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The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.

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.