This method was tested on three proteins that are nuclear and found in protein complexes (histone H2B, telomere repeat-binding factor 2 [TRF2], and CENP-A) and two that are cytoplasmic (cyclin B1 and polo-like kinase 4 [PLK4]). The proteins have a rapid t1/2 (average of 19 minutes) with quantitative loss within 80 minutes, and the degradation was inhibited by the proteasome inhibitor MG132 (see Figure 2). Even the usually long-lived centromere-bound protein CENP-A was rapidly degraded by this technique.
Additionally, for histone H2B, it was shown that the degradation was efficient in all cell cycle phases. The effect was shown to be reversible, and for PLK4 new protein was detected within just 10 minutes of washout of the TIR1 inducer indole-3-acetic acid (IAA). In contrast, RNAi is not readily reversible.
The authors point out that it is not yet clear if this method can be used in animals due to possible toxicity of the IAA induction system. An alternate approach to reduce the levels of tagged protein in cells, called the HaloTag approach, has been shown previously to be effective in mice and in zebrafish embryos (Neklesa et al., Nat Chem Biol 2011;7:538–543.). This approach involves expressing a protein fused to a bacterial dehalogenase protein (i.e., the HaloTag), and then a small molecule that binds to this tag is added. The small molecule contains a large hydrophobic motif, such as adamantyl, that mimics the partially denatured state of a protein and leads to the degradation of the protein.
For a protein for which a small molecule cell-permeable inhibitor exists, it will be interesting to compare the various techniques (RNAi, HaloTag, small molecule inhibition) to the AID method described in this article because it is possible that the phenotypes could differ depending on how rapidly and completely the protein of interest can be depleted or inactivated.