The Galactose Switch in Kluyveromyces lactis Depends on Nuclear Competition between Gal4 and Gal1 for Gal80 Binding

Abstract

The Gal4 protein represents a universally functional transcription activator, which in yeast is regulated by protein-protein interaction of its transcription activation domain with the inhibitor Gal80. Gal80 inhibition is relieved via galactose-mediated Gal80-Gal1-Gal3 interaction. The Gal4-Gal80-Gal1/3 regulatory module is conserved between Saccharomyces cerevisiae and Kluyveromyces lactis. Here we demonstrate that K. lactis Gal80 (KlGal80) is a nuclear protein independent of the Gal4 activity status, whereas KlGal1 is detected throughout the entire cell, which implies that KlGal80 and KlGal1 interact in the nucleus. Consistently KlGal1 accumulates in the nucleus upon KlGAL80 overexpression. Furthermore, we show that the KlGal80-KlGal1 interaction blocks the galactokinase activity of KlGal1 and is incompatible with KlGal80-KlGal4-AD interaction. Thus, we propose that dissociation of KlGal80 from the AD forms the basis of KlGal4 activation in K. lactis. Quantitation of the dissociation constants for the KlGal80 complexes gives a much lower affinity for KlGal1 as compared with Gal4. Mathematical modeling shows that with these affinities a switch based on competition between Gal1 and Gal4 for Gal80 binding is nevertheless efficient provided two monomeric Gal1 molecules interact with dimeric Gal80. Consistent with such a mechanism, analysis of the sedimentation behavior by analytical ultracentrifugation demonstrates the formation of a heterotetrameric KlGal80-KlGal1 complex of 2:2 stoichiometry.Regulation of transcription requires coupling of gene-specific transcription activators to regulatory signals. The galactose switch, which induces the synthesis of galactose metabolic enzymes in response to this sugar in the environment, serves as a textbook model of how such coupling can be achieved in a eukaryotic cell (for reviews see Refs.

Understanding a Transcriptional Paradigm at the Molecular Level

“…Note that both of the proposed binding sites for Gal4p and Gal1p (or ScGal3p) are on the same side of the Gal80p dimer (Fig. 3b), and this is in keeping with the recent and elegant experiments of Anders et al (7), which suggest that the binding sites on Gal80p for Gal1p or Gal4p are overlapping.…”

“…4b). However, the interaction between KlGal80p and KlGal1p would appear to occur via a 2:1 complex, although it is possible that, at high concentration, a dimer of Gal80p interacts with two molecules of Gal1p (7). The proposed binding sites for both Gal4p and Gal1p in KlGal80p are distinct but are located on the same side of the molecule.…”

“…ScGal3p is predominately, if not exclusively, cytoplasmic (5,6). Recently, KlGal80p has been identified as an exclusively nuclear protein while KlGal1p is present both in the nucleus and the cytoplasm (7). On the basis of these and other data it has been suggested that the S. cerevisiae GAL switch is activated when galactose and ATP bind to Gal3p in the cytoplasm.…”

“…This traps Gal80p in the cytoplasm thereby freeing Gal4p from its repressing effects and allowing transcriptional activation to occur (8). In K. lactis, the GAL switch appears to be controlled by competition in the nucleus between KlGal1p and KlGal4p for KlGal80p binding (7). In either model, Gal80p must interact with two very different proteins: Gal4p, via its carboxyl-terminal activation domain, and Gal3p (Gal1p in K. lactis) when galactose and ATP are bound to the ligand sensor.…”

The Expanding Landscape of Moonlighting Proteins in Yeasts

“…Moonlighting action may or may not affect the canonical activity of the protein; binding of galactokinase to the inhibitory protein Gal80 blocks galactokinase activity (136), but binding of arginase to ornithine transcarbamylase does not abolish arginase activity (137).…”