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

Abstract

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 firstorder 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.proteasome | SCF | ubiquitin

A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans

“…One method for achieving this that was recently adapted for C. elegans (Zhang et al, 2015) is based on transplanting the auxin-induced protein degradation system from plants (Holland et al, 2012;Nishimura et al, 2009). In this system, addition of the small molecule auxin activates a plant-specific F-box protein, TIR1, that serves as a substrate recognition component for a Skp1-Cullin-Fbox (SCF) E3 ubiquitin ligase.…”

Repurposing an endogenous degradation system for rapid and targeted depletion ofC. elegansproteins

“…Even during the relatively prolonged 2-day larval phase of development, crucial morphogenetic and signaling events, such as uterine-vulval specification and morphogenesis, occur within short time periods (Kimble, 1981;Sharma-Kishore et al, 1999;Sherwood and Sternberg, 2003;Wang and Sternberg, 2000). An inducible protein degradation system has been described that can deplete proteins in less than 1 hour, but this method requires the addition of auxin, which may not be permeable to many living embryos and organisms (Holland et al, 2012;Nishimura et al, 2009). Therefore, existing protein-degradation strategies are poorly suited to study rapid developmental events in vivo.…”

DNA Damage to a Single Chromosome End Delays Anaphase Onset

“…Cell Lines and Cell Culture-Long nosed potoroo (rat kangaroo), P. tridactylus (PtK2 (male) and PtK1 (female)) kidney epithelial cells (American Type Culture Collection (ATCC), CCL 56 and CCL 35) and TRF2-AID-EYFP PtK2 (where AID represents an auxin-inducible degron that has been shown to degrade the AIDtagged target protein upon the addition of a plant hormone; in these experiments, activation of AID was not induced) (37) were grown in Invitrogen Advanced Minimum Essential Medium (Invitrogen) supplemented with L-glutamine, 4% fetal bovine serum (FBS), and antibiotics. PtK1 cells stably expressing a green fluorescent protein (GFP)-tagged Nbs1 previously generated (27) were incubated with Advanced F-12/DMEM supplemented with L-glutamine, 4% FBS, and antibiotics.…”

“…Generation of Stable PtK Cell Lines-To generate PtK2 cells stably expressing eGFP-53BP1 or TRF2-AID-EYFP, we transiently transfected retroviral plasmids eGFP-53BP1 pLPC (kindly donated by the Denchi laboratory, Scripps Research Institute) and pBABEneo TRF2-AID-EYFP (kindly donated by the Cleveland laboratory, University of California, San Diego) (37) into Phoenix amphotropic packing cell line, using Effectene transfection reagent (Qiagen) according to the manufacturer's instructions. Viral particles were generated using a modified protocol (38,39).…”