Trehalose-6-phosphate synthase/phosphatase complex from bakers’ yeast: purification of a proteolytically activated form

Londesborough, John; Vuorio, Outi E.

doi:10.1099/00221287-137-2-323

Cited by 79 publications

(49 citation statements)

References 24 publications

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“…Gel filtration experiments showed that the molecular mass of the complex is around 630-800 kDa (Londesborough and Vuorio, 1991;Bell et al, 1992). As the sum of the molecular masses of the four putative subunits only adds up to Ϸ400 kDa, either some or all of the subunits must exist in more than one copy in the complex.…”

Section: Discussionmentioning

confidence: 99%

Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Reinders

Bürckert

Hohmann

et al. 1997

Molecular Microbiology

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SummarySynthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while Tsl1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, Tsl1, and Tps3, a protein with high homology to Tsl1, revealed that both Tsl1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that Tsl1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/ phosphatase complex in exponentially growing, heatshocked cells.

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Section: Discussionmentioning

confidence: 99%

Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Reinders

Bürckert

Hohmann

et al. 1997

Molecular Microbiology

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“…TCT AAG GGC AAT ATC GTT TAC ATC ATG AAC TCA TTT CCA AAG CCC ATT CTG GAA AAT CTT TAC AGT CGT GTG CAA seemed to result from the adventitious truncation of the 123-kDa polypeptide during the purification (Londesborough and Vuorio, 1991), we attempted to convert the intact enzyme into the truncated form by treatment with trypsin. Fig.…”

Section: Truncation Of Intact Trehalose Synthase With Trypsin Causingmentioning

confidence: 99%

“…The 56-kDa, 86-kDa and 93-kDa polypeptides of truncated trehalose synthase were separated by SDSPAGE, digestejd on nitrocellulose blots and fractionated by HPLC as described in Londesborough and Vuorio (1991). These polypeptides and polypeptides of molecular mass 56, 102 and 123 kDa, immunoprecipitated with anti-(trehalose synthase) serum from high-speed supernatants of yeast extracts, were also separated by SDSPAGE and digested in the gel with lysylendopeptidase C. The derived peptides were separated by HPLC using a DEAE-cellulose pre-column before the reverse-phase column essentially as described by Kawasaki and Suzuki (1990).…”

Section: Sequencing Of Peptidesmentioning

confidence: 99%

“…Tre6P synthase and Tre6Pase activities were assayed as described by Londesborough and Vuorio (1991) except that the standard Tre6P synthase assay mixture contained 5 mM fructose 6-phosphate (Fru6P). Protein was estimated according to Warburg and Christian (1941).…”

Section: Enzyme and Protein Assays And Trehalose Determinationmentioning

confidence: 99%

“…These activities co-elute through several chromatographic procedures, suggesting that they are part of a single bifunctional protein (Vandercammen et al, 1989 ;Londesborough and Vuorio, 1991). A protein complex, called trehalose synthase, has been purified in a specifically proteolysed form (Londesborough and Vuorio, 1991) and an apparently intact form (Vuorio et al …”

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confidence: 99%

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Cloning of two related genes encoding the 56‐kDa and 123‐kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae

Vuorio¹,

Kalkkinen

Londesborough³

1993

European Journal of Biochemistry

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143

127

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Preparations of intact trehalose synthase contain three polypeptides with molecular masses of 56, 102 and 123 kDa. We have cloned the genes TSSl and TSLl coding for the 56-and 123-kDa subunits, respectively. These genes are located on chromosomes I1 (TSSI) and XI11 (TSLI). The TSSl gene was found to be identical with ClFl, a gene required for normal growth on glucose. The product of the entire TSSl gene exibits 37% identity with a 502-amino-acid stretch from the middle of the TSLl product. Disruption of the TSSl gene in yeast eliminates both trehalose 6-phosphate synthase (Tre6P synthase) and trehalose 6-phosphate phosphatase (Tre6Pase) activities, and reintroduction of this gene restores these activities. Transformation of Escherichia coli with TSSl increases its Tre6P synthase activity. Specific proteolytic degradation of the 123-kDa polypeptide from the N-terminus greatly influences the Tre6P synthase activity, decreasing its inhibition by phosphate and activatability by fructose 6-phosphate but has little effect on the Tre6Pase activity. These results suggest that this N-terminal part confers regulatory properties upon the Tre6P synthase activity.Trehalose, a non-reducing disaccharide of glucose, occurs in Saccharomyces cerevisiae during periods of reduced growth, such as during starvation of cells for nitrogen, phosphate or sulfur, as well as during the stationary phase after growth on glucose (Lillie and Pringle, 1980). It is also accumulated in cells exposed to extremes of temperature, dehydration or hazardous chemicals (Attfield, 1987 ;Hottiger et al., 1987;Hino et al., 1990). Experiments using mutants producing varying amounts of trehalose and the increases in intracellular trehalose concentration caused by heat-shock treatment, have shown that heat tolerance, as well as desiccation tolerance, are closely related to the trehalose content of the yeast cell (Hottiger et al., 1987).The formation of trehalose is catalysed by the sequential action of trehalose-6-phosphate synthase (Tre6P synthase) and trehalose-6-phosphate phosphatase (Tre6Pase) activities. These activities co-elute through several chromatographic procedures, suggesting that they are part of a single bifunctional protein (Vandercammen et al., 1989 ;Londesborough and Vuorio, 1991). A protein complex, called trehalose synthase, has been purified in a specifically proteolysed form (Londesborough and Vuorio, 1991) and an apparently intact form (Vuorio et al

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

Ernandes

Meirsman

Rolland

et al. 1998

Yeast

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In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (GGS1FDP1BYP1CIF1GLC6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.

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Trehalose-6-phosphate synthase/phosphatase complex from bakers’ yeast: purification of a proteolytically activated form

Cited by 79 publications

References 24 publications

Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Structural analysis of the subunits of the trehalose‐6‐phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock

Cloning of two related genes encoding the 56‐kDa and 123‐kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae

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

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