The mIAA7 degron improves auxin-mediated degradation inCaenorhabditis elegans

Abstract

Auxin-inducible degradation (AID) is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the AID system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in C. elegans, as previously reported in human cells. We tested depletion of a series of proteins with various sub-cellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for five out of six proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for two of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system (C.e.AID2), which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the AID system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

Cell cycle perturbation uncouples mitotic progression and invasive behavior in a post-mitotic cell

“…2B,F). These ambiguities could be addressed in future studies by creating and/or combining conditional null alleles using the auxin-inducible degron (AID) system (Ashley et al, 2021; Hills-Muckey et al, 2021; Martinez et al, 2020; Negishi et al, 2021; Sepers et al, 2022; Xiao et al, 2023; Zhang et al, 2015) or an analogous approach (Nance and Frøkjær-Jensen, 2019).…”

Reevaluating the relationship between EGL-43 (EVI1) and LIN-12 (Notch) during C. elegans anchor cell invasion

“…The auxin-inducible degron (AID) system enables rapid degradation of C. elegans proteins ( Ashley et al, 2021 ; Hills-Muckey et al, 2021 ; Martinez et al, 2020 ; Negishi et al, 2022 ; Sepers et al, 2022 ; Zhang et al, 2015 ). It requires a minimal AID tag on the protein of interest (POI), expression of the Arabidopsis F-box protein TIR1, and exogenous exposure to the plant hormone auxin.…”

Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron

“…In Caenorhabditis elegans , higher protein degradation efficiency was seen with mIAA7 than with AID* ( Sepers et al, 2022 ). Though derived from different A. thaliana IAA response transcription repressor proteins (IAA7 vs. IAA17), and differ in primary sequences, both mIAA7 (IAA7 37-104) and AID* (IAA17 71-114) degrons contain the conserved domain II F-box protein binding motif, yet the former has a longer amino-terminal extension (which was reported to be important to TIR1-mediated protein degradation in plants) and contains no putative ubiquitination sites (and hence the degron tag itself is not ubiquitinated) ( Sepers et al, 2022 ). The mIAA7 degron was first reported by Li et al (2019) and was shown to be an optimal degron to pair with the A. thaliana AFB2 F-box protein to enable protein degradation.…”

“…The mIAA7 degron was first reported by Li et al (2019) and was shown to be an optimal degron to pair with the A. thaliana AFB2 F-box protein to enable protein degradation. Sepers et al (2022) used mIAA7 in combination with AtOsTIR1 F79G in their AID system. In the present study, we showed that mIAA7 worked well in combination with OsTIR1 F74A and either 5-Ad-IAA or 5-Ph-IAA in inducing rapid auxin-dependent protein degradation in Y. lipolytica .…”