Abstract
Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).
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References
Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479
Rakhit R, Navarro R, Wandless TJ (2014) Chemical biology strategies for posttranslational control of protein function. Chem Biol 21(9):1238–1252
Kanemaki MT (2013) Frontiers of protein expression control with conditional degrons. Pflugers Arch 465(3):419–425
Ravid T, Hochstrasser M (2008) Diversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol 9(9):679–690
Jariel-Encontre I, Bossis G, Piechaczyk M (2008) Ubiquitin-independent degradation of proteins by the proteasome. Biochim Biophys Acta 1786(2):153–177
Jungbluth M, Renicke C, Taxis C (2010) Targeted protein depletion in Saccharomyces cerevisiae by activation of a bidirectional degron. BMC Syst Biol 4:176
Gautier A, Gauron C, Volovitch M, Bensimon D, Jullien L, Vriz S (2014) How to control proteins with light in living systems. Nat Chem Biol 10(7):533–541
Zhang K, Cui B (2015) Optogenetic control of intracellular signaling pathways. Trends Biotechnol 33(2):92–100
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):619–626
Bonger KM, Rakhit R, Payumo AY, Chen JK, Wandless TJ (2014) General method for regulating protein stability with light. ACS Chem Biol 9(1):111–115
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 8:128
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):6359–6370
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):947–962
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):339–346
Taxis C, Knop M (2006) System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques 40(1):73–78
Taxis C, Knop M (2012) TIPI: TEV protease-mediated induction of protein instability. Methods Mol Biol 832:611–626
Ausubel FM, Kingston RE, Seidman FG, Struhl K, Moore DD, Brent R, Smith FA (eds) (1995) Current protocols in molecular biology. Wiley, New York
Yaffe MP, Schatz G (1984) Two nuclear mutations that block mitochondrial protein import in yeast. Proc Natl Acad Sci U S A 81(15):4819–4823
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
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):4350–4354
Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth K, Schiebel E (1999) Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15(10B):963–972
Acknowledgements
We thank D. Störmer for her excellent technical assistance. This work was supported by the DFG grant TA320/3-1 and the DFG-funded graduate school GRK1216.
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Lutz, A.P., Renicke, C., Taxis, C. (2016). Controlling Protein Activity and Degradation Using Blue Light. In: Kianianmomeni, A. (eds) Optogenetics. Methods in Molecular Biology, vol 1408. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3512-3_5
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DOI: https://doi.org/10.1007/978-1-4939-3512-3_5
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