title: Small-molecule control of protein degradation using split adaptors.
abstract: Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Davis
authors.forename: Tania A
authors.initials: TA
authors.lastname: Baker
authors.forename: Robert T
authors.initials: RT
authors.lastname: Sauer
mesh_terms: D049071:Endopeptidase Clp / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D056968:Lac Repressors / Q000378:metabolism; D008970:Molecular Weight; D015202:Protein Engineering; D059748:Proteolysis / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D029968:Escherichia coli Proteins; C489310:FtsA protein, E coli; D056968:Lac Repressors; C539064:LacI protein, E coli; D011993:Recombinant Fusion Proteins; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D020123:Sirolimus