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, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, 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 supramolecular 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.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylasenegative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1␣ subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malatedependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.Pyruvate is a key intermediate in sugar metabolism. Three major pathways of pyruvate metabolism in the yeast Saccharomyces cerevisiae have been described (for a review, see Ref. 1) (Fig. 1). During fermentative growth, pyruvate is decarboxylated into acetaldehyde by pyruvate decarboxylase, which is, in its turn, reduced into ethanol in the cytosol by ADH1, one of the four known alcohol dehydrogenase isoenzymes (2, 3). This sequence of reactions allows the reoxidation of NADH, which is produced at the level of the glyceraldehyde-3-phosphate dehydrogenase. During respiratory metabolism, pyruvate can enter the mitochondria by a specific carrier (4, 5) and is decarboxylated and oxidized into acetyl-CoA by pyruvate dehydrogenase, a multienzyme complex located in the matrix (6). In addition, a pyruvate dehydrogenase bypass located in the cytosol converts pyruvate into acetyl-CoA by the action of the following enzymes: pyruvate decarboxylase (7), cytosolic acetaldehyde dehydrogenase (8, 9), and acetyl-CoA synthetases (10, 11). AcetylCoA synthesized in the cytosol is either directly used for the biosynthetic pathways or enters the mitochondria via the carnitine acetyltransferase system (12, 13). It has been proposed that this system works unidirectionally; i.e. acetyl-CoA can only move from the outside into inside (13). In contrast, direct oxidative decarboxylation of pyruvate into acetyl-CoA by the pyruvate dehydrogenase complex does not require ATP hydrolysis, since the energy required for the thioester formation is provided by oxidation of the carbonyl into carboxyl groups (Fig. 1). It is generally assumed that in wild-type S. cerevisiae grown under glucose limitation, the pyruvate dehydrogenase complex is primarily responsible for pyruvate c...

When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.