Identification of Gal4 activation domain-binding proteins in the 26S proteasome by periodate-triggered cross-linking.
A common occurrence in biology is that a regulatory peptide, protein, or small molecule regulates the activity of a large multi-protein complex through direct interactions with a protein(s) in that complex. To characterize the direct receptor of the regulatory molecule, one would ideally like to study the native system. We report here that periodate-triggered cross-linking of catechol-containing regulatory factors, followed by two-dimensional electrophoresis and Western blotting, is an effective method for the characterization of regulatory factor--protein interactions in the context of large multi-protein complexes. We demonstrate the utility of this methodology by identifying the Rpt6/Sug1 and Rpt4/Sug2 proteins as the direct targets of transcriptional activation domains in the 26S proteasome.
Archer
Chase T
CT
Division of Translation Research, University of Texas Southwestern Medical Center Dallas, TX 75390-9185, USA.
Burdine
Lyle
L
Kodadek
Thomas
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004295:Dihydroxyphenylalanine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004295:Dihydroxyphenylalanine; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease