title: Identification of CED-3 substrates by a yeast-based screening method.
abstract: Identifying cellular substrates repertoire of individual proteases will facilitate our understanding of their physiological and pathological roles. In this article, we employed a yeast-based screening method to isolate CED-3 substrates. This method uses a transcription factor anchored to the plasma membrane by fusion to a library of cellular protein sequences. When a fusion protein is cleaved by CED-3, the transcription factor is released from the plasma membrane and enters the nucleus where it turns on the expression of reporter genes. We identified seven candidate clones by screening a genomic library using this method. Of these seven clones, two were cleaved by purified CED-3 in vitro. Therefore, the method described here may be generally used for genomewide screening to isolate potential substrates of specific proteases.
authors.affiliation: Department of Life Science, National Research Laboratory of Proteolysis, Kwangju Institute of Science and Technology (K-JIST), 1 Oryong-dong, Puk-gu, Kwangju, 500-712, Korea.
authors.forename: Sung Yun
authors.initials: SY
authors.lastname: Kim
authors.forename: Marcela
authors.initials: M
authors.lastname: Valencia
authors.forename: Eui Seung
authors.initials: ES
authors.lastname: Lee
authors.forename: Daeho
authors.initials: D
authors.lastname: Park
authors.forename: Myungsok
authors.initials: M
authors.lastname: Oh
authors.forename: Ding
authors.initials: D
authors.lastname: Xue
authors.forename: Woo Jin
authors.initials: WJ
authors.lastname: Park
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D020169:Caspases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D015723:Gene Library; D008967:Molecular Biology / Q000379:methods*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism; D000099160:LexA Repressor Protein
chemical_list: D001426:Bacterial Proteins; D029742:Caenorhabditis elegans Proteins; D020169:Caspases; D003546:Cysteine Endopeptidases; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C089223:ced-3 protein, C elegans