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In the yeast Saccharomyces cerevisiae, TPS1-encoded trehalose-6-phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6-phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild-type, tps1Δ and tps2Δ (Tre6P phosphatase) strains. For the tps1Δ strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Δ strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Δ spheroplasts to those of the wild-type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Δ strain compared to a wild-type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1-mediated protein–protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also Tps2 appears to play a role in the control of hexokinase activity.
In the yeast Saccharomyces cerevisiae, four genes encode the trehalose-6-phosphate synthase (TPS) complex. TPS1 encodes TPS [1–4], TPS2 encodes trehalose-6-phosphate phosphatase  and TSL1 and TPS3 redundantly encode a regulatory subunit [4,6,7]. Several mutants unable to grow on glucose were found to be allelic with TPS1. The fdp1, cif1 and byp1 mutants were originally isolated as completely or partially glucose-negative mutants [8–13]. The inability to grow on glucose, or related rapidly fermented sugars, was shown to be due to a severe deregulation of glycolysis, as evidenced by hyperaccumulation of Glu6P, Fru6P and Fru(1,6)P2, and depletion of ATP, Pi and all intermediates of glycolysis downstream of glyceraldehyde-3-phosphate dehydrogenase. These mutants were also deficient in a wide range of glucose-induced regulatory effects at the post-translational and transcriptional level and in other seemingly unrelated properties such as sporulation capacity and glycogen accumulation [3,8,9,14]. Deficient trehalose accumulation was also identified as one of the phenotypic properties [3,13,15]. However, in these glucose-negative mutants, it was not recognized as a primary cause of the inability to grow on glucose, because in itself this would be rather unexpected, and also because other mutants had already been identified, such as those with elevated activity of the cAMP–protein kinase A pathway, that lacked trehalose accumulation, but grew normally on glucose [16,17].
The identification of the TPS1 gene as being identical to the CIF1, FDP1, BYP1 and also GLC6 genes, unexpectedly raised the novel issue of how mutations in a seemingly ordinary house-keeping enzyme like TPS could cause such a pleiotropic phenotype. A first hint was given by the demonstration that deletion of HXK2 restored both growth of the tps1Δ mutant on glucose and the glucose-induced regulatory effects . This appeared to indicate that the deficiency in glucose-induced signalling was a side-effect of the deregulation of glycolysis. Moreover, it also indicated that the essential control function exerted by Tps1 might be restriction of hexokinase activity. Stimulation of glycerol production by overexpression of the Fps1 glycerol facilitator or GPD1 encoded glycerol-3-phosphate dehydrogenase suppressed the growth defect of the tps1 mutants on glucose-containing medium [15,20]. Subsequently, it was discovered that trehalose 6-phosphate (Tre6P) inhibits hexokinase activity in vitro in a competitive manner with glucose. The Tre6P level in vivo appeared to be in the same range as the Tre6P level required for inhibition of hexokinase activity in vitro.
As a consequence of these findings different models have been proposed to explain the connection between Tps1 and the restriction of glucose influx into glycolysis. These models have been discussed in detail previously . A central question is whether Tre6P is the sole mediator of Tps1 inhibition of hexokinase or whether the Tps1 protein itself also plays a regulatory role in this process. Recently, it has been demonstrated that yeast cell extracts also contain Tps1, which does not form part of the trehalose synthase complex, and that this form displays a different regulation . In a tps2Δ mutant, which has a constitutively elevated level of Tre6P, a clear reduction in sugar phosphate accumulation after addition of glucose was observed . This appears to support the Tre6P inhibition model, although deletion of TPS2 might also shift the equilibrium distribution between different pools of Tps1. A second important issue is the competitive character of Tre6P inhibition of hexokinase in vitro with respect to glucose. If this would also be true in vivo any elevation of the free glucose level in the cell would tend to cause deregulation of glycolysis by lifting the inhibition on hexokinase. Other important issues are why strong overexpression of hexokinase does not cause the tps1 phenotype  and why deletion of TPS2 in a strain (byp1) with reduced level of Tps1 restores growth and fermentation with glucose but not proper control of glucose influx into glycolysis .
The Tps1 control stands out among the many other control mechanisms proposed for yeast glycolysis, e.g. activation by Fru(2,6)P2, allosteric controls on fructose-1,6-bisphosphatase  and on phosphofructokinase , futile cycles and ATP inhibition . It is the only control for which it is clear that elimination causes a dramatic deregulation of glycolysis. However, whether the role of this control is restricted to glycolytic metabolite homeostasis or whether it also plays a role in overall flux control is unclear.
Tre6P-sensitive hexokinases have also been found in other fungi, but deletion of Tre6P synthase activity does not always reside in a clear glucose-negative phenotype as in S. cerevisiae[30–34]. It has not been possible to relate the sensitivity of hexokinase for Tre6P in the different fungi to the phenotype of the Tre6P synthase deletion mutant for growth on glucose . Hence, also in other fungi, the relevance and the precise mechanism by which TPS activity might control hexokinase is unclear.
In this paper we focus on three specific questions concerning the Tps1 control on glycolysis. (a) Is Tre6P the sole mediator of hexokinase inhibition? (b) Is there more than one action point of Tps1 in control of yeast glycolysis? (c) Is the inhibition of hexokinase by Tre6P also competitive with respect to glucose in vivo? To answer the first two questions we have reconstituted ethanolic fermentation in permeabilized spheroplasts of wild-type, tps1Δ and tps2Δ strains of S. cerevisiae with glucose and different intermediates of glycolysis as substrates. The permeabilized spheroplasts were prepared using a very gentle and strictly controlled permeabilization protocol which only affects the plasma membrane and not the mitochondrial membranes . To answer the third question we have investigated the effect of Tre6P on glycolysis and hexokinase activity in the permeabilized spheroplasts with an excess of glucose as substrate.