Ubiquitin Family Modifiers and the Proteasome

Volume 832 of the series Methods in Molecular Biology pp 349-362


Disulfide Engineering to Map Subunit Interactions in the Proteasome and Other Macromolecular Complexes

  • Mark HochstrasserAffiliated withDepartment of Molecular Biophysics and Biochemistry, Yale University Email author 
  • , Minoru FunakoshiAffiliated withDepartment of Molecular Biophysics and Biochemistry, Yale UniversityGraduate School of Natural Science and Technology, Okayama University

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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.

Key words

Disulfide engineering Proteasome Site-directed mutagenesis Nondenaturing polyacrylamide gel electrophoresis (native PAGE) Substrate overlay Saccharomyces cerevisiae Yeast Immunoblotting