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 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.

Fig. 1.

Auxin-inducible degradation system for controlling protein stability in human cells. (A) Schematic illustration of the SCF ubiquitin ligase. The variable F-box protein is involved in substrate recruitment. (B) Schematic illustration of the auxin-induced degradation system. Ectopically expressed TIR1 F-box protein incorporates into the SCF complex in human cells. In the presence of the IAA, TIR1 associates with the AID fused to the protein of interest (POI). SCF^TIR1 recruits an E2 ligase and polyubiquitinates the AID, resulting in the degradation of the POI by the proteasome. (C) Parental and TIR1-9Myc expressing DLD-1 and RPE1 were treated with or without IAA for 3 d and processed for flow cytometry. Note expression of TIR1-9Myc and/or treatment with IAA does not cause alterations in the cell cycle profile. (D) DLD-1 or RPE1 cells stably expressing TIR1-9Myc were cotransfected with histone H2B^mRFP or histone H2B^AID-YFP; after 24 h, they were treated with (+) or without (−) IAA for a further 24 h. Fluorescent images show the presence or absence of histone H2B^mRFP and histone H2B^AID-YFP. (Scale bars = 5 μm.)

Fig. 2.

Auxin-induced degradation system is capable of controlling the stability of substrates localized to different regions of cells. (A) AID-YFP–tagged proteins were induced for 8–24 h. Fluorescence images show the appropriate subcellular localization of doxycycline (Dox)-induced transgenes, histone-H2B^AID-YFP (nucleus), Plk4^AID-YFP (centrosome), TRF2^AID-YFP (telomere), CENP-A^AID-YFP (centromere), and cyclin B1^AID-YFP (cytoplasm). (Scale bars = 5 μm.) (B) AID-YFP–tagged proteins were induced for 24 h and subsequently purified from cell lysates using GFP-binder protein (GBP)-coupled beads. Control purifications were performed in parallel from parental cells that do not contain an inducible AID-YFP transgene. GBP-purified protein complexes were analyzed by immunoblotting.

Fig. 3.

Complete depletion of target proteins can be achieved with the auxin-induced degradation system. (A) AID-YFP–tagged proteins were induced for 24 h, and cells were treated with (+) or without (−) IAA for a further 24 h. Remaining protein levels were analyzed by immunoblotting. (B) AID-YFP–tagged proteins were induced for 8–24 h and treated with (+) or without (−) IAA for a further 24 h. Fluorescence images show the localization and level of AID-YFP–tagged target proteins. Bar graphs show the quantification of EYFP levels in fluorescence images. Bars represent the mean of >20 cells per condition. Error bars represent the SEM. (Scale bars = 5 μm.)

Fig. 4.

Protein degradation occurs rapidly following IAA addition. AID-YFP–tagged proteins were induced for 8–24 h, and cells were treated with IAA in the presence or absence of the proteasome inhibitor MG132. EYFP levels were monitored by fluorescence time-lapse microscopy beginning 9–10 min after drug addition. (Left) Graphs show EYFP fluorescence at various times after drug addition. (Right) Graphs show the logarithmic plot of EYFP fluorescence at various time points after filming began. Graphs represent the mean of 7–20 cells per condition. Error bars represent the SEM.

Fig. 5.

Protein degradation occurs during all phases of the cell cycle. (A) Histone H2B^AID-YFP was induced for 8 h, and cells were treated with IAA in the presence or absence of the proteasome inhibitor MG132. EYFP levels were monitored by fluorescence time-lapse microscopy beginning 10 min after drug addition. (Upper) Graph shows EYFP fluorescence at various times after drug addition. (Lower) Graph shows the logarithmic plot of EYFP fluorescence at various time points after filming began. Graphs represent the mean of 19 (IAA + MG132) or 32 (IAA) cells per condition, and error bars represent the SEM. Representative images show the level of histone H2B^AID-YFP and corresponding differential interference contrast (DIC) images at the indicated times. (B) Same as in A except that in one condition, cells were arrested in mitosis with nocodazole for 6 h before the beginning of filming. Destruction was quantified in mitotically arrested cells, which can be distinguished by their highly condensed chromosomes. Graphs represent the mean of 10 (mitosis) or 32 (interphase) cells per condition. Error bars represent the SEM. (Scale bars = 5 μm.)

Fig. 6.

Protein depletion is readily reversible on removal of IAA. (A) Plk4^AID-YFP expression was induced for 16 h, and cells were treated with IAA for 20 min. Cells were washed to remove IAA and supplemented with doxycycline (DOX) for a further 2 h. Cells were fixed at the indicated times after drug addition, and the localization of Plk4^AID-YFP and TIR1-9Myc was determined by fluorescent microscopy. In the presence of IAA, TIR1-9Myc is reversibly recruited to the centrosome. (B) Plk4^AID-YFP expression was induced for 16 h, and cells were treated with IAA for a further 2 h. Cells were washed to remove IAA and supplemented with doxycycline, and levels of Plk4^AID-YFP fluorescence were monitored by fluorescence time-lapse microscopy. The graph represents the mean of 10 cells, and error bars represent the SEM. Representative images show the level of Plk4^AID-YFP and corresponding differential interference contrast (DIC) images at the indicated times. (C) Expression of CENP-A^AID-YFP was induced for 8 h, and cells were treated with IAA for a further 2 h. Cells were washed to remove IAA and supplemented with doxycycline, and recruitment of CENP-A^AID-YFP to the centromere was monitored by fluorescence time-lapse microscopy. Note that although newly synthesized CENP-A^AID-YFP accumulates diffusely in the nuclei of cells, loading of CENP-A^AID-YFP onto centromeres is delayed until late telophase/early G1. (Scale bars = 5 μm.)

Fig. 7.

Functional replacement of siRNA-depleted BubR1 with an AID-tagged BubR1 transgene. (A) Schematic outlines the experimental strategy used to replace endogenous BubR1 with ^GFP-AIDBubR1. (B) Immunoblot shows the levels of various proteins at 48 h after siRNA depletion of endogenous BubR1. (C) Graph shows the fraction of cells remaining in mitosis at various times after addition of nocodazole. There were >95 cells per condition from two independent experiments. Dox, doxycycline.

Fig. P1.

Schematic illustration of the auxin-induced degradation system. Ectopically expressed TIR1 F-box protein is incorporated into the SCF complex in human cells. In the presence of the auxin hormone indole-3-acetic acid (IAA), TIR1 associates with the AID fused to the protein of interest (POI). SCF^TIR1 recruits an E2 ligase and adds a chain of ubiquitin molecules to the AID, resulting in the degradation of the POI by the proteasome.