Genetics. Published Articles Ahead of Print: February 3, 2008, Copyright © 2008
doi:10.1534/genetics.107.074799


A more recent version of this article appeared on February 1, 2008.

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Genetic evidence for sites of interaction between the Gal3 and Gal80 proteins of the Saccharomyces cerevisiae GAL gene switch

Cuong Q. Diep 1, Xiaorong Tao 2, Vepkhia Pilauri 3, Mandy Losiewicz 4, T. Eric Blank 5 and James E. Hopper 2*

1 Massachusetts General Hospital/Harvard Medical School
2 Ohio State University
3 Mount Sinai School of Medicine
4 Pennsylvania State University, College of Medicine
5 USAMRIID

* To whom correspondence should be addressed. E-mail: hopper.65{at}osu.edu.

Submitted on April 19, 2007
Revised on May 16, 2007
Accepted on 17 November 2007


Abstract

Galactose-activated transcription of the Saccharomyces cerevisiae GAL genes occurs when Gal3 binds the Gal4 inhibitor, Gal80. Non-interacting variants of Gal3 or Gal80 render the GAL genes non-inducible. To identify the binding determinants for Gal3's interaction with Gal80 we carried out GAL3-GAL80 intergenic suppression analyses and selected for new GAL3 mutations that impair the Gal3-Gal80 interaction. We show that a GAL3C-D368V mutation can suppress the non-inducibility due to a GAL80S-1-G323R mutation, and a GAL80-M350C mutation can suppress the non-inducibility due to a gal3-D111C mutation. A yeast reverse two-hybrid selection for GAL3 mutations that impair the Gal3-Gal80 interaction yielded 12 single amino acid substitutions at residues that are surface-exposed on a Gal3 homology model. The majority of the affected Gal3 residues localized to a composite surface that includes D111 and an insertion motif containing D368, which has been implicated in interaction with Gal80. The striking co-localization of intergenic suppressor residues and Gal80 non-binder residues identifies a candidate Gal3 surface that interacts with Gal80.

Key Words: GAL gene switch, Gal3, Gal80, Intergenic suppression