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

Alexander Anders,Hauke Lilie,Kathlen Franke,Lutz Kapp,Jörg Stelling,Ernst D Gilles,Karin D Breunig

doi:10.1074/jbc.m604271200

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.

Highlights

A textbook model of how such coupling can be achieved in a eukaryotic cell
KlGAL1 can substitute for GAL3 in S. cerevisiae [23], neither Gal3 nor ScGal1 can complement the noninducible phenotype of a K. lactis gal1 deletion mutant unless the KlGAL80 gene is exchanged by ScGAL80 [15]
In the K. lactis Gal80 (KlGal80) variant N-terminal His6-tagged KlGal80 variant (NHGal80), the His6 tag was fused to the N terminus of KlGal80, and in IHGal80 the tag was inserted at an internal position, in the so-called linker region

Summary

Introduction

A textbook model of how such coupling can be achieved in a eukaryotic cell (for reviews see Refs. 1–3). Competitive Mechanism of the Galactose Switch in K. lactis pressed phenotype (GAL80s) on ScGal4 [30] has a similar effect in K. lactis upon substitution of equivalent amino acids in KlGAL80 [31] These data indicate differences between both yeasts either in the Gal1/Gal3-Gal interface and/or in the mechanism that relieves Gal inhibition. Attempts to map subdomains of Gal sufficient for binding to either one of its interaction partners have been unsuccessful so far Both Gal orthologs are remarkably sensitive to mutations, and many single amino acid exchanges in ScGal simultaneously eliminate binding to Gal, Gal, and homodimerization [36, 37].5. Close proximity of Gal3/Gal and Gal binding interfaces on Gal is suggested by the few amino acid substitutions that selectively influence Gal or Gal3/Gal binding activity, like G302R (KlGal binding deficiency) and G311E (Gal binding deficiency) [31]

Methods

Results

Conclusion