Constitutive expression of yeast phospholipid biosynthetic genes by variants of Ino2 activator defective for interaction with Opi1 repressor.
Regulated expression of structural genes involved in yeast phospholipid biosynthesis is mediated by inositol/choline-responsive element (ICRE) upstream motifs, bound by the heterodimeric activator complex Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring an intact Opi1 repressor which binds to Ino2. For a better understanding of interactions among regulators, we mapped an 18 aa repressor interaction domain (RID, aa 118-135) within Ino2 necessary and sufficient for binding by Opi1. By alanine scanning mutagenesis of the entire RID we were able to identify nine residues critical for Opi1-dependent repression of Ino2 function. Consequently, the corresponding dominant Ino2 variants conferred constitutive expression of an ICRE-dependent reporter gene and were no longer inhibited even by overproduction of Opi1. Interestingly, Ino2 RID partially overlaps with transcriptional activation domain TAD2. As certain mutations exclusively affect repression while others affect both repression and activation, both functions of Ino2 can be functionally uncoupled. Correspondingly, we mapped the RID-binding activator interaction domain (AID, aa 321-380) at the C-terminus of Opi1 and introduced missense mutations at selected positions. An Opi1 variant simultaneously mutated at three highly conserved positions showed complete loss of repressor function, confirming RID-AID interaction as the crucial step of regulated expression of ICRE-dependent genes.
Heyken
Willm-Thomas
WT
Institut für Mikrobiologie, Abteilung Genetik und Biochemie, Ernst-Moritz-Arndt Universität Greifswald, Jahnstr. 15a, D-17487 Greifswald, Germany.
Repenning
Antje
A
Kumme
Jacqueline
J
Schüller
Hans-Joachim
HJ
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002794:Choline / Q000378:metabolism; D017124:Conserved Sequence; D015966:Gene Expression Regulation, Fungal; D007294:Inositol / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000096:biosynthesis* / Q000235:genetics; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D000409:Alanine; D051792:Basic Helix-Loop-Helix Proteins; D002794:Choline; D007294:Inositol; D010743:Phospholipids; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C095723:INO2 protein, S cerevisiae; C096105:OPI1 protein, S cerevisiae