title: Intracellular context affects levels of a chemically dependent destabilizing domain.
abstract: 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.
authors.affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, United States of America.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Sellmyer
authors.forename: Ling-chun
authors.initials: LC
authors.lastname: Chen
authors.forename: Emily L
authors.initials: EL
authors.lastname: Egeler
authors.forename: Rishi
authors.initials: R
authors.lastname: Rakhit
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
mesh_terms: D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: D004268:DNA-Binding Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D014415:Tunicamycin