Fig. 3
From: Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy
Degradation rates and subcellular localization of fluorescent proteins and the influence of tags. (a) Structure of eGFP31 (PDB 5MA3), commonly referred to as Clontech-eGFP, with native N- and C-termini (MVSK… and …LYK, respectively, see sequence in Supplementary Fig. S18b) indicated as blue and red spheres. Lysines, representing possible ubiquitination sites are shown in blue. Tags investigated are listed next to the structure with names and sequences. (b-h) Bars indicate the mean degradation rate constants from fitting an exponential decay model on the single-cell level, with error bars showing the standard deviation. Pairwise statistical analysis of differences between rate averages was performed using Student’s t-test, as shown in Supplementary Fig. S3-S7. Additional MG132 controls are shown in Supplementary Fig. S22. The purple bar refers to the reference construct GS-eGFP (starting with GSMVSK). Since the y-axes of the panels are different, a purple dashed line indicates this degradation rate. (b) Comparison of fluorescent proteins, starting either with glycine-serine (group 1), or preceded by His8-biotinylated Avi-tag (group 2, with biotinylation being indicated by red star). Successful purification was confirmed as shown in Supplementary Fig. S11 and 14. (c) Influence of tags. Samples are denoted as shown in a, with sample quality represented in Supplementary Fig. S12. (d) Tags introducing free cysteines for subsequent maleimide-thiol conjugation with or without the conjugated dyes TMR, Cy5-2 and Cy5-432. Quality control is shown in Supplementary Fig. S13. (e) Influence of the N-terminal amino acid X on eGFP degradation, where the sequence starts with XSMVSK. The variants were produced by TEV cleavage in vitro before injection. Quality control is represented in Supplementary Fig. S14. (f) Influence of N-terminal acetylation on XS-eGFP variants. The presence of the N-terminal acetyl group was verified using mass spectrometry (Supplementary Fig. S15). (g) Degradation rate analysis of eGFP without N-terminal GSM, and without initiator methionine, thus starting with VSK (termed [-M]-eGFP). Proteasomal inhibition was performed using MG132. The rightmost [-M]-eGFP sample was expressed and purified from the cytosol of HEK293 cells using anion-exchange chromatography, while all others were purified from the cytosol of E. coli. (h) Co-injection of degradation-inducing bioPROTAC (see main text) with indicated samples. (i) Subcellular localization of fluorescent proteins, determined by fluorescence confocal microscopy two hours after injection, showing homogenous signal distribution, except for untagged GS-mNG and GS-mR3. Time-resolved export of GS-mNG upon microinjection is shown in accompanying Supplementary Figs. S19 and S20.
