Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity

The Mig1 repressor is a zinc finger protein that mediates glucose repressio n in yeast. Previous work in Saccharomyces cerevisiae has shown that two do mains in Mig1p are required for repression: the N-terminal zinc finger regi on and a C-terminal effector domain. Both domains are also conserved in Mig 1p homologs from the distantly related yeasts Kluyveromyces lactis and K. m arxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-ter minal effector domain in Mig1p from K. marxianus, using expression in S. ce revisiae to monitor its function. First, a series of small deletions were m ade within the effector domain. Second, an alanine scan mutagenesis was car ried out across the effector domain. Third, double, triple and quadruple mu tants were made that affect certain residues within the effector domain. Ou r results show that four conserved residues within the effector domain, thr ee 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 ninefold 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 cor responding mutations were made in ScMig1p. Moreover, we found that mutation s in these residues affect the interaction between Mig1p and the general co repressor 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.