Leu343Phe substitution in the Malx3 protein of Saccharomyces cerevisiae increases the constitutivity and glucose insensitivity of MAL gene expression

• Published in: Current genetics 1999-06, Vol.35 (5), p.491-498
• Main Authors: Higgins, V.J, Braidwood, M, Bissinger, P, Dawes, I.W, Attfield, P.V
• Format: Article
• Language: English
• Subjects: alpha-glucosidase; alpha-Glucosidases - genetics; alpha-Glucosidases - metabolism; Amino Acid Sequence; amino acid sequences; Amino Acid Substitution; Base Sequence; enzyme activity; Fungal Proteins - genetics; Fungal Proteins - metabolism; genbank/af002703; genbank/af002704; gene expression; Gene Expression Regulation, Fungal; genetic regulation; glucose; Glucose - metabolism; Leucine; maltose; maltose permease; malx1 gene; malx2 gene; malx3 gene; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; messenger RNA; Molecular Sequence Data; Monosaccharide Transport Proteins; mutants; Mutation; nucleotide sequences; nutrient availability; phenotype; Phenylalanine; promoter regions; proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Sequence Analysis; site-directed mutagenesis; Trans-Activators - genetics; Trans-Activators - metabolism; transcription (genetics); Transcription Factors; transcriptional activators
• ISSN: 0172-8083; 1432-0983
• DOI: 10.1007/s002940050444

To utilise maltose as a carbon source Saccharomyces cerevisiae needs one or more functional MAL loci that contain the MALx1 gene encoding maltose permease, MALx2 encoding maltase, and MALx3 encoding a transcriptional activator. Maltose causes a rapid MALx3-dependent induction of MAL gene transcription, and glucose represses this activation via Mig1p. A MALx3 gene conveying high MAL gene expression in the absence of maltose in a malx3 laboratory mutant strain has been isolated from baker's yeast. The construction of hybrid genes between the isolated gene and a highly regulated MALx3 gene showed that constitutivity was the result of multiple amino-acid alterations throughout the structural gene. The combined effect of these amino-acid alterations was shown to be stronger than the sum of their individual effects on constitutivity. Analysis in glucose-repressed conditions confirmed that increased MALx3 transcript levels increased the glucose insensitivity of MAL gene expression but did not affect constitutivity. Analysis of four mutations between aa 343 and 375, lying within a proposed negative regulatory domain, showed that the single mutation of Leu343Phe increased the glucose insensitivity of MAL gene expression by 30-fold. These results demonstrate that not only Mig1p modulation of MALx3 expression, but also the MALx3 protein structure, is involved in the glucose-insensitive expression of the MAL genes.