Fig. 1: Principles and validation of the yGPS-peptidome method.
From: Conserved degronome features governing quality control associated proteolysis
a Schematic representation of yGPS-peptide library (yGPS-Plib) where a DNA library comprised of 51-mer tiled DNA fragments from 326 proteins, represented by different colors, are cloned in-frame downstream to GFP in the yGPS-P vector. The single mRNA product consists of yeCherry-IRES-yeGFP-peptide. DNA and protein sequences illustrate the cloning site that pursues GFP and the pentameric linker. b Flowchart of the degron screen. yGPS-Plib is transformed into yeast, followed by flow cytometry or FACS. c Flow cytometry histograms of CL1 and DegAB degrons appended to yGPS-P, in wild type and doa10∆ cells. Fluorescence emissions of 10,000 cells were determined for each condition. Stability scale: Median value of the yeG/yeC ratio in empty vector (EV) control was set as one. All other histograms were distributed accordingly. d Immunoblot analysis of the levels of CL1- and DegAB- appended GFP compared to Cherry loading control. This analysis was repeated three times. yeG: yeGFP. e Flow cytometry histogram of normalized yeG/yeC in yGPS-Plib compared to empty vector control. Stability scale was set as explicated in Fig. 1c. f, g Proteasome dependence of GFP-appended peptides. Cells expressing yGPS-Plib were treated with 10 µM Bortezomib (BZ) for 4 h or with DMSO vehicle control. Cells were subjected to immunoblotting with anti-ubiquitin Abs (Fig. 1f), or to flow cytometry analysis (Fig. 1g). The immunoblot was repeated two times. h Flow cytometry analysis of a pre-selected degron library composed of top10% degrons, with or without BZ. Stability scale was set as explicated in Fig. 1c. Source data for panels c-h are provided as a Source Data file.
