Open AccessUbiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast
Author(s) -
Gupta Ronish,
Kus Bart,
Fladd Christopher,
Wasmuth James,
Tonikian Raffi,
Sidhu Sachdev,
Krogan Nevan J,
Parkinson John,
Rotin Daniela
Publication year - 2007
Publication title -
molecular systems biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 8.523
H-Index - 148
ISSN - 1744-4292
DOI - 10.1038/msb4100159
Subject(s) - ubiquitin ligase , biology , saccharomyces cerevisiae , proteome , protein microarray , ubiquitin protein ligases , ubiquitin , yeast , proteomics , computational biology , protein array analysis , tandem affinity purification , dna microarray , ubiquitin conjugating enzyme , protein–protein interaction , ddb1 , biochemistry , enzyme , gene , gene expression , affinity chromatography
Ubiquitin‐protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast ( Saccharomyces cerevisiae ) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets.