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Purification of 26S Proteasomes and Their Subcomplexes from Plants

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Isolation of Plant Organelles and Structures

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1511))

Abstract

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type plants, and another that employs the genetic replacement of individual subunits with epitope-tagged variants combined with affinity purification. In addition to these purification protocols, we describe methods commonly used to analyze the activity and composition of the complex.

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Acknowledgements

The authors wish to thank Peizhen Yang, Adam J. Book, and Joseph M. Walker for their extensive work on the initial development of these protocols. We also wish to thank Erin Gemperline for critical reading of the manuscript. This work, R.S.M. and D.C.G. were supported by the U.S. Department of Energy Office of Science; Office of Basic Energy Sciences; Chemical Sciences, Geosciences and Biosciences Division (grant number DE-FG02-88ER13968).

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    Authors and Affiliations

    1. Department of Genetics, University of Wisconsin-Madison, 425-G Henry Mall, Madison, WI, USA

      Richard S. Marshall, David C. Gemperline & Richard D. Vierstra

    2. Department of Biology, Washington University in St Louis, Campus Box 1137, One Brookings Drive, St. Louis, MO, 63130, USA

      Richard S. Marshall & Richard D. Vierstra

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    1. Richard S. Marshall
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    2. David C. Gemperline
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    Correspondence to Richard D. Vierstra .

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    1. The University of Western Australia, Perth, West Australia, Australia

      Nicolas L. Taylor

    2. The University of Western Australia, Perth, West Australia, Australia

      A. Harvey Millar

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    Marshall, R.S., Gemperline, D.C., Vierstra, R.D. (2017). Purification of 26S Proteasomes and Their Subcomplexes from Plants. In: Taylor, N., Millar, A. (eds) Isolation of Plant Organelles and Structures. Methods in Molecular Biology, vol 1511. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6533-5_24

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    • DOI: https://doi.org/10.1007/978-1-4939-6533-5_24

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