-008-0395-1) contains supplementary material, which is available to authorized users. P.-C. Lin A. Puhar J. Steuber (&) Biochemisches Institut, Universita¨t Zu¨rich, 8057 Zurich, Switzerland e-mail: [email protected] Present Address: A. Puhar Unite´ de Pathoge´nie Microbienne Mole´culaire, Institut Pasteur, 75724 Paris Cedex 15, France
Keywords Na+ transport NADH dehydrogenase Respiration Mitochondria Abbreviations Smp Submitochondrial particle Q Ubiquinone
Introduction Mitochondria are vital for the synthesis of ATP, the regulation of programmed cell death, and the modulation of intracellular Ca2+ concentration. These processes are governed by mitochondrial respiration which creates a proton motive force (Dp) consisting of DW, the transmembrane voltage, and DpH, the proton concentration gradient (Mitchell 1961). Respiring mitochondria maintain a Dp of 170 mV which essentially consists of DW as DpH contributes less than 10 mV (Murphy and Brand 1987). Three electron transfer reactions from NADH to quinone, quinole to ferricytochrome c, and ferrocytochrome c to O2 provide the driving force for the generation of Dp by the respiratory complexes I, III and IV, respectively. The stoichiometries of translocated protons per transferred electrons in each of these respiratory segments were determined with intact mitochondria and inside-out vesicles of mitochondrial membranes, or submitochondrial particles (SMPs). H+/estoichiometries were also studied with the purified complexes reconstituted into artificial membrane systems. By this approach, consistent ratios for the mitochondrial comple
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