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The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.

Galactose-activated transcription of the Saccharomyces cerevisiae GAL genes occurs when Gal3 binds the Gal4 inhibitor, Gal80. Noninteracting variants of Gal3 or Gal80 render the GAL genes noninducible. 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 GAL3 C -D368V mutation can suppress the noninducibility due to a GAL80 S-1 -G323R mutation, and a GAL80-M350C mutation can suppress the noninducibility due to a gal3-D111C mutation. A reverse two-hybrid selection for GAL3 mutations that impair the Gal3-Gal80 interaction yielded 12 singleamino-acid substitutions at residues that are predicted to be surface exposed on Gal3. The majority of the affected Gal3 residues localized to a composite surface that includes D111 and a sequence motif containing D368, which has been implicated in interaction with Gal80. The striking colocalization of intergenic suppressor residues and Gal80 nonbinder residues identifies a Gal3 surface that likely interacts with Gal80.

Gal4-mediated activation of GAL gene transcription in Saccharomyces cerevisiae requires the interaction of Gal3 with Gal80, the Gal4 inhibitor protein. While it is known that galactose and ATP activates Gal3 interaction with Gal80, neither the mechanism of activation nor the surface that binds to Gal80 is known. We addressed this through intragenic suppression of GAL3 C alleles that cause galactose-independent Gal3-Gal80 interaction. We created a new allele, GAL3 SOC , and showed that it suppressed a new GAL3 C allele. We tested the effect of GAL3 SOC on several newly isolated and existing GAL3 C alleles that map throughout the gene. All except one GAL3 C allele, D368V, were suppressible by GAL3 SOC. GAL3 SOC and all GAL3 C alleles were localized on a Gal3 homology model that is based on the structure of the highly related Gal1 protein. These results provide evidence for allosterism in the galactose-and ATP-activation of Gal3 binding to Gal80. In addition, because D368V and residues corresponding to Gal80-nonbinder mutations colocalized to a domain that is absent in homologous proteins that do not bind to Gal80, we suggest that D368 is a part of the Gal80-binding surface.