Real-time monitoring of ubiquitination in living cells by BRET

Julie Perroy, Stephanie Pontier, Pascale G. Charest, Muriel Aubry, Michel Bouvier

Research output: Contribution to journalArticle

126 Scopus citations

Abstract

Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET), we measured the ubiquitination of β-arrestin 2, a regulatory protein implicated in the modulation of G protein–coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of β-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.

Original languageEnglish (US)
Pages (from-to)203-208
Number of pages6
JournalNature Methods
Volume1
Issue number3
DOIs
StatePublished - Dec 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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