NOVEL GAL3 PROTEINS SHOWING ALTERED GAL80P BINDING CAUSE CONSTITUTIVETRANSCRIPTION OF GAL4P-ACTIVATED GENES IN SACCHAROMYCES-CEREVISIAE

Gal4p-mediated activation of galactose gene expression in Saccharomyce s cerevisiae normally requires both galactose and the activity of Gal3 p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Gal80p, an inhibitor of the transcriptional acti vator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independent ly of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Ga l3p. The level of constitutive GAL gene expression in cells bearing di fferent GAL3(c) alleles varies over more than a fourfold range and inc reases in response to galactose. Utilizing glutathione S-transferase-G al3p fusions, we determined that the mutant Gal3p proteins show altere d Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding abil ity. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that incre ases either its ability to bind to Gal80p or its access to Gal80p. Wit h the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for st udying the induction mechanism through both in vivo and in vitro metho ds.