Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50%

ABSTRACT In Saccharomyces cerevisiae cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zerotrans-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.

[1]  F. Azam,et al.  Glucose‐6‐phosphate as regulator of monosaccharide transport in baker's yeast , 1969, FEBS letters.

[2]  M. Walsh,et al.  Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose , 1994, Journal of bacteriology.

[3]  P. Postma,et al.  High‐affinity glucose uptake in Saccharomyces cerevisiae is not dependent on the presence of glucose‐phosphorylating enzymes , 1996, Yeast.

[4]  F. Opperdoes,et al.  Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux. , 1991, The Journal of biological chemistry.

[5]  J. Bailey,et al.  Fermentation pathway kinetics and metabolic flux control in suspended and immobilized Saccharomyces cerevisiae , 1990 .

[6]  E. Boles,et al.  Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. , 1997, European journal of biochemistry.

[7]  J. Thevelein,et al.  Evidence for trehalose‐6‐phosphate‐dependent and ‐independent mechanisms in the control of sugar influx into yeast glycolysis , 1996, Molecular microbiology.

[8]  R. Lagunas,et al.  The low-affinity component of Saccharomyces cerevisiae maltose transport is an artifact , 1992, Journal of bacteriology.

[9]  H V Westerhoff,et al.  Sustained oscillations in free‐energy state and hexose phosphates in yeast , 1996, Yeast.

[10]  Carlos Gancedo,et al.  Trehalose‐6‐phosphate, a new regulator of yeast glycolysis that inhibits hexokinases , 1993, FEBS letters.

[11]  J. van Steveninck,et al.  The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae. , 1988, Biochimica et biophysica acta.

[12]  A. Kruckeberg,et al.  The hexose transporter family of Saccharomyces cerevisiae , 1996, Archives of Microbiology.

[13]  V. P. Cirillo,et al.  Glucose transport in a kinaseless Saccharomyces cerevisiae mutant , 1987, Journal of bacteriology.

[14]  K. van Dam,et al.  A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH. , 1992, Analytical biochemistry.

[15]  M. Carlson,et al.  The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. François,et al.  On the mechanism by which a heat shock induces trehalose accumulation in Saccharomyces cerevisiae. , 1992, The Biochemical journal.

[17]  A. Kotyk [34] Kinetic studies of transprot in yeast , 1989 .

[18]  F. Gamo,et al.  The low‐affinity component of the glucose transport system in Saccharomyces cerevisiae is not due to passive diffusion , 1995, Folia microbiologica.

[19]  P. Postma,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. , 1985, Microbiological reviews.

[20]  D. Fraenkel,et al.  Functional studies of yeast glucokinase , 1993, Journal of bacteriology.

[21]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[22]  J. D. de Winde,et al.  Differential requirement of the yeast sugar kinases for sugar sensing in establishing the catabolite-repressed state. , 1996, European journal of biochemistry.

[23]  E. Querfurth,et al.  In vivo investigations of glucose transport in Saccharomyces cerevisiae , 2000, Biotechnology and bioengineering.

[24]  A. Betz,et al.  Membrane transport as controlling pacemaker of glycolysis in Saccharomyces carlsbergensis. , 1972, Biochimica et biophysica acta.

[25]  H. Kacser,et al.  The control of flux. , 1995, Biochemical Society transactions.

[26]  R. Lagunas Sugar transport in Saccharomyces cerevisiae. , 1993, FEMS microbiology reviews.

[27]  L. Gustafsson,et al.  The extent to which the glycolytic flux in Saccharomyces cerevisiae is controlled by the glucose transport system varies with the extracellular glucose concentration , 1996 .

[28]  Hans V. Westerhoff,et al.  Control of the glycolytic flux in Trypanosoma brucei: why control can shift suddenly , 1996 .

[29]  M. Walsh,et al.  Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells, but not the activity of glucose transport , 1994, Yeast.

[30]  L. Bisson,et al.  Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Kruckeberg,et al.  Yeast sugar transporters. , 1993, Critical reviews in biochemistry and molecular biology.

[32]  A. H. Rose Energy-Yielding Metabolism , 1968 .

[33]  S. Wölfl,et al.  Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Steveninck The mechanism of transmembrane glucose transport in yeast: Evidence for phosphorylation, associated with transport☆ , 1969 .

[35]  A. Kotyk Mobility of the free and of the loaded monosaccharide carrier in Saccharomyces cerevisiae. , 1967, Biochimica et biophysica acta.

[36]  G R Jacobson,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. , 1993, Microbiological reviews.

[37]  M. Walsh,et al.  Glucose sensing and signalling properties in Saccharomyces cerevisiae require the presence of at least two members of the glucose transporter family , 1996, Journal of bacteriology.

[38]  J. Thevelein,et al.  Trehalose synthase: guard to the gate of glycolysis in yeast? , 1995, Trends in biochemical sciences.

[39]  K. Entian,et al.  Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII. , 1991, European journal of biochemistry.

[40]  L. Bisson,et al.  The C‐terminal Domain of Snf3p is Sufficient to Complement the Growth Defect of snf3 Null Mutations in Saccharomyces cerevisiae: SNF3 Functions in Glucose Recognition , 1997, Yeast.

[41]  R. Boulton,et al.  Computer-assisted nonlinear regression analysis of the multicomponent glucose uptake kinetics of Saccharomyces cerevisiae , 1995, Journal of bacteriology.

[42]  T. Fukasawa,et al.  Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Wilfred D. Stein,et al.  Transport and Diffusion Across Cell Membranes , 1986 .

[44]  M. Ciriacy,et al.  Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic flux , 1995, Molecular microbiology.

[45]  J. van Steveninck The mechanism of transmembrane glucose transport in yeast: evidence for phosphorylation, associated with transport. , 1969, Archives of biochemistry and biophysics.