The C′ AID-GFP library has robust responsiveness across cellular compartments. (A) Summary schematics of the expansion of the AID system to a whole genome collection. The original C′ SWAT library was used to generate the C′ AID-GFP library using an automated process of mating, selection, sporulation, and SWATing. In the final library, every strain expresses the OsTIR1(F74G) protein, and a different gene is fused to the AID-GFP tag. Fluorescence was detected in 60% of the strains under standard laboratory conditions (log-phase cells in rich media). Of those, 86% responded to the induction with a measurable protein depletion. (B) Dot plot showing the mean GFP fluorescence intensity per cell for each strain before or after 24 h of induction time. The background autofluorescence noted A.F. calculated as the mean fluorescence of control strains is marked in dotted lines with a confidence interval of two standard deviations in each direction. The diagonal line indicates the expected location for irresponsive strains. Many strains die after induction (triangles), making their fluorescence signal not informative, while most of the irresponsive strains encode for mitochondrial proteins (green edge). (C) Fluorescent images of selected proteins were imaged after no (0 min), short (30 min), or long (24 h) periods of induction. In the case of dually localized proteins, induction depletes one of the subpopulations with faster kinetics. Gdp1 remains visible in peroxisomes, Vma1 in endosomes and Rex2 in mitochondria while their respective cytosolic, vacuolar, or nuclear subpopulations are decreased. In cases where the signal is too dim, the outline of the cells is depicted with a dotted line. Scale bar: 5 µm. (D and E) Schematics of a yeast cell in panel D and a mitochondrion in panel E displaying the responsiveness per subcellular compartment. Proteins in membrane-bound organelles are more protected from degradation but the responsiveness across subcellular compartments is still high.