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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Vydáno v:Proceedings of the National Academy of Sciences - PNAS Ročník 109; číslo 49; s. E3350
Hlavní autoři: Holland, Andrew J, Fachinetti, Daniele, Han, Joo Seok, Cleveland, Don W
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 04.12.2012
Témata:
Cell Line, Tumor
Gene Expression Regulation - genetics
Half-Life
Humans
Indoleacetic Acids - metabolism
Kinetics
M Phase Cell Cycle Checkpoints - genetics
M Phase Cell Cycle Checkpoints - physiology
Molecular Biology - methods
Proteins - metabolism
Proteolysis
RNA Interference
SKP Cullin F-Box Protein Ligases - metabolism
ISSN:1091-6490, 1091-6490
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Shrnutí: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.
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ISSN:1091-6490
1091-6490
DOI:10.1073/pnas.1216880109