scientific report (PTP1B) is a protein-tyrosine phosphatase predominantly localized on intracellular membranes by means of a hydrophobic carboxy-terminal targeting sequence (Frangioni et al., 1992). Evidence for the involvement of PTP1B in insulin action has been provided by studies showing that increased insulin sensitivity is associated with higher levels of tyrosine phosphorylation of the IR and one of its substrates, IR substrate 1 (IRS1), in the liver and skeletal muscle of insulintreated Ptp1B knockout mice (Elchebly et al., 1999). Because PTP1B could be a potential therapeutic target, a better understanding of the interaction between the IR and PTP1B is an important requirement for the development of compounds to improve insulin sensitivity. However, although PTP1B has been shown to interact physically with the IR (Bandyopadhyay et al., 1997), nothing is known about the dynamics of this interaction in living cells.Bioluminescence resonance energy transfer (BRET) allows the study of protein-protein interactions in intact living cells (Angers et al., 2000;Xu et al., 1999). To study the interaction between two proteins, one of them is fused to Renilla luciferase (Rluc) and the other to a yellow fluorescent protein (YFP). The luciferase is excited by a substrate (coelenterazine). If the two proteins are less than 100 Å apart, energy transfer occurs between the luciferase and the YFP, and a signal emitted by the YFP can be detected. We previously showed that this methodology can be used to monitor insulin-induced conformational The dynamics of the interaction of the insulin receptor with a substrate-trapping mutant of protein-tyrosine phosphatase 1B (PTP1B) were monitored in living human embryonic kidney cells using bioluminescence resonance energy transfer (BRET). Insulin dosedependently stimulates this interaction, which could be followed in real time for more than 30 minutes. The effect of insulin on the BRET signal could be detected at early time-points (30 seconds), suggesting that in intact cells the tyrosine-kinase activity of the insulin receptor is tightly controlled by PTP1B. Interestingly, the basal (insulin-independent) interaction of the insulin receptor with PTP1B was much weaker with a soluble form of the tyrosinephosphatase than with the endoplasmic reticulum (ER)-targeted form. Inhibition of insulin-receptor processing using tunicamycin suggests that the basal interaction occurs during insulin-receptor biosynthesis in the ER. Therefore, localization of PTP1B in this compartment might be important for the regulation of insulin receptors during their biosynthesis.

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...