Functional Domains in the Mig1 Repressor

Abstract

Mig1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. It is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 proteins and binds to a GC-rich motif that resembles the GC boxes recognized by these proteins. We have performed deletion mapping in order to identify functional domains in Mig1. We found that a small C-terminal domain comprising the last 24 amino acids mediates Mig1-dependent repression of a reporter gene. This effector domain contains several leucine-proline dipeptide repeats. We further found that inhibition of Mig1 activity in the absence of glucose is mediated by two internal elements in the Mig1 protein. A Mig1-VP16 hybrid activator was used to further investigate how Mig1 is regulated. Mig1-VP16 can activate transcription from promoters containing Mig1-binding sites and suppresses the inability of Snf1-deficient cells to grow on certain carbon sources. We found that a deletion of the SNF1 gene increases the activity of Mig1-VP16 fivefold under derepressing conditions but not in the presence of glucose. This shows that the hybrid activator is under negative control by the Snf1 protein kinase. Deletion mapping within Mig1-VP16 revealed that regulation of its activity by Snf1 is conferred by the same internal elements in the Mig1 sequence that mediate inhibition of Mig1 activity in the absence of glucose.In yeasts and other fungi, a large number of genes are turned off in the presence of glucose. This response is called glucose repression (9,14,28,36). A key effector in glucose repression is a zinc finger protein known as Mig1 (26). Mig1 is a transcriptional repressor which belongs to the GC box-binding proteins, a subfamily of evolutionarily related zinc finger proteins that also includes the Sp1, Krox/Egr, and Wilms' tumor proteins. Like these mammalian proteins, Mig1 binds to a GC box, which is present in many genes that are repressed by glucose (20,24).Mutations in Mig1 show partial epistasis over mutations in Snf1 (or Cat1), a protein kinase which is required for expression of all glucose-repressed genes (6, 31). This suggests that one function of the Snf1 kinase is to prevent Mig1 from repressing its target genes in the absence of glucose. The mechanism by which Mig1 represses transcription is not clear, but it is thought that several yeast repressors, including Mig1, act by recruiting a general corepressor complex containing the Ssn6 (or Cyc8) and Tup1 proteins to their target promoters (16,35,38,42). Recent evidence suggests that Tup1 is the active subunit within this complex (37,38).An important question is whether Mig1 contains distinct domains that mediate transcriptional repression and regulation by glucose. We have therefore performed an extensive deletion analysis of Mig1 in order to identify such domains within the protein. A large number of deletions were tested for their ability to repress a target promoter both in the presence and in the absence of glucose. We found that a small domain comprising the 24 C-terminal residues in M...

Regulated nuclear translocation of the Mig1 glucose repressor.

“…Two smaller deletions define the domain further: deletion of amino acids 174-391 also results in a partially constitutive repressor ( Table 4, pBM3255) that is constitutively in the nucleus ( Figure 6A, parts 3 and 4); deletion of amino acids 97-173 has little effect on glucose regulation of transcriptional repression (Table 4, pBM3350) and no apparent effect on regulation of nuclear localization ( Figure 6A, parts 5 and 6). This region of the protein contains two stretches very rich in the basic amino acids characteristic of nuclear localization signals, one of which is conserved in the Migl homologs of Kluyveromyces lactis and Kluyveromyces marxianus and is very similar to the well-characterized nuclear localization signal of the yeast transcription factor Swi5 (Cassart et al, 1995;Ostling et al, 1996). However, these sequences are not required for the nuclear localization of Migl ( Figure 6A, parts 5 and 6) and thus are unlikely to be involved in nuclear localization.…”

“…The ssn6 mutation itself does not affect nuclear localization of Migl (Figure 5,E and F). In this double mutant, Migl is localized to the nucleus even in cells shifted to glycerol for 2 h ( Figure 5, G and H sion domain within the carboxyl-terminal 25 amino acids that probably mediates interaction with Ssn6 and Tupl (Ostling et al, 1996). Neither of these domains is required for regulated nuclear translocation ( Figure 6A, parts 7 and 8).…”

“…It is probably this domain that causes the internal deletions described above to be constitutively localized to the nucleus. The carboxyl terminus, which is thought to interact with the Ssn6-Tupl complex (Ostling et al, 1996), does not confer nuclear localization to the chimera: it is found in both the cytoplasm and the nucleus, as though it has no localization signal ( Figure 7A, parts 9 and 10; though it is also possible that the Migl sequences are cleaved from this chimeric protein). The same results are seen in regl ( Figure 7A, part 11) and snfl mutants (our unpublished observation).…”

“…Internal deletions that remove the region including amino acids 261-400 produce a protein that is no longer responsive to glucose (it is a constitutive repressor) and is always in the nucleus. Ostling et al (1996) also showed that removal of regions of Migl including amino acids 261-400 convert it into a constitutive repressor. Internally truncated proteins are targeted to the nucleus by an unregulated nuclear localization signal in the amino-terminal 92 amino acids of the protein.…”

“…Repression is 1990); regl::hisG, pREG1-4 cut with SacI and SalI (Hoekstra, perprobably not regulated: replacement of the repression sonal communication). domain of Migl with the activation domain of VP16 results in a transcriptional activator whose function is Plasmids inhibited by Snfl (Ostling et al, 1996). We have dis-Standard procedures for the manipulation of plasmid DNA and covered that the nuclear localization of Migl is regu-transformation of bacteria were followed (Sambrook et al, 1989).…”