A double role of the Gal80 N terminus in activation of transcription by Gal4p.

The yeast galactose switch operated by the Gal4p-Gal80p-Gal3p regulatory module is a textbook model of transcription regulation in eukaryotes. The Gal80 protein inhibits Gal4p-mediated transcription activation by binding to the transcription activation domain. In Saccharomyces cerevisiae, inhibition is relieved by formation of an alternative Gal80-Gal3 complex. In yeasts lacking a Gal3p ortholog, such as Kluyveromyces lactis, the Gal1 protein (KlGal1p) combines regulatory and enzymatic activity. The data presented here reveal a yet unknown role of the KlGal80 N terminus in the mechanism of Gal4p activation. The N terminus contains an NLS, which is responsible for nuclear accumulation of KlGal80p and KlGal1p and for KlGal80p-mediated galactokinase inhibition. Herein, we present a model where the N terminus of KlGal80p reaches the catalytic center of KlGal1p causing enzyme inhibition in the nucleus and stabilization of the KlGal1-KlGal80p complex. We corroborate this model by genetic analyses and structural modelling and provide a rationale for the divergent evolution of the mechanism activating Gal4p.