Ligand-switchable Substrates for a Ubiquitin-Proteasome System

• Published in: The Journal of biological chemistry Vol. 286; no. 36; pp. 31328 - 31336
• Main Authors: Egeler, Emily L., Urner, Lorenz M., Rakhit, Rishi, Liu, Corey W., Wandless, Thomas J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.09.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Line; Humans; Ligands; Mice; Proteasome; Proteasome Endopeptidase Complex - metabolism; Protein Degradation; Protein Folding; Protein Quality Control; Protein Stability; Protein Synthesis and Degradation; Protein Turnover; Protein Unfolding; Substrate Specificity; Tacrolimus Binding Protein 1A - metabolism; Tacrolimus Binding Proteins; Technology Development; Ubiquitin - metabolism; Ubiquitination; Protein Quality Control; Ubiquitination; Protein Turnover; Protein Stability; Technology Development; Protein Degradation; Protein Folding; Proteasome
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.264101

Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.