Synthetic Protein Switches pp 241-255 | Cite as
Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light
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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.
Key words
Optogenetics Protein degradation Proteasome Ubiquitin-independent degradation Protein stability Synthetic biology LOV2 domain Blue light DegronNotes
Acknowledgments
I thank D Störmer for her excellent technical assistance as well as S Hepp, C Renicke, and J Trauth for helpful comments on the manuscript. A Batschauer is acknowledged for the opportunity to use his photobiology equipment. This work was supported by the DFG grant TA320/3-1 and the DFG-funded graduate school GRK1216.
References
- 1.Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479CrossRefGoogle Scholar
- 2.Pickart CM (2001) Ubiquitin enters the new millennium. Mol Cell 8(3):499–504CrossRefGoogle Scholar
- 3.Ravid T, Hochstrasser M (2008) Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 9(9):679–690CrossRefGoogle Scholar
- 4.Dohmen RJ, Wu P, Varshavsky A (1994) Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science 263(5151):1273–1276CrossRefGoogle Scholar
- 5.Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ (2006) A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126(5):995–1004CrossRefGoogle Scholar
- 6.Taxis C, Stier G, Spadaccini R, Knop M (2009) Efficient protein depletion by genetically controlled deprotection of a dormant N-degron. Mol Syst Biol 5:267CrossRefGoogle Scholar
- 7.Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12):917–922CrossRefGoogle Scholar
- 8.Jungbluth M, Mosch HU, Taxis C (2012) Acetate regulation of spore formation is under the control of the Ras/cyclic AMP/protein kinase A pathway and carbon dioxide in Saccharomyces cerevisiae. Eukaryot Cell 11(8):1021–1032CrossRefGoogle Scholar
- 9.Renicke C, Schuster D, Usherenko S, Essen LO, Taxis C (2013) A LOV2 domain-based optogenetic tool to control protein degradation and cellular function. Chem Biol 20(4):619–626CrossRefGoogle Scholar
- 10.Christie JM, Salomon M, Nozue K, Wada M, Briggs WR (1999) LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci U S A 96(15):8779–8783CrossRefGoogle Scholar
- 11.Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropin light switch. Science 301(5639):1541–1544CrossRefGoogle Scholar
- 12.Erales J, Coffino P (2014) Ubiquitin-independent proteasomal degradation. Biochim Biophys Acta 1843(1):216–221CrossRefGoogle Scholar
- 13.Takeuchi J, Chen H, Coffino P (2007) Proteasome substrate degradation requires association plus extended peptide. EMBO J 26(1):123–131CrossRefGoogle Scholar
- 14.Takeuchi J, Chen H, Hoyt MA, Coffino P (2008) Structural elements of the ubiquitin-independent proteasome degron of ornithine decarboxylase. Biochem J 410(2):401–407CrossRefGoogle Scholar
- 15.Jungbluth M, Renicke C, Taxis C (2010) Targeted protein depletion in Saccharomyces cerevisiae by activation of a bidirectional degron. BMC Syst Biol 4:176CrossRefGoogle Scholar
- 16.Usherenko S, Stibbe H, Musco M, Essen LO, Kostina EA, Taxis C (2014) Photo-sensitive degron variants for tuning protein stability by light. BMC Syst Biol 8:128CrossRefGoogle Scholar
- 17.Pereira G, Tanaka TU, Nasmyth K, Schiebel E (2001) Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex. EMBO J 20(22):6359–6370CrossRefGoogle Scholar
- 18.Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41CrossRefGoogle Scholar
- 19.van Leeuwen J, Andrews B, Boone C, Tan G (2015) Rapid and efficient plasmid construction by homologous recombination in yeast. Cold Spring Harb Protoc 2015(9): pdb prot085100Google Scholar
- 20.Renicke C, Spadaccini R, Taxis C (2013) A tobacco etch virus protease with increased substrate tolerance at the P1' position. PLoS One 8(6):e67915CrossRefGoogle Scholar
- 21.Ausubel FM, Kingston, R.E., Seidman, F.G., Struhl, K., Moore, D.D., Brent, R., and Smith, F.A. (eds) (1995) Current protocols in molecular biology. John Wiley and Sons, New York, NYGoogle Scholar
- 22.Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16(5–6):339–346CrossRefGoogle Scholar
- 23.Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21(11):947–962CrossRefGoogle Scholar
- 24.Taxis C, Knop M (2006) System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques 40(1):73–78CrossRefGoogle Scholar
- 25.Taxis C, Knop M (2012) TIPI: TEV protease-mediated induction of protein instability. Methods Mol Biol 832:611–626CrossRefGoogle Scholar
- 26.Lutz AP, Renicke C, Taxis C (2016) Controlling protein activity and degradation using blue light. Methods Mol Biol 1408:67–78CrossRefGoogle Scholar
- 27.Yaffe MP, Schatz G (1984) Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci U S A 81(15):4819–4823CrossRefGoogle Scholar
- 28.Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685CrossRefGoogle Scholar
- 29.Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9):4350–4354CrossRefGoogle Scholar
- 30.Ninnemann H, Butler WL, Epel BL (1970) Inhibition of respiration in yeast by light. Biochim Biophys Acta 205(3):499–506CrossRefGoogle Scholar
- 31.Yee EF, Diensthuber RP, Vaidya AT, Borbat PP, Engelhard C, Freed JH, Bittl R, Moglich A, Crane BR (2015) Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue. Nat Commun 6:10079CrossRefGoogle Scholar