A 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.
The split-ubiquitin assay detects protein interactions in 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 proteins in vivo. A library of genomic Saccharomyces 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 yeast S. cerevisiae.
Laser
H
H
Max-Delbrück-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
Bongards
C
C
Schüller
J
J
Heck
S
S
Johnsson
N
N
Lehming
N
N
D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D024001:HMGN Proteins; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D024001:HMGN Proteins; C086471:NHP6A protein, S cerevisiae; C086472:NHP6B protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors