Function and regulation of the Yeast Mig1 repressor
1998 (English)Doctoral thesis, comprehensive summary (Other academic)
Glucose repression is a global regulatory response in the yeast Saccharomyces cerevisiae. Thus, in the presence of glucose, the preferred carbon source, expression of many genes encoding proteins required for metabolism of other carbon sources are turned off.
An important mediator of glucose repression is the Mig1 repressor. It is a DNA binding transcription factor that has two zinc fingers of the C2H2 type. When glucose is available, Mig1 binds to the promotors of glucose repressed genes and is then thought to recruit a large co-repressor complex, consisting of the Cyc8/Ssn6 and Tup1 proteins. Mig1 is negatively regulated by the Snf1 protein kinase, a protein which is essential for the expression of all glucose repressed genes in yeast.
In this work, domains within the Mig1 repressor were characterized that mediate its carbon source regulation and its function as a repressor. An extensive deletion analysis of Mig1 revealed that an effector domain of 24 amino acid residues in the extreme C-terminus of the protein is important for repression. The C-terminal effector domain is structurally and functionally conserved also in the Mig1 homologues from the distantly related yeasts Kluyveromyces lactis and K. marxianus. A combined deletion analysis and alanine scanning mutagenesis of the effector domain in K. marxianus revealed that three leucines and one isoleucine are particularly important for its function in vivo.
The deletion analysis of the Mig1 from S. cerevisiae also revealed that two internal domains, R1 and R2, are important for the regulation of Mig1 by glucose. Thus, a deletion of either R1 or R2 converts Mig1 to a constitutive repressor. Further experiments were conducted with a Mig1-VP16 hybrid activator in which the C-terminal effector domain was replaced by the activating, domain from the viral protein VP16 The analysis revealed that this hybrid activator is negatively regulated by Snf1. We further found that Mig1-VP16 is phosphorylated in the absence of glucose, and that this phosphorylation is dependent on Snf1. Point mutations in Mig1 -VP16 identified three serines that are important both for Snf1 control of Mig1-VP16 activity and for phosphorylation in the absence of glucose. Two of the serines are located in the R1 domain within sequence motifs similar to a consensus in vitro target site for Snf1. The third serine, which does not resemble the Snf1 consensus target site, is located near the zinc fingers adjacent to a basic domain, the B-domain, which is essential both for the function of Mig1-VP16 as an activator and for the function of Mig1 as a repressor.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 1998. , , 52 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 738
Biochemistry, glucose repression, Mig1, zinc finger protein, Saccharomyces cerevisiae, Kluyveromyces marxianus, Snf1
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject Molecular Cellbiology
IdentifiersURN: urn:nbn:se:uu:diva-140ISBN: 91-554-4132-7OAI: oai:DiVA.org:uu-140DiVA: diva2:160915
1997-02-20, Lecture hall, B42, Biomedical Center, Uppsala University, Uppsala, 13:15