Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells

Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functi...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 109; no. 49; pp. E3350 - E3357
Main Authors Holland, Andrew J, Fachinetti, Daniele, Han, Joo Seok, Cleveland, Don W
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 04.12.2012
National Acad Sciences
SeriesPNAS Plus
Subjects
auxins
Biodegradation
Biological Sciences
Cell cycle
Cell Line, Tumor
Gene Expression Regulation - genetics
Genotype & phenotype
Half-Life
Humans
Indoleacetic Acids - metabolism
Kinetics
M Phase Cell Cycle Checkpoints - genetics
M Phase Cell Cycle Checkpoints - physiology
Mammals
mitosis
Molecular Biology - methods
phenotype
PNAS Plus
Proteins
Proteins - metabolism
Proteolysis
Ribonucleic acid
RNA
RNA Interference
SKP Cullin F-Box Protein Ligases - metabolism
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Summary:Inducible degradation is a powerful approach for identifying the function of a specific protein or protein complex. Recently, a plant auxin-inducible degron (AID) system has been shown to degrade AID-tagged target proteins in nonplant cells. Here, we demonstrate that an AID-tagged protein can functionally replace an endogenous protein depleted by RNAi, leading to an inducible null phenotype rapidly after auxin addition. The AID system is shown to be capable of controlling the stability of AID-tagged proteins that are in either nuclear or cytoplasmic compartments and even when incorporated into protein complexes. Induced degradation occurs rapidly after addition of auxin with protein half-life reduced to as little as 9 min and proceeding to completion with first-order kinetics. AID-mediated instability is demonstrated to be rapidly reversible. Induced degradation is shown to initiate and continue in all cell cycle phases, including mitosis, making this system especially useful for identifying the function(s) of proteins of interest during specific points in the mammalian cell cycle.
Bibliography:http://dx.doi.org/10.1073/pnas.1216880109
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Contributed by Don W. Cleveland, October 9, 2012 (sent for review August 29, 2012)
Author contributions: A.J.H., D.F., J.S.H., and D.W.C. designed research; A.J.H., D.F., and J.S.H. performed research; A.J.H., D.F., J.S.H., and D.W.C. analyzed data; and A.J.H., D.F., and D.W.C. wrote the paper.
1A.J.H. and D.F. contributed equally to this work.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.1216880109