Tre~lo~6-phosphate (P) ~mpetitively inhibited the hexokinases from ~~cc~omyee~ cerevisiae. The strongest inhibition was observed upon hexokinase II, with a K, of 40 FM, while in the case of hexoklnase I the K, was 200 NM. Glucokinase was not inhibited by trehalose-6-P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose-6-P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia iipolytica, was particularly sensitive to the inhibition by trehalose-6-P: when assayed with 2 mM fructose an apparent K, of 5 PM was calculated. Two 5. cerevisiae mutants with abnormal levels of trehalose-6-P exhibited defects in glucose metabolism. It is concluded that trehalose-6-P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.Trehalose-6-phosphate; Hexokinase; Glycolysis; Yeast PRODUCTIONControl of the glycolytic flux in Succhurowryces cerevisicze has been considered to occur mainly at the level of phosphofructokinase and pyruvate kinase. Phosphofructokinase is regulated through activation by fructose-2,6-bisphosphate, phosphate (P) and AMP, and inhibition by ATP, while pyruvate kinase activity is modulated by its activation by fructose-l,(i-bisphosphate (for a review see [l]). However, since phosphof~~tokinase does not catalyze the first irreversible step in the utilization of glucose, its regulation is not sufficient to control the rate of glucose utilization, and thus some mechanism should exist to regulate the rate of glucose transport, phosphorylation, or both. Regulation of glucose transport by glucose-6-P was suggested by Sols [2], but experimental results obtained using mutants affected in phosphoglucose isomerase activity did not support this idea [3,4]. Besides, none of the yeast kinases that phosphorylate glucose are sensitive to glucose-6-P, in contrast with the mammalian hexokinase PI-The need for a regulation of the first steps of yeast glycolysis is illustrated by the pattern of ambulation of metabolites in certain yeast mutants upon addition of glucose [6-81. Yeast strains carrying the mutations fdpl [6] or cifl [7] do not grow on glucose, although the glycolytic enzymes are operative. These mutations turned out to be allelic [9], and strains bearing them become depleted of ATP upon addition of glucose and accumulate fructose-l ,6-bisphosphate up to 20 mM [6,7,10], suggesting that the rate of the first glycolytic steps exceeds the capacity of the glycolytic pathway. The sequence of the CIFZ gene [lo] encodes the small subunit of the trehalose-6-P synthase/trehalose-6-P phosphatase complex [ 11,123. A plausible explanation for the growth behaviour and the metabolic defects of cifl strains could be that either trehalose or trehalose-6-P play a role in the regulation of the yeast glycolytic flux. We show in this article that trehalose-6-P inhibits sugar phospho~lation.The stron~st inh...

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The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.Sugar transporters are integral plasma membrane proteins that catalyze the first rate-limiting step of glycolysis in Saccharomyces cerevisiae (11). Several strategies are used by this organism to adjust the activities of these proteins to different environmental conditions (22). One of these strategies is the irreversible inactivation of the transporters, which occurs when protein synthesis is impaired upon exhaustion of a nitrogen source in the medium (3-5, 9, 23, 27) and which results in dramatic physiological effects (20,21,23). This inactivation, known as catabolite inactivation (17), affects mainly the V max of the transporters, follows first-order kinetics, and is an energy-dependent process stimulated by fermentable substrates (3,4,9). By using polyclonal antibodies against a recombinant maltose transporter protein, it has been shown that catabolite inactivation is due to proteolysis (26). The experiments reported here attempt to establish the location and mechanism of the maltose transporter degradation. We investigated the inactivation of the maltose transporter by measuring the rate of maltose uptake with radioactive sugar as well as by determining the cellular content of the transporter with polyclonal antibodies. Possible loci investigated are the plasma membrane, which is the locus of transporter action, the cytoplasm, and the vacuole after internalization of the transporter by endocytosis. In this study, we used strains defective in the internalization step of endocytosis as well as strains that show a defect either in the ''chymotrypsin-like'' activity of the proteasome complex or in the two main vacuolar endopeptidases.