Binding of ubiquitin conjugates to proteasomes as visualized with native gels

Methods Mol Biol. 2012;832:403-22. doi: 10.1007/978-1-61779-474-2_28.

Abstract

The proteasome is an ATP-dependent molecular machine that degrades proteins through the concerted activity of dozens of subunits. It is the yin to the ribosome's yang, and together these entities mold the protein landscape of the cell. Native gels are generally superior to conventional and affinity purifications for the analytical resolution proteasomal variants, and have thus become a staple of proteasome work. Here, we describe the technique of using native gels to observe proteasomes in complex with ubiquitin conjugates. We discuss the consequences of ubiquitin conjugate length and concentration on the migration of these complexes, the use of this mobility shift to evaluate the relative affinity of mutant proteasomes for ubiquitin conjugates, and the effects of deubiquitinating enzymes and competing ubiquitin-binding proteins on the interactions of ubiquitin conjugates with the proteasome.

MeSH terms

  • Electrophoresis, Polyacrylamide Gel / methods*
  • Electrophoretic Mobility Shift Assay / methods*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Mutation
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteolysis
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / metabolism
  • Ubiquitin / metabolism*

Substances

  • Ubiquitin
  • Proteasome Endopeptidase Complex