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

Abstract

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

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

“…A variety of biochemical data suggest that Tps1, Tps2, and Tsl1 interact intimately with each other to form the Tre6P synthase/phosphatase complex (Bell et al, 1992;De Virgilio et al, 1993;Vuorio et al, 1993;Londesborough and Vuorio, 1993). Based on its homology to Tsl1, a fourth protein, Tps3, may also be part of this complex (De Virgilio et al, 1993).…”

“…The biochemical and genetic data available suggest that the three proteins Tps1, Tps2, and Tsl1 form a multimeric protein complex (Bell et al, 1992;De Virgilio et al, 1993;Vuorio et al, 1993;Londesborough and Vuorio, 1993). In the present study we have performed a series of experiments to further elucidate the structural composition of the Tre6P synthase complex as well as the specific role of its potential subunits.…”

“…This complex, the Tre6P synthase/phosphatase complex, is composed of at least three different subunits with apparent molecular masses of 56, 102, and 123 kDa. The corresponding genes, TPS1 (encoding the 56 kDa subunit; Bell et al, 1992), TPS2 (encoding the 102 kDa subunit; De Virgilio et al, 1993), and TSL1 (encoding the 123 kDa subunit; Vuorio et al, 1993), have been identified and sequenced. Based on its homology to Tsl1 (55% identity upon optimal alignment over the entire amino acid sequence; Manning et al, 1992), Tps3 may constitute a fourth subunit of the complex.…”

Dehydration-induced tps gene transcripts from an anhydrobiotic nematode contain novel spliced leaders and encode atypical GT-20 family proteins

“…Genes encoding the first of these enzymes (tps) have been identified in a wide range of organisms, including plants (e.g., Selaginella lepidophylla [8]), invertebrates (e.g., Caenorhabditis elegans [9]), yeast (e.g., Saccharomyces cerevisiae [10,11]) and bacteria (e.g., Escherichia coli [12]); trehalose-6-phosphate phosphatase genes have also been characterised for many micro-organisms. Although some of these organisms are anhydrobiotic (e.g., Se.…”

Trehalose synthesis is important for the acquisition of thermotolerance in Schizosaccharomyces pombe

“…Synthesis of trehalose is mediated by the trehalose-6-phosphate (Tre6P) synthase-phosphatase complex, which, in S. cerevisiae, consists of at least four subunits carrying either Tre6P synthase (Tps1), Tre6P phosphatase (Tps2) or regulatory activities (Tps3 and Tsl1; Bell et al, 1992;De Virgilio et al, 1993;Vuorio et al, 1993;Reinders et al, 1997). In accordance with the observation that heat-induced accumulation of trehalose is partially dependent on protein synthesis in S. cerevisiae (De Virgilio et al, 1991), both catalytic subunits (Tps1 and Tps2) have been shown to be hsps, and expression of all four genes encoding the subunits of the Tre6P synthase complex (TPS1, TPS2, TPS3 and TSL1 ) has been found to be dramatically enhanced under heat-shock conditions (De Virgilio et al, 1993;Winderickx et al, 1996).…”