The MIG1 repressor from <i>Kluyveromyces lactis</i>: cloning, sequencing and functional analysis in <i>Saccharomyces cerevisiae</i>

Cassart, Jean-Pol; Georis, Isabelle; Östling, Jonas; Ronne, Hans; Vandenhaute, Jean

doi:10.1016/0014-5793(95)00909-s

Cited by 37 publications

(46 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, it lacks the PXXP core motif, which is thought to be crucial for SH3 binding (27). The proline-rich motif is conserved in Mig1 from K. lactis (5), which is consistent with the notion that it plays an important role in vivo. Similar motifs are also found in the more distantly related Mig1 homolog CREA (7) and in the Rox1 repressor (2).…”

Section: Discussionsupporting

confidence: 76%

See 1 more Smart Citation

Functional Domains in the Mig1 Repressor

Östling

Carlberg

Ronne

1996

Molecular and Cellular Biology

105

111

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionsupporting

confidence: 76%

“…Significantly, the C-terminal part of the effector domain including the leucine-proline repeats and the RSL motif is conserved in Mig1 from Kluyveromyces lactis (5), which suggests that these residues are functionally important ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

Functional Domains in the Mig1 Repressor

Östling

Carlberg

Ronne

1996

Molecular and Cellular Biology

105

111

View full text Add to dashboard Cite

show abstract

“…The respective situation has been much less explored in glucose-repressible strains of K. lactis; however, the existence of an ScMig1 homolog (KlMig1) has been inferred from homology considerations, and complementation of a mig1 mutant of S. cerevisiae by the homologous K. lactis gene has shown that the function of Mig1 is conserved in these distantly related yeasts (64). Keeping in mind both the considerable degree of sequence identity between KlHxk1 and ScHxk2 (73%) (22), with a striking conservation of amino acids in the intersubunit interface including the in vivo phosphorylation site, Ser-15, and the comparably high degree of identity (70.6% in an 85-residues overlap) in the N-terminal part of the KlMig1 and ScMig1 proteins, a mechanism of Mig1-hexokinase interaction similar to that proposed for S. cerevisiae may also be anticipated for K. lactis.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kuettner

Kettner

Keim

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ringshaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. The enzymes of the hexokinase family catalyze the intracellular trapping and the initiation of metabolism of glucose, fructose, and mannose. In addition to their role in glycolysis, an increasing number of yeast, plant, and mammalian hexokinases have been found to represent multifunctional proteins that are implicated in glucose sensing and signaling (1-4), whereas their glycolytic sugar substrate plays a dual role as a carbon source and hormone-like regulator (4, 5). The molecular basis underlying the involvement of hexokinases in the transcriptional control of glucose metabolism and in glucose homeostasis is their ability to interact with mitochondria and to reversibly translocate to nuclei (3, 6 -9).In the Crabtree-positive yeast Saccharomyces cerevisiae, glucose abundance is accompanied by the translocation of the cytosolic hexokinase isoenzyme 2 (ScHxk2) 4 and the transcriptional repressor Mig1 (ScMig1) into the nucleus, where both proteins participate in the formation of a hetero-oligomeric complex that suppresses the transcription of ScMig1 target genes like SUC2 encoding invertase (9). The mechanism of glucose signaling in glucose-repressible strains of the Crabtree-negative yeast Kluyveromyces lactis, used increasingly as a model organism in comparative functional genomics (10 -12), is largely unknown; however, the unique hexokinase KlHxk1 encoded by the RAG5 gene, the expression of glucose transporters, and the capacity for glucose transport seem to be involved (13)(14)(15).Contrary to the situation in bakers' yeast, glucose and fructose limitation causes the translocation of the mammalian hexokinase isoenzyme IV (also referred to as "glucokinase" or hexokinase D) to the nucleus of the liver parenchymal cell where it binds to its regulatory protein, GKRP, and retranslocates when glucose is abundantly av...

show abstract

“…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.…”

Section: Nuclear Localization Of Migl Is Regulated By Glucosementioning

confidence: 99%

Regulated nuclear translocation of the Mig1 glucose repressor.

Vít

Waddle

Johnston

1997

MBoC

321

182

View full text Add to dashboard Cite

Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae). Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to their promoters and recruiting the general repression complex Ssn6-Tup1. We have found that the subcellular localization of Mig1 is regulated by glucose. Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. This regulated nuclear localization requires components of the glucose repression signal transduction pathway. An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export. Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation. Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.

show abstract

The MIG1 repressor from Kluyveromyces lactis: cloning, sequencing and functional analysis in Saccharomyces cerevisiae

Cited by 37 publications

References 29 publications

Functional Domains in the Mig1 Repressor

Functional Domains in the Mig1 Repressor

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Regulated nuclear translocation of the Mig1 glucose repressor.

Contact Info

Product

Resources

About