Abstract
Ubiquitin ligases (E3s) function by binding to both a protein substrate and to ubiquitin-conjugating enzymes (E2s) bound to ubiquitin. E3s facilitate the transfer of ubiquitin from the E2 active site to an E3-bound substrate. Thus, the affinity of the interaction of an E2 with its E3 partner is of considerable interest. The purpose of this work is to (1) provide protocols for the purification of the human E2 Cdc34, as well as for some additional protein components needed for the assays described here whose purification protocols haven’t been described elsewhere in detail; (2) provide the researcher with critical information regarding the proper long-term storage of these enzymes to retain maximal activity; (3) provide a protocol to benchmark Cdc34 activity with previously described activity levels in the literature; and (4) provide a simple and rapid means of measuring E2 affinity for an E3.
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References
Yau R, Rape M (2016) The increasing complexity of the ubiquitin code. Nat Cell Biol 18(6):579–586. https://doi.org/10.1038/ncb3358
Zheng N, Shabek N (2017) Ubiquitin ligases: structure, function, and regulation. Annu Rev Biochem 86:129–157. https://doi.org/10.1146/annurev-biochem-060815-014922
Swatek KN, Komander D (2016) Ubiquitin modifications. Cell Res 26(4):399–422. https://doi.org/10.1038/cr.2016.39
Mirzaei H, Rogers RS, Grimes B, Eng J, Aderem A, Aebersold R (2010) Characterizing the connectivity of poly-ubiquitin chains by selected reaction monitoring mass spectrometry. Mol BioSyst 6(10):2004–2014. https://doi.org/10.1039/c005242f
Das R, Liang YH, Mariano J, Li J, Huang T, King A, Tarasov SG, Weissman AM, Ji X, Byrd RA (2013) Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine. EMBO J 32(18):2504–2516. https://doi.org/10.1038/emboj.2013.174
Buetow L, Gabrielsen M, Anthony NG, Dou H, Patel A, Aitkenhead H, Sibbet GJ, Smith BO, Huang DT (2015) Activation of a primed RING E3-E2-ubiquitin complex by non-covalent ubiquitin. Mol Cell 58(2):297–310. https://doi.org/10.1016/j.molcel.2015.02.017
Eletr ZM, Kuhlman B (2007) Sequence determinants of E2-E6AP binding affinity and specificity. J Mol Biol 369(2):419–428. https://doi.org/10.1016/j.jmb.2007.03.026
Wright JD, Mace PD, Day CL (2016) Secondary ubiquitin-RING docking enhances Arkadia and Ark2C E3 ligase activity. Nat Struct Mol Biol 23(1):45–52. https://doi.org/10.1038/nsmb.3142
Kleiger G, Saha A, Lewis S, Kuhlman B, Deshaies RJ (2009) Rapid E2-E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell 139(5):957–968. https://doi.org/10.1016/j.cell.2009.10.030
Eletr ZM, Huang DT, Duda DM, Schulman BA, Kuhlman B (2005) E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat Struct Mol Biol 12(10):933–934. https://doi.org/10.1038/nsmb984
Huang DT, Schulman BA (2005) Expression, purification, and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex. Methods Enzymol 398:9–20. https://doi.org/10.1016/S0076-6879(05)98002-6
Chiba T (2005) In vitro systems for NEDD8 conjugation by Ubc12. Methods Enzymol 398:68–73. https://doi.org/10.1016/S0076-6879(05)98007-5
Beaudenon S, Huibregtse JM (2005) High-level expression and purification of recombinant E1 enzyme. Methods Enzymol 398:3–8. https://doi.org/10.1016/S0076-6879(05)98001-4
Zheng M, Liu J, Yang Z, Gu X, Li F, Lou T, Ji C, Mao Y (2010) Expression, purification and characterization of human ubiquitin-activating enzyme, UBE1. Mol Biol Rep 37(3):1413–1419. https://doi.org/10.1007/s11033-009-9525-3
Lorick KL, Jensen JP, Weissman AM (2005) Expression, purification, and properties of the Ubc4/5 family of E2 enzymes. Methods Enzymol 398:54–68. https://doi.org/10.1016/S0076-6879(05)98006-3
Li T, Pavletich NP, Schulman BA, Zheng N (2005) High-level expression and purification of recombinant SCF ubiquitin ligases. Methods Enzymol 398:125–142. https://doi.org/10.1016/S0076-6879(05)98012-9
Saha A, Deshaies RJ (2008) Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol Cell 32(1):21–31. https://doi.org/10.1016/j.molcel.2008.08.021
Pierce NW, Kleiger G, Shan SO, Deshaies RJ (2009) Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462(7273):615–619. https://doi.org/10.1038/nature08595
Acknowledgments
This work, including the efforts of Spencer Hill, Connor Hill, and Gary Kleiger, was funded by HHS | National Institutes of Health (NIH) (R15 GM117555- 01).
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Hill, S., Hill, C., Kleiger, G. (2018). Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for Their Ubiquitin Ligase Partners. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_4
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