Abstract
In studies of protein complexes for which high-resolution structural data are unavailable, it is often still possible to determine both nearest-neighbor relationships between subunits and atomic-resolution details of these interactions. The eukaryotic 26S proteasome, a ∼2.5 MDa protein complex with at least 33 different subunits, is a prime example. Important information about quaternary organization and assembly of proteasomes has been gained using a combination of sequence alignments with related proteins of known tertiary structure, molecular modeling, and disulfide engineering to allow oxidative cross-linking between predicted polypeptide neighbors. Here, we provide detailed protocols for engineered cysteine cross-linking of yeast proteasome subunits in whole-cell extracts, in active 26S proteasome complexes first isolated by native polyacrylamide gel electrophoresis, and in subcomplexes that function as potential assembly intermediates.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Chen P, Hochstrasser M (1996) Autocatalytic subunit processing couples active site formation in the 20S proteasome to completion of assembly. Cell 86:961–972.
Arendt CS, Hochstrasser M (1997) Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc. Natl. Acad. Sci. U.S.A. 94:7156–7161.
Velichutina I, Connerly PL, Arendt CS et al (2004) Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast. Embo J 23:500–510.
Kusmierczyk AR, Kunjappu MJ, Funakoshi M, Hochstrasser M (2008) A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nat Struct Mol Biol 15:237–244.
Tomko RJ, Jr, Funakoshi M, Schneider K et al (2010) Heterohexameric ring arrangement of the eukaryotic proteasomal ATPases: implications for proteasome structure and assembly. Mol Cell 38:393–403.
Hochstrasser M (1996) Ubiquitin-dependent protein degradation. Annual Review of Genetics 30:405–439.
Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiological Reviews 82:373–428.
Pickart CM, Cohen RE (2004) Proteasomes and their kin: proteases in the machine age. Nat Rev Mol Cell Biol 5:177–187.
Marques AJ, Palanimurugan R, Matias AC et al (2009) Catalytic mechanism and assembly of the proteasome. Chem Rev 109:1509–1536.
Chen P, Hochstrasser M (1995) Biogenesis, structure, and function of the yeast 20S proteasome. EMBO J. 14:2620–2630.
Groll M, Ditzel L, Löwe J et al (1997) Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 386:463–471.
Fu H, Reis N, Lee Y et al (2001) Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome. EMBO Journal 20:7096–7107.
Zhang F, Hu M, Tian G et al (2009) Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol Cell 34:473–484.
Djuranovic S, Hartmann MD, Habeck M et al (2009) Structure and activity of the N-terminal substrate recognition domains in proteasomal ATPases. Mol Cell 34:580–590.
Verma R, Chen S, Feldman R et al (2000) Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 11:3425–3439.
Hough R, Pratt G, Rechsteiner M (1987) Purification of two high molecular weight proteases in rabbit reticulocyte lysate. J. Biol. Chem. 262:8303–8313.
Glickman MH, Rubin DM, Fried VA, Finley D (1998) The regulatory particle of the Saccharomyces cerevisiae proteasome. Mol. Cell. Biol. 18:3149–3162.
Elsasser S, Schmidt M, Finley D (2005) Characterization of the proteasome using native gel electrophoresis. Methods Enzymol 398:353–363.
Acknowledgments
We thank Robb Tomko and Mary Kunjappu for critical reading of the manuscript. Work from our laboratory that led to the development of these cross-linking methods was supported by NIH grants GM046904 and GM083050.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hochstrasser, M., Funakoshi, M. (2012). Disulfide Engineering to Map Subunit Interactions in the Proteasome and Other Macromolecular Complexes. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_24
Download citation
DOI: https://doi.org/10.1007/978-1-61779-474-2_24
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-473-5
Online ISBN: 978-1-61779-474-2
eBook Packages: Springer Protocols