73 Molecular and Cellular Biochemistry 256/257: 73–81, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae Michel Rigoulet, 1 Hugo Aguilaniu, 1,3 Nicole Avéret, 1 Odile Bunoust, 1 Nadine Camougrand, 1 Xavier Grandier-Vazeille, 1 Christer Larsson, 3 Inga-Lill Pahlman, 2 Stephen Manon 1 and Lena Gustafsson 2,3 1 Institut de Biochimie et Génétique Cellulaires, Université Victor Segalen-CNRS, Bordeaux cedex, France; 2 Department of Cellular and Molecular Biology-Microbiology, Lundberg Laboratory, Gothenburg University; 3 Department of Molecular Biotechnology, Lundberg Laboratory, Chalmers University of Technology, Gothenburg, Sweden Abstract Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, com- prising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpd1p, and the mitochondrial glycerol-3-phosphate dehydro- genase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supra- molecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed. (Mol Cell Biochem 256/257: 73–81, 2004) Key words: yeast, in situ mitochondria, NADH dehydrogenases, respiratory chain, channeling and kinetic regulation, supra- molecular complexes Synthesis of one mole of glycerol, the second major by-prod- uct of glucose fermenting cells of S. cerevisiae, results in consumption of one mole of NADH while other by-products like acetate lead to the production of cytosolic NADH. How- ever, the largest part of excess cytosolic NADH formation is connected to biomass production [3, 5]. Synthesis of proteins, nucleic acids and even the highly reduced lipids are associ- ated with the assimilatory NADH production. In particular, NADH is generated in the biosynthetic pathways of amino acid synthesis. Anaerobically, the only means by which S. cerevisiae can reoxidize surplus production of NADH is by glycerol production [2, 4, 6]. Aerobically, several systems for conveying excess cytosolic NADH to the mitochondrial elec- Address for offprints: M. Rigoulet, IBGC-CNRS, UMR 5095, 1 rue Camille Saint-Saëns, 33077, Bordeaux cedex, France (E-mail: michel.rigoulet@ibgc.u- bordeaux2.fr) Introduction The yeast Saccharomyces cerevisiae lacks transhydrogenase activity [1, 2] and the redox couples NAD + /NADH and NADP + / NADPH cannot pass the mitochondrial membrane. Hence, to maintain the redox balances, the reduced coenzymes must be reoxidized in the compartment in which they are produced. In contrast to NADPH turnover, which occurs essentially in the cytosol [3, 4], systems for NADH turnover in mitochon- dria as well as in the cytosol are required both during aero- bic and anaerobic conditions. The reason being that several processes result in production of NADH, i.e. several proc- esses are contrary to ethanol fermentation not redox neutral.