Identification of CED-3 substrates by a yeast-based screening method.
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.
Kim
Sung Yun
SY
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.
Valencia
Marcela
M
Lee
Eui Seung
ES
Park
Daeho
D
Oh
Myungsok
M
Xue
Ding
D
Park
Woo Jin
WJ
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
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