Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake.
In the yeast Saccharomyces cerevisiae the GGS1 gene is essential for growth on glucose or other readily fermentable sugars. GGS1 is the same gene as TPS1 which was identified as encoding a subunit of the trehalose-6-phosphate synthase/phosphatase complex and it is allelic to the fdp1, byp1, glc6 and cif1 mutations. Its precise function in the regulation of sugar catabolism is unknown. We have cloned the GGS1 homologue from the distantly related yeast Kluyveromyces lactis. The KlGGS1 gene is 74% and 79% identical at the nucleotide and amino acid sequence level, respectively, to the S. cerevisiae counterpart. We also compared the sequence with the partly homologous products of the S. cerevisiae genes TPS2 and TSL1 which code for the larger subunits of the trehalose synthase complex and with a TSL1 homologue, TPS3, of unknown function. Multiple alignment of these sequences revealed several particularly well conserved elements. Disruption of GGS1 in K. lactis caused the same pleiotropic phenotype as in S. cerevisiae, i.e. inability to grow on glucose or fructose and strongly reduced trehalose content. We have also studied short-term glucose-induced regulatory effects related to cAMP and cAMP-dependent protein kinase, i.e. the cAMP signal, trehalase activation, trehalose mobilization and inactivation of fructose-1,6-bisphosphatase. These effects occur very rapidly in S. cerevisiae and are absent in the Scggs1 mutant. In K. lactis all these effects were much slower and largely unaffected by the Klggs1 mutation. On the other hand, glucose strongly induced pyruvate decarboxylase and activated the potassium transport system in K. lactis and both effects were absent in the Klggs1 mutant. Addition of glucose to galactose-grown cells of the Klggs1 mutant caused, as in S. cerevisiae, intracellular accumulation of free glucose and of sugar phosphates and a rapid drop of the ATP and inorganic phosphate levels. Glucose transport kinetics were the same for the wild type and the Klggs1 mutant in both derepressed cells and in cells incubated with glucose. We have isolated phenotypic revertants of the Klggs1 mutant for growth on fructose. The suppressors that we characterized had, to different extents, diminished glucose uptake in derepressed cells but cells incubated in glucose showed very different characteristics. The suppressor mutations prevented deregulation of glycolysis in the Klggs1 mutant but not the accumulation of free glucose. The mutants with higher residual uptake activity showed partially restored induction of pyruvate decarboxylase and activation of potassium transport.(ABSTRACT TRUNCATED AT 400 WORDS)
Luyten
K
K
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Flanders, Belgium.
de Koning
W
W
Tesseur
I
I
Ruiz
M C
MC
Ramos
J
J
Cobbaert
P
P
Thevelein
J M
JM
Hohmann
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005632:Fructose / Q000378:metabolism*; D005800:Genes, Fungal; D016153:Genes, Suppressor; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics*; D006019:Glycolysis; D007716:Kluyveromyces / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D005632:Fructose; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; D005947:Glucose