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

124 Citations (Scopus)

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 beta-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 beta-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
JournalNat Methods
Volume1
Issue number3
StatePublished - Dec 2004
Externally publishedYes

Fingerprint

Bioluminescence
Ubiquitination
Energy Transfer
Energy transfer
Cells
Arrestin
Monitoring
Proteins
Mammals
G-Protein-Coupled Receptors
Ubiquitin
Yeast
Assays
Modulation
Protein Stability
Kinetics
Post Translational Protein Processing
Yeasts
Kidney

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Cite this

Perroy, J., Pontier, S., Charest, P. G., Aubry, M., & Bouvier, M. (2004). Real-time monitoring of ubiquitination in living cells by BRET. Nat Methods, 1(3), 203-208.

Real-time monitoring of ubiquitination in living cells by BRET. / Perroy, Julie; Pontier, Stephanie; Charest, Pascale G; Aubry, Muriel; Bouvier, Michel.

In: Nat Methods, Vol. 1, No. 3, 12.2004, p. 203-208.

Research output: Contribution to journalArticle

Perroy, J, Pontier, S, Charest, PG, Aubry, M & Bouvier, M 2004, 'Real-time monitoring of ubiquitination in living cells by BRET.', Nat Methods, vol. 1, no. 3, pp. 203-208.
Perroy J, Pontier S, Charest PG, Aubry M, Bouvier M. Real-time monitoring of ubiquitination in living cells by BRET. Nat Methods. 2004 Dec;1(3):203-208.
Perroy, Julie ; Pontier, Stephanie ; Charest, Pascale G ; Aubry, Muriel ; Bouvier, Michel. / Real-time monitoring of ubiquitination in living cells by BRET. In: Nat Methods. 2004 ; Vol. 1, No. 3. pp. 203-208.
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