Molecular analysis of the neutral trehalase gene from Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry 1993-03, Vol.268 (7), p.4766-4774
• Main Authors: Kopp, M, Muller, H, Holzer, H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Base Sequence; binding sites; Biological and medical sciences; Cloning, Molecular; DNA, Fungal; DNA-binding proteins; enzyme activity; Fundamental and applied biological sciences. Psychology; genbank/x65925; gene expression; genes; Genes, Fungal; Genes. Genome; Hot Temperature; messenger RNA; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis; mutants; neutral trehalase; NTH1 gene; nucleotide sequence; nucleotide sequences; Plasmids; predictions; promoter regions; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sequence Homology, Amino Acid; structural genes; TATA box; trehalase; Trehalase - genetics; Trehalase - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)53463-3

Neutral trehalase (EC 3.2.1.28) is a trehalose hydrolyzing enzyme of the yeast Saccharomyces cerevisiae (App, H., and Holzer, H. (1989) J. Biol. Chem. 264, 17583-17588). The gene df neutral trehalase was cloned by complementation of a neutral trehalase-deficient yeast mutant which was obtained by ethylmethanesulfonate mutagenesis. Three mutants without detectable neutral trehalase activity were obtained and characterized by tetrad analysis and found to belong to the same complementation group. The mutants were transformed with a S. cerevisiae genomic library in YEp24. Two overlapping plasmids were isolated, containing the neutral trehalase gene NTH1 with an open reading frame of 2079 base pairs (bp), encoding a protein of 693 amino acids, corresponding to a molecular mass of 79,569 Da. Several putative TATA boxes were found in the 5'-nontranslated region of the NTH1 gene. In positions -652 to -641 a possible binding sequence for the MIG1 protein, a multicopy inhibitor of the GAL1 promotor, which also binds to the promotor sequences of the SUC2 and the FBP1 gene, was found. The start codon of the neutral trehalase is located about 2500 bp upstream of the centromere 4 consensus sequence elements I, II, and III (Mann, C., and Davis, R. W. (1986) Mol. Cell. Biol. 6, 241-245). Vicinity to a centromere is known to have a depressing influence on the number of plasmid copies per cell. This probably explains why transformation with pNTH does not lead to overexpression of neutral trehalase. The four consensus sequences AATAAA contained in the centromeric elements and reconfirmed by our sequencing data might be polyadenylation signals for NTH1-mRNA transcription termination. Northern blot analysis yielded a single mRNA species of approximately 2.3 kilobase(s). The neutral trehalase protein has a putative cAMP-dependent phosphorylation consensus sequence RRGS from amino acid positions 22-25. Therefore, the previously described activation of neutral trehalase by cAMP-dependent phosphorylation is probably due to phosphorylation of serine 25. Three potential N-glycosylation sites (Asn-X-Ser/Thr) occur in the open reading frame of the neutral trehalase gene. However, no evidence for glycosylation could be detected by Western blotting. The amino acid sequence of neutral trehalase from S. cerevisiae points to significant similarity in three domains with the osmoregulated treA gene encoding the periplasmic trehalase from E. coli K12 (Gutierrez, C., Ardourel, M., Bremer, E.,