Hills-Muckey, Kelly, Martinez, Michael AQ, Stec, Natalia, Hebbar, Shilpa, Saldanha, Joanne, Medwig-Kinney, Taylor N, Moore, Frances EQ, Ivanova, Maria, Morao, Ana, Ward, JD, Moss, Eric G, Ercan, Sevinc, Zinovyeva, Anna Y, Matus, David Q, Hammell, Christopher M (October 2021) An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans. Genetics: a periodical record of investigations bearing on heredity and variation. ISSN 1943-2631
Abstract
The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.
| Item Type: | Paper |
|---|---|
| Subjects: | organism description > plant > Arabidopsis organism description > animal > C elegans organism description > animal bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > auxin organism description > plant |
| CSHL Authors: | |
| Communities: | CSHL labs > Hammell C. lab CSHL Cancer Center Program CSHL Cancer Center Program > Gene Regulation and Inheritance Program |
| SWORD Depositor: | CSHL Elements |
| Depositing User: | CSHL Elements |
| Date: | 12 October 2021 |
| Date Deposited: | 10 Nov 2021 17:59 |
| Last Modified: | 09 Feb 2024 18:56 |
| PMCID: | PMC9097248 |
| URI: | https://repository.cshl.edu/id/eprint/40417 |
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