During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

Authors

  • José Roberto Ernandes,

    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    Current affiliation:
    1. Department of Biochemistry, Institute of Chemistry, Sao Paulo State University, C.P. 355, 14800-900 Araraquara, SP, Brazil
    Search for more papers by this author
  • Catherine De Meirsman,

    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    Search for more papers by this author
  • Filip Rolland,

    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    Search for more papers by this author
  • Joris Winderickx,

    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    Search for more papers by this author
  • Johannes De Winde,

    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    Search for more papers by this author
  • Rogelio Lopes Brandão,

    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    Current affiliation:
    1. Laboratorio de Bioquimica e Fisiologia de Microrganismos, Escola de Farmacia, Universidade Federal de Ouro Preto, Rua Costa Sena 171, 35.400.00 Ouro Preto, MG, Brazil
    Search for more papers by this author
  • Johan M. Thevelein

    Corresponding author
    1. Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium
    • Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal, Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium. Tel: (+32) 16 321507; fax (+32) 16 321979
    Search for more papers by this author

Abstract

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 ([DOUBLE BOND]GGS1[DOUBLE BOND]FDP1[DOUBLE BOND]BYP1[DOUBLE BOND]CIF1[DOUBLE BOND]GLC6[DOUBLE BOND]TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d-glucose and an equal concentration of radiolabelled l-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d-glucose measured (intracellular+periplasmic/extracellular) and the total l-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm-glucose to 0·5–2 mm in the wild-type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2–3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.

Ancillary