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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Nat Struct Mol Biol. 2015 Dec 14;23(1):59–66. doi: 10.1038/nsmb.3146

Figure 3.

Figure 3

Use of the auxin-inducible degron (AID) system to selectively degrade essential exocyst proteins from yeast. (a) Schematic of the AID system. The auxin-inducible degron (AID) tag from Arabidopsis thaliana was fused to the C-terminus of exocyst subunits at their genomic locus in yeast strains constitutively expressing OsTIR1 (F-box transport inhibitor response 1) protein. Upon treatment with the natural plant hormone auxin (IAA=Indole 3-acetic acid), the SCF-OsTIR1 E3 Ubiquitin ligase complex is activated, which then recruits E2 Ubiquitin ligases for polyubiquitination of the AID-tagged protein. The AID-tagged protein is then rapidly degraded by the proteasome41,42. (b) AID-tagged exocyst strains were tested for growth by serial dilution growth assay on YPD plates containing the indicated amount of IAA. Suppressor colonies can be seen in some dilutions. (c) Degradation of exocyst subunits in these strains was confirmed by western blotting lysates from NaOH/SDS lysis. (−) denotes untreated strains and (+) treated with IAA. All subunits were degraded to <10–12% of starting protein level. Asterisks indicate the AID-tagged exocyst subunit in blots where antibodies also bind non-exocyst subunits.