Characterization of the 56‐kDa subunit of yeast trehalose‐6‐phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation

Abstract

Trehalose‐6‐phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose‐6‐phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose‐6‐phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose‐6‐phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation

“…Of the genes involved in trehalose synthesis, only TPS1 demonstrated an increase in transcription (at 60 h), while TPS2 demonstrated a clear reduction in transcript levels throughout the sampling period. Trehalose is synthesised by the α,α-trehalose-phosphate synthase complex, made up of the products of TPS1, TPS2, TSL1 and TPS3 genes (Bell et al, 1992). Why the genes responded differently to the same conditions is not clear, as they are known to be regulated similarly by stress conditions and repressed via the RAS-cAMP pathway (Winderickx et al, 1996).…”

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.…”

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

“…Since then, several studies have dealt with the purification and kinetic properties of Tre6P synthase and Tre6P phosphatase in yeast (Panek et al, 1987;Vandercammen et al, 1989;Bell et al, 1992;De Virgilio et al, 1993;Londesborough and Vuorio, 1993). The overall picture that emerged from these studies is that both enzymes are part of a multimeric protein complex with an approximate molecular mass of 630-800 kDa.…”

“…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.…”