Identification of CED-3 substrates by a yeast-based screening method

Sung Yun Kim; Marcela Valencia; Eui Seung Lee; Daeho Park; Myungsok Oh; Ding Xue; Woo Jin Park

doi:10.1385/MB:27:1:01

Identification of CED-3 substrates by a yeast-based screening method

Mol Biotechnol. 2004 May;27(1):1-6. doi: 10.1385/MB:27:1:01.

Authors

Sung Yun Kim¹, Marcela Valencia, Eui Seung Lee, Daeho Park, Myungsok Oh, Ding Xue, Woo Jin Park

Affiliation

¹ 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.

PMID: 15122042
DOI: 10.1385/MB:27:1:01

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Caenorhabditis elegans Proteins / analysis
Caenorhabditis elegans Proteins / isolation & purification
Caenorhabditis elegans Proteins / metabolism*
Caspases / analysis
Caspases / isolation & purification
Caspases / metabolism*
Cysteine Endopeptidases / analysis
Cysteine Endopeptidases / isolation & purification
Cysteine Endopeptidases / metabolism*
Gene Library
Molecular Biology / methods*
Receptors, Mating Factor
Receptors, Peptide / genetics
Receptors, Peptide / metabolism
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Serine Endopeptidases / genetics
Serine Endopeptidases / metabolism
Substrate Specificity
Transcription Factors / genetics
Transcription Factors / metabolism
Yeasts / genetics*
Yeasts / metabolism

Substances

Bacterial Proteins
Caenorhabditis elegans Proteins
LexA protein, Bacteria
Receptors, Mating Factor
Receptors, Peptide
Recombinant Fusion Proteins
Transcription Factors
Serine Endopeptidases
Caspases
Cysteine Endopeptidases
ced-3 protein, C elegans