title: Self-association of the Gal4 inhibitor protein Gal80 is impaired by Gal3: evidence for a new mechanism in the GAL gene switch.
abstract: 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.
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Onur
authors.initials: O
authors.lastname: Egriboz
authors.forename: Sudip
authors.initials: S
authors.lastname: Goswami
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Tao
authors.forename: Kathleen
authors.initials: K
authors.lastname: Dotts
authors.forename: Christie
authors.initials: C
authors.lastname: Schaeffer
authors.forename: Vepkhia
authors.initials: V
authors.lastname: Pilauri
authors.forename: James E
authors.initials: JE
authors.lastname: Hopper
mesh_terms: D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005800:Genes, Fungal; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; C099289:Gal3 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D005690:Galactose