Abstract
Proteasomes are complex molecular machines that consist of 66 subunits. The assembly of these complexes is highly coordinated in a process that requires at least ten proteasome-specific molecular chaperones. One of the challenges in studying assembly intermediates is their relatively low abundance as compared to the proteasome holoenzyme. Therefore, superior separating techniques are crucial for analyses of proteasomal complexes in general and studies in the assembly in particular. For this reason, native gel analyses have been abundantly used in studying proteasomes, as they provide a high resolution. Native gels are very versatile and can be used in various combinatorial approaches. In this chapter, we outline two approaches to prepare samples for native gels. The first is a yeast cryogrinding method and the second a core particle (CP)-base reconstitution approach. We describe the native gel electrophoresis, as well as various downstream analyses, including 2D native-SDS-PAGE. These techniques and approaches can all be used, often in parallel, to gain a variety of information about activity and composition of the complexes separated by native gel. The potential combined approaches are discussed in this review.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kressler D, Hurt E, Bassler J (2017) A puzzle of life: crafting ribosomal subunits. Trends Biochem Sci 42(8):640–654. https://doi.org/10.1016/j.tibs.2017.05.005
Ellis RJ (2013) Assembly chaperones: a perspective. Philos Trans R Soc Lond B Biol Sci 368(1617):20110398. https://doi.org/10.1098/rstb.2011.0398
Budenholzer L, Cheng CL, Li Y, Hochstrasser M (2017) Proteasome structure and assembly. J Mol Biol 429(22):3500–3524. https://doi.org/10.1016/j.jmb.2017.05.027
Wani PS, Rowland MA, Ondracek A, Deeds EJ, Roelofs J (2015) Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association. Nat Commun 6:6384. https://doi.org/10.1038/ncomms7384
Ramos PC, Hockendorff J, Johnson ES, Varshavsky A, Dohmen RJ (1998) Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell 92(4):489–499. https://doi.org/10.1016/S0092-8674(00)80942-3
Kock M, Nunes MM, Hemann M, Kube S, Dohmen RJ, Herzog F, Ramos PC, Wendler P (2015) Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone. Nat Commun 6:6123. https://doi.org/10.1038/ncomms7123
Shi Y, Chen X, Elsasser S, Stocks BB, Tian G, Lee BH, Shi Y, Zhang N, de Poot SA, Tuebing F, Sun S, Vannoy J, Tarasov SG, Engen JR, Finley D, Walters KJ (2016) Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science 351(6275):aad9421. https://doi.org/10.1126/science.aad9421
Park S, Roelofs J, Kim W, Robert J, Schmidt M, Gygi SP, Finley D (2009) Hexameric assembly of the proteasomal ATPases is templated through their C termini. Nature 459(7248):866–870. https://doi.org/10.1038/nature08065
Roelofs J, Park S, Haas W, Tian G, McAllister FE, Huo Y, Lee BH, Zhang F, Shi Y, Gygi SP, Finley D (2009) Chaperone-mediated pathway of proteasome regulatory particle assembly. Nature 459(7248):861–865. https://doi.org/10.1038/nature08063
Saeki Y, Toh EA, Kudo T, Kawamura H, Tanaka K (2009) Multiple proteasome-interacting proteins assist the assembly of the yeast 19S regulatory particle. Cell 137(5):900–913. https://doi.org/10.1016/j.cell.2009.05.005
Funakoshi M, Tomko RJ Jr, Kobayashi H, Hochstrasser M (2009) Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base. Cell 137(5):887–899. https://doi.org/10.1016/j.cell.2009.04.061
Bedford L, Paine S, Sheppard PW, Mayer RJ, Roelofs J (2010) Assembly, structure, and function of the 26S proteasome. Trends Cell Biol 20(7):391–401. https://doi.org/10.1016/j.tcb.2010.03.007
Tomko RJ Jr, Hochstrasser M (2013) Molecular architecture and assembly of the eukaryotic proteasome. Annu Rev Biochem 82:415–445. https://doi.org/10.1146/annurev-biochem-060410-150257
Higashitsuji H, Liu Y, Mayer RJ, Fujita J (2005) The oncoprotein gankyrin negatively regulates both p53 and RB by enhancing proteasomal degradation. Cell Cycle 4(10):1335–1337. https://doi.org/10.4161/cc.4.10.2107
Tomko RJ Jr, Funakoshi M, Schneider K, Wang J, Hochstrasser M (2010) Heterohexameric ring arrangement of the eukaryotic proteasomal ATPases: implications for proteasome structure and assembly. Mol Cell 38(3):393–403. https://doi.org/10.1016/j.molcel.2010.02.035
Kusmierczyk AR, Kunjappu MJ, Funakoshi M, Hochstrasser M (2008) A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nat Struct Mol Biol 15(3):237–244. https://doi.org/10.1038/nsmb.1389
Tomko RJ Jr, Taylor DW, Chen ZA, Wang HW, Rappsilber J, Hochstrasser M (2015) A single alpha helix drives extensive remodeling of the proteasome lid and completion of regulatory particle assembly. Cell 163(2):432–444. https://doi.org/10.1016/j.cell.2015.09.022
Estrin E, Lopez-Blanco JR, Chacon P, Martin A (2013) Formation of an intricate helical bundle dictates the assembly of the 26S proteasome lid. Structure 21(9):1624–1635. https://doi.org/10.1016/j.str.2013.06.023
Kaneko T, Murata S (2012) Using siRNA techniques to dissect proteasome assembly pathways in mammalian cells. Methods Mol Biol 832:433–442. https://doi.org/10.1007/978-1-61779-474-2_30
Tallec BL, Peyroche A (2012) Using DNA damage sensitivity phenotypes to characterize mutations affecting proteasome function. Methods Mol Biol 832:363–371. https://doi.org/10.1007/978-1-61779-474-2_25
Barrault MB, Richet N, Godard C, Murciano B, Le Tallec B, Rousseau E, Legrand P, Charbonnier JB, Le Du MH, Guerois R, Ochsenbein F, Peyroche A (2012) Dual functions of the Hsm3 protein in chaperoning and scaffolding regulatory particle subunits during the proteasome assembly. Proc Natl Acad Sci U S A 109(17):E1001–E1010. https://doi.org/10.1073/pnas.1116538109
Leggett DS, Glickman MH, Finley D (2005) Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast. Methods Mol Biol 301:57–70. https://doi.org/10.1385/1-59259-895-1:057
Park S, Li X, Kim HM, Singh CR, Tian G, Hoyt MA, Lovell S, Battaile KP, Zolkiewski M, Coffino P, Roelofs J, Cheng Y, Finley D (2013) Reconfiguration of the proteasome during chaperone-mediated assembly. Nature 497(7450):512–516. https://doi.org/10.1038/nature12123
Le Tallec B, Barrault MB, Guerois R, Carre T, Peyroche A (2009) Hsm3/S5b participates in the assembly pathway of the 19S regulatory particle of the proteasome. Mol Cell 33(3):389–399. https://doi.org/10.1016/j.molcel.2009.01.010
Li F, Tian G, Langager D, Sokolova V, Finley D, Park S (2017) Nucleotide-dependent switch in proteasome assembly mediated by the Nas6 chaperone. Proc Natl Acad Sci U S A 114(7):1548–1553. https://doi.org/10.1073/pnas.1612922114
Verdoes M, Florea BI, Menendez-Benito V, Maynard CJ, Witte MD, van der Linden WA, van den Nieuwendijk AM, Hofmann T, Berkers CR, van Leeuwen FW, Groothuis TA, Leeuwenburgh MA, Ovaa H, Neefjes JJ, Filippov DV, van der Marel GA, Dantuma NP, Overkleeft HS (2006) A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo. Chem Biol 13(11):1217–1226. https://doi.org/10.1016/j.chembiol.2006.09.013
Waite KA, De-La Mota-Peynado A, Vontz G, Roelofs J (2016) Starvation induces proteasome autophagy with different pathways for core and regulatory particles. J Biol Chem 291(7):3239–3253. https://doi.org/10.1074/jbc.M115.699124
Enenkel C (2012) Using native gel electrophoresis and phosphofluoroimaging to analyze GFP-tagged proteasomes. Methods Mol Biol 832:339–348. https://doi.org/10.1007/978-1-61779-474-2_23
Lee SY, De la Mota-Peynado A, Roelofs J (2011) Loss of Rpt5 protein interactions with the core particle and Nas2 protein causes the formation of faulty proteasomes that are inhibited by Ecm29 protein. J Biol Chem 286(42):36641–36651. https://doi.org/10.1074/jbc.M111.280875
De La Mota-Peynado A, Lee SY, Pierce BM, Wani P, Singh CR, Roelofs J (2013) The proteasome-associated protein Ecm29 inhibits proteasomal ATPase activity and in vivo protein degradation by the proteasome. J Biol Chem 288(41):29467–29481. https://doi.org/10.1074/jbc.M113.491662
Kleijnen MF, Roelofs J, Park S, Hathaway NA, Glickman M, King RW, Finley D (2007) Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nat Struct Mol Biol 14(12):1180–1188. https://doi.org/10.1038/nsmb1335
Li Y, Tomko RJ Jr, Hochstrasser M (2015) Proteasomes: Isolation and activity assays. Curr Protoc Cell Biol 67:3.43. 1–3.43.20. https://doi.org/10.1002/0471143030.cb0343s67
Hochstrasser M, Funakoshi M (2012) Disulfide engineering to map subunit interactions in the proteasome and other macromolecular complexes. Methods Mol Biol 832:349–362. https://doi.org/10.1007/978-1-61779-474-2_24
Acknowledgments
Many of these techniques described above have been developed or optimized and tweaked by many experts in the field, and the power of their use has been displayed in numerous excellent papers. We apologize for not being able to provide a comprehensive reference to all those contributions in this publication. This work was supported in part by grants to J.R. from NIH (R01-GM118660 and R15-GM112142).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Roelofs, J., Suppahia, A., Waite, K.A., Park, S. (2018). Native Gel Approaches in Studying Proteasome Assembly and Chaperones. 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_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8706-1_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8705-4
Online ISBN: 978-1-4939-8706-1
eBook Packages: Springer Protocols