An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans

Kelly Hills-Muckey, Michael A.Q. Martinez, Natalia Stec, Shilpa Hebbar, Joanne Saldanha, Taylor N. Medwig-Kinney, Frances E.Q. Moore, Maria Ivanova, Ana Morao, J. D. Ward, Eric G. Moss, Sevinc Ercan, Anna Y. Zinovyeva, David Q. Matus, Christopher M. Hammell

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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.

Original languageEnglish (US)
Article numberiyab174
JournalGenetics
Volume220
Issue number2
DOIs
StatePublished - Feb 2022

All Science Journal Classification (ASJC) codes

  • Genetics

Fingerprint

Dive into the research topics of 'An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans'. Together they form a unique fingerprint.

Cite this