Genetic evidence for sites of interaction between the Gal3 and Gal80 proteins of the Saccharomyces cerevisiae GAL gene switch.
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 single-amino-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.
Diep
Cuong Q
CQ
Department of Biochemistry and Molecular Biology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA.
Tao
Xiaorong
X
Pilauri
Vepkhia
V
Losiewicz
Mandy
M
Blank
T Eric
TE
Hopper
James E
JE
D019943:Amino Acid Substitution; D001665:Binding Sites; D002455:Cell Division; D017931:DNA Primers; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D005812:Genes, Switch; D005838:Genotype; D009154:Mutation; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D004271:DNA, Fungal; C055002:GAL80 protein, S cerevisiae; C099289:Gal3 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D005690:Galactose