title: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron.
abstract: Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology.
authors.affiliation: EMBL, Cell Biology and Biophysics Unit, Meyerhofstr. 1, Heidelberg, Germany.
authors.forename: Christof
authors.initials: C
authors.lastname: Taxis
authors.forename: Gunter
authors.initials: G
authors.lastname: Stier
authors.forename: Roberta
authors.initials: R
authors.lastname: Spadaccini
authors.forename: Michael
authors.initials: M
authors.lastname: Knop
mesh_terms: D010450:Endopeptidases / Q000235:genetics*; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C099488:TEV protease