Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast - PubMed (original) (raw)
Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast
David S Leggett et al. Methods Mol Biol. 2005.
Abstract
The proteasome is a highly complex, ATP-dependent protease, consisting of over 30 subunits, and dedicated mainly to the degradation of ubiquitin-protein conjugates. Proteasomes are evolutionarily conserved in the eukaryotic kingdom, and those of yeast are well suited to serve as a general model. We describe techniques for the purification of proteasomes from budding yeast in milligram amounts via conventional and affinity-based strategies. While both approaches yield highly purified material, the affinity method is faster and easier. In addition, the affinity method is more suitable for identifying proteasome-associated proteins. We also describe methods for purifying the major subassemblies of the proteasome, such as the CP, the RP, the lid, and the base. A variety of activity assays and native gel procedures are available to evaluate purified proteasomes functionally. When coupled with the genetic methods available in yeast, these biochemical procedures allow for detailed functional analysis of this unique protein complex.
Similar articles
- Purification, crystallization, and X-ray analysis of the yeast 20S proteasome.
Groll M, Huber R. Groll M, et al. Methods Enzymol. 2005;398:329-36. doi: 10.1016/S0076-6879(05)98027-0. Methods Enzymol. 2005. PMID: 16275340 - Large- and small-scale purification of mammalian 26S proteasomes.
Hirano Y, Murata S, Tanaka K. Hirano Y, et al. Methods Enzymol. 2005;399:227-40. doi: 10.1016/S0076-6879(05)99015-0. Methods Enzymol. 2005. PMID: 16338359 - Purification and proteomic analysis of 20S proteasomes from human cells.
Bousquet-Dubouch MP, Uttenweiler-Joseph S, Ducoux-Petit M, Matondo M, Monsarrat B, Burlet-Schiltz O. Bousquet-Dubouch MP, et al. Methods Mol Biol. 2008;432:301-20. doi: 10.1007/978-1-59745-028-7_21. Methods Mol Biol. 2008. PMID: 18370027 - Comprehensive mass spectrometric analysis of the 20S proteasome complex.
Huang L, Burlingame AL. Huang L, et al. Methods Enzymol. 2005;405:187-236. doi: 10.1016/S0076-6879(05)05009-3. Methods Enzymol. 2005. PMID: 16413316 Review. - [Biogenesis of the 26S proteasome in the yeast Saccharomyces cerevisiae].
Isono E, Toh-e A. Isono E, et al. Tanpakushitsu Kakusan Koso. 2006 Aug;51(10 Suppl):1224-9. Tanpakushitsu Kakusan Koso. 2006. PMID: 16922378 Review. Japanese. No abstract available.
Cited by
- Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1.
Wu X, Du Y, Liang LJ, Ding R, Zhang T, Cai H, Tian X, Pan M, Liu L. Wu X, et al. Nat Commun. 2024 Feb 10;15(1):1266. doi: 10.1038/s41467-024-45635-y. Nat Commun. 2024. PMID: 38341401 Free PMC article. - Global organization and function of mammalian cytosolic proteasome pools: Implications for PA28 and 19S regulatory complexes.
Shibatani T, Carlson EJ, Larabee F, McCormack AL, Früh K, Skach WR. Shibatani T, et al. Mol Biol Cell. 2006 Dec;17(12):4962-71. doi: 10.1091/mbc.e06-04-0311. Epub 2006 Sep 20. Mol Biol Cell. 2006. PMID: 16987959 Free PMC article. - Structure of the Blm10-20 S proteasome complex by cryo-electron microscopy. Insights into the mechanism of activation of mature yeast proteasomes.
Iwanczyk J, Sadre-Bazzaz K, Ferrell K, Kondrashkina E, Formosa T, Hill CP, Ortega J. Iwanczyk J, et al. J Mol Biol. 2006 Oct 27;363(3):648-59. doi: 10.1016/j.jmb.2006.08.010. Epub 2006 Aug 9. J Mol Biol. 2006. PMID: 16952374 Free PMC article. - Proteomic profiling of yeast heterochromatin connects direct physical and genetic interactions.
Zukowski A, Phillips J, Park S, Wu R, Gygi SP, Johnson AM. Zukowski A, et al. Curr Genet. 2019 Apr;65(2):495-505. doi: 10.1007/s00294-018-0889-6. Epub 2018 Oct 12. Curr Genet. 2019. PMID: 30310994 Free PMC article. - Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients.
Henneberg F, Chari A. Henneberg F, et al. Life (Basel). 2021 Nov 24;11(12):1289. doi: 10.3390/life11121289. Life (Basel). 2021. PMID: 34947821 Free PMC article. Review.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Miscellaneous