title: GAL4 is a substrate for caspases: implications for two-hybrid screening and other GAL4-based assays.
abstract: Yeast two-hybrid technology as well as mammalian reporter assays use fusions between a protein of interest and the GAL4 DNA-binding domain (GAL4DB). We demonstrate that expression of a GAL4DB/caspase-1 chimeric protein in yeast leads to autoproteolytic cleavage of GAL4DB. Moreover, recombinant GAL4DB is a good in vitro substrate for recombinant caspase-1 and several other caspases. Cleavage sites map at the C-terminus of GAL4DB and result in release of the fused protein. The finding that GAL4DB can be cleaved by caspases has important implications for the use of caspases in two-hybrid analysis and in the interpretation of mammalian assays based on GAL4-dependent reporter gene expression.
authors.affiliation: Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Belgium.
authors.forename: W
authors.initials: W
authors.lastname: van Criekinge
authors.forename: S
authors.initials: S
authors.lastname: Cornelis
authors.forename: M
authors.initials: M
authors.lastname: Van De Craen
authors.forename: P
authors.initials: P
authors.lastname: Vandenabeele
authors.forename: W
authors.initials: W
authors.lastname: Fiers
authors.forename: R
authors.initials: R
authors.lastname: Beyaert
mesh_terms: D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D020170:Caspase 1