Auxin-Inducible Degron System for Depletion of Proteins in Saccharomyces cerevisiae

Abstract

The auxin-inducible degron (AID) is a powerful tool that is used for depletion of proteins to study their function in vivo. This method can conditionally induce the degradation of any protein by the proteasome simply by the addition of the plant hormone auxin. This approach is particularly valuable to study the function of essential proteins. The protocols provided here describe the steps to construct the necessary strains and to optimize auxin-inducible depletion in Saccharomyces cerevisiae. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Construction of TIR1-expressing strains by transformation Basic Protocol 2: Tagging a yeast protein of interest with an auxin-inducible degron Support Protocol: Construction of depletion strains by genetic crosses Basic Protocol 3: Optimization for depletion of the auxin-inducible-degron-tagged protein.

Keywords: auxin; conditional depletion; degron; yeast.

Figure 1.

Depletion of protein by the AID system. The protein of interest (POI) is fused to an AID tag in a yeast strain that expresses the TIR1 protein. Addition of auxin promotes the interaction between TIR1-containing Skp1-F-box-Cullin E3 ubiquitin ligase, which recruits the E2 ubiquitin conjugating enzyme. Together they polyubiquitinate the AID tag, thereby targeting the AID-tagged protein for degradation by the proteasome. Image created with BioRender.

Figure 2.

pTIR1 plasmid and restriction fragment for transformation of yeast. The pTIR1 plasmid (9719 bp) has regions homologous (gray) to 646 bp upstream and 525 bp downstream of the S. cerevisiae LEU2 gene. Between these homology regions is the Candida glabrata LEU2 (Cg LEU2) gene with its own promoter and terminator, and the OsTIR1 gene is expressed under the control of the S. cerevisiae GPD1 promoter. The Cg LEU2 gene complements the S. cerevisiae leu2Δ mutation, thus allowing selection of yeast containing the plasmid fragment on medium lacking leucine. The fragment (6221 bp) used for transformation is released by digestion with Pme1. The arrows indicate the direction of genes. Os: Oryza sativae, Ca tADH1: Candida albicans ADH1 terminator. Image created with BioRender.

Figure 3.

pScAID2 plasmid. The cassette carrying 3xV5-IAA7-KanMX6 is amplified with two primers as indicated. The 3’ part of the primers has 40 base pairs of homologous sequence to the site where the tag is to be integrated. GOI: gene of interest. Image created with BioRender.

Figure 4.

Checking the AID tag by colony PCR. A PCR reaction with three primers is set up. Primers ‘a’ and ‘b’ will give two different product sizes for the two possible different alleles (with and without the AID sequence), whereas primers ‘a’ and ‘c’ will generate a product only for the correct fusion. GOI: gene of interest. Image created with BioRender.

Figure 5.

Time course experiment to determine the time required for depletion of the protein of interest. The Spn1–3xV5-AID tagged strain was grown in YPD and treated with 100 μM IAA for the indicated time (in minutes), and protein extracts were prepared. The levels of Spn1 protein were analyzed by western blotting using anti-Spn1 antisera. Pgk1 protein was used as a loading control.