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Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_15, © Springer Science+Business Media LLC 2017
Chapter 15
Development of a Synthetic Switch to Control Protein
Stability in Eukaryotic Cells with Light
Christof Taxis
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 lightactivated, 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.
Key words Optogenetics, Protein degradation, Proteasome, Ubiquitin-independent degradation,
Protein stability, Synthetic biology, LOV2 domain, Blue light, Degron
1 Introduction
Regulated proteolysis is involved in virtually each and every regulatory process in eukaryotic cells. Continuously, protein levels are
adjusted during passage through the cell cycle or as reaction on
changing environmental conditions, which results in the degradation of selected proteins by the ubiquitin-proteasome system.
Ubiquitin is a small protein that serves among other things as a
marker for proteolysis by the proteasome. In general, an enzymatic cascade of ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2) and ubiquitin-protein ligase (E3)
modifies a protein substrate with several moieties of ubiquitin in
the form of a linear chain, which targets the protein for destruction
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_15, © Springer Science+Business Media LLC 2017
Chapter 15
Development of a Synthetic Switch to Control Protein
Stability in Eukaryotic Cells with Light
Christof Taxis
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 lightactivated, 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.
Key words Optogenetics, Protein degradation, Proteasome, Ubiquitin-independent degradation,
Protein stability, Synthetic biology, LOV2 domain, Blue light, Degron
1 Introduction
Regulated proteolysis is involved in virtually each and every regulatory process in eukaryotic cells. Continuously, protein levels are
adjusted during passage through the cell cycle or as reaction on
changing environmental conditions, which results in the degradation of selected proteins by the ubiquitin-proteasome system.
Ubiquitin is a small protein that serves among other things as a
marker for proteolysis by the proteasome. In general, an enzymatic cascade of ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2) and ubiquitin-protein ligase (E3)
modifies a protein substrate with several moieties of ubiquitin in
the form of a linear chain, which targets the protein for destruction