Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast.
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.
Gupta
Ronish
R
Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.
Kus
Bart
B
Fladd
Christopher
C
Wasmuth
James
J
Tonikian
Raffi
R
Sidhu
Sachdev
S
Krogan
Nevan J
NJ
Parkinson
John
J
Rotin
Daniela
D
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D019151:Peptide Library; D040081:Protein Array Analysis; D011485:Protein Binding; D020543:Proteome; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*
D019151:Peptide Library; D020543:Proteome; D012269:Ribosomal Proteins; D025801:Ubiquitin; C030831:ribosomal protein S5