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. 2019;618:135-147.
doi: 10.1016/bs.mie.2018.12.032. Epub 2019 Feb 1.

Detection of Ubiquitination Activity and Identification of Ubiquitinated Substrates Using TR-TUBE

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Detection of Ubiquitination Activity and Identification of Ubiquitinated Substrates Using TR-TUBE

Yukiko Yoshida et al. Methods Enzymol. .

Abstract

Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).

Keywords: Polyubiquitin chain; Substrate identification; TR-TUBE; Ubiquitin ligase; Ubiquitin-binding protein; Ubiquitination.

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