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

To utilise maltose as a carbon source Saccharomyces cerevisiae needs one or more functional MAL loci that contain the MALx1 gene encoding maltose perm ease, MALx2 encoding maltase, and MALx3 encoding a transcriptional activato r. Maltose causes a rapid MALx3-dependent induction of MAL gene transcripti on, and glucose represses this activation via Mig1p. A MALx3 gene conveying high MAL gene expression in the absence of maltose in a malx3 laboratory m utant strain has been isolated from baker's yeast. The construction of hybr id genes between the isolated gene and a highly regulated MALx3 gene showed that constitutivity was the result of multiple amino-acid alterations thro ughout the structural gene. The combined effect of these amino-acid alterat ions was shown to be stronger than the sum of their individual effects on c onstitutivity. Analysis in glucose-repressed conditions confirmed that incr eased MALx3 transcript levels increased the glucose insensitivity of MAL ge ne 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 insens itivity of MAL gene expression by 30-fold. These results demonstrate that n ot only Mig Ip modulation of MALx3 expression, but also the MALx3 protein s tructure, is involved in the glucose-insensitive expression of the MAL gene s.