Figure 2.
Efficient protein degradation with genome-wide AID libraries. (a) Features of the auxin-inducible degron system in the AID libraries. A protein of interest (POI) with a C-terminal AID* tag can be ubiquitinated and degraded in the presence of 5-Ph-IAA upon expression of the F-box protein OsTir1. (b) Different tags used in the AID libraries. Expression of OsTIR1 is controlled by the galactose-inducible GAL1 promoter. (c) Characterization of the AID-v1 library with fluorescence measurements of colony arrays. Top, for each ORF, mNG-AID*-3myc-tagged strains with (+) or without (−) the GAL1pr-OsTIR1 construct were placed next to each other. A reference strain (ref, with an abundant mNG-tagged protein, for correction of spatial and plate effects) and a strain without a fluorescent tag (bkg, for background correction) were included in each 4 × 4 group. Middle, colony arrays were grown on galactose medium with 1 μM 5-Ph-IAA. Bottom, example heatmap of mNG fluorescence intensities. (d) Protein levels (mNG signal in units of background fluorescence, mNG/bkg) in the AID-v1 library determined according to c. Not detected, strains without a detectable mNG signal in the absence of OsTIR1 (mNG/bkg(OsTIR1−) ≤ 1.2). Detectable proteins were classified by the extent of OsTIR1-dependent degradation (Materials and methods). Top and right, normalized frequency distributions of protein levels in each category. (e) Frequency of OsTIR1-dependent degradation phenotypes in the AID-v1 library according to protein localization, location of the C-terminus (cytosol – in, lumen of organelles or extracellular – out) for membrane proteins according to (Kim et al., 2006) or abundance of the mNG-AID*-3myc-tagged proteins.

Efficient protein degradation with genome-wide AID libraries. (a) Features of the auxin-inducible degron system in the AID libraries. A protein of interest (POI) with a C-terminal AID* tag can be ubiquitinated and degraded in the presence of 5-Ph-IAA upon expression of the F-box protein OsTir1. (b) Different tags used in the AID libraries. Expression of OsTIR1 is controlled by the galactose-inducible GAL1 promoter. (c) Characterization of the AID-v1 library with fluorescence measurements of colony arrays. Top, for each ORF, mNG-AID*-3myc-tagged strains with (+) or without (−) the GAL1pr-OsTIR1 construct were placed next to each other. A reference strain (ref, with an abundant mNG-tagged protein, for correction of spatial and plate effects) and a strain without a fluorescent tag (bkg, for background correction) were included in each 4 × 4 group. Middle, colony arrays were grown on galactose medium with 1 μM 5-Ph-IAA. Bottom, example heatmap of mNG fluorescence intensities. (d) Protein levels (mNG signal in units of background fluorescence, mNG/bkg) in the AID-v1 library determined according to c. Not detected, strains without a detectable mNG signal in the absence of OsTIR1 (mNG/bkg(OsTIR1−) ≤ 1.2). Detectable proteins were classified by the extent of OsTIR1-dependent degradation (Materials and methods). Top and right, normalized frequency distributions of protein levels in each category. (e) Frequency of OsTIR1-dependent degradation phenotypes in the AID-v1 library according to protein localization, location of the C-terminus (cytosol – in, lumen of organelles or extracellular – out) for membrane proteins according to (Kim et al., 2006) or abundance of the mNG-AID*-3myc-tagged proteins.

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