title: Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity.
abstract: The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Miglp are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Miglp homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an alpha-helix.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala Biomedical Center, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Ostling
authors.forename: J P
authors.initials: JP
authors.lastname: Cassart
authors.forename: J
authors.initials: J
authors.lastname: Vandenhaute
authors.forename: H
authors.initials: H
authors.lastname: Ronne
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D007930:Leucine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D016335:Zinc Fingers
chemical_list: C065455:CYC8 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C085976:MIG1 protein, S cerevisiae; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D007930:Leucine; D005947:Glucose; D000409:Alanine