Open AccessA new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter
Author(s) -
Heike Laser,
Christine Bongards,
Jutta Schüller,
Stephanie Heck,
Nils Johnsson,
Norbert Lehming
Publication year - 2000
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.250400997
Subject(s) - saccharomyces cerevisiae , ubiquitin , ubiquitin conjugating enzyme , biology , biochemistry , yeast , dna binding protein , dna , ubiquitin ligase , genetics , microbiology and biotechnology , transcription factor , gene
The split-ubiquitin assay detects protein interactionsin vivo . To identify proteins interacting with Gal4p and Tup1p, two transcriptional regulators, we converted the split-ubiquitin assay into a generally applicable screen for binding partners of specific proteinsin vivo . A library of genomicSaccharomyces cerevisiae DNA fragments fused to the N-terminal half of ubiquitin was constructed and transformed into yeast strains carrying either Gal4p or Tup1p as a bait. Both proteins were C-terminally extended by the C-terminal half of ubiquitin followed by a modified Ura3p with an arginine in position 1, a destabilizing residue in the N-end rule pathway. The bait fusion protein alone is stable and enzymatically active. However, upon interaction with its prey, a native-like ubiquitin is reconstituted. RUra3p is then cleaved off by the ubiquitin-specific proteases and rapidly degraded by the N-end rule pathway. In both screens, Nhp6B was identified as a protein in close proximity to Gal4p as well as to Tup1p. Direct interaction between either protein and Nhp6B was confirmed by coprecipitation assays. Genetic analysis revealed that Nhp6B, a member of the HMG1 family of DNA-binding proteins, can influence transcriptional activation as well as repression at a specific locus in the chromosome of the yeastS. cerevisiae .