title: Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.
abstract: In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.
authors.affiliation: Department of Biology/Genetics, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35043, Marburg, Germany. taxis@staff.uni-marburg.de.|Department of Chemistry/Biochemistry, Philipps-Universität Marburg, Hans-Meerwein-Strasse 4, 35043, Marburg, Germany. taxis@staff.uni-marburg.de.
authors.forename: Christof
authors.initials: C
authors.lastname: Taxis
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D008027:Light; D009955:Ornithine Decarboxylase / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D055550:Protein Stability; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D010750:Phosphoproteins; C103646:NPH1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase
keywords: Blue light; Degron; LOV2 domain; Optogenetics; Proteasome; Protein degradation; Protein stability; Synthetic biology; Ubiquitin-independent degradation