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Protocol to synthesize the auxin analog 5-Ph-IAA for conditional protein depletion in C. elegans using the AID2 system.

ABSTRACT: The auxin-inducible degron (AID) system is a broadly used tool for spatiotemporal and reversible control of protein depletion in multiple experimental model systems. AID2 technology relies on a synthetic ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA), for improved specificity and efficiency of protein degradation. Here, we provide a protocol for cost-effective 5-Ph-IAA synthesis utilizing the Suzuki coupling of 5-chloroindole and phenylboronic acid. We describe steps for evaluating the quality of lab-synthesized 5-Ph-IAA using a C. elegans AID2 tester strain.

SUBMITTER: Sural S

PROVIDER: S-EPMC10884774 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Protocol to synthesize the auxin analog 5-Ph-IAA for conditional protein depletion in C. elegans using the AID2 system.

Sural Surojit S Botero Juan Quintero JQ Hobert Oliver O Tekle-Smith Makeda M

STAR protocols 20240219 1

The auxin-inducible degron (AID) system is a broadly used tool for spatiotemporal and reversible control of protein depletion in multiple experimental model systems. AID2 technology relies on a synthetic ligand, 5-phenyl-indole-3-acetic acid (5-Ph-IAA), for improved specificity and efficiency of protein degradation. Here, we provide a protocol for cost-effective 5-Ph-IAA synthesis utilizing the Suzuki coupling of 5-chloroindole and phenylboronic acid. We describe steps for evaluating the quality ...[more]

PMID: 38377002

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Conditional immobilization for live imaging Caenorhabditis elegans using auxin-dependent protein depletion.

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The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans.

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Jasmonoyl-L-Tryptophan Disrupts IAA Activity through the AUX1 Auxin Permease.

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| S-EPMC5420569 | biostudies-literature

TIR1 auxin receptors are implicated in the differential response to 4-Cl-IAA and IAA in developing pea fruit.

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| S-EPMC6382345 | biostudies-literature

Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction.

Project description:Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.

| S-EPMC5467235 | biostudies-literature

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