Monitoring stress-induced autophagic engulfment and degradation of the 26S proteasome in mammalian cells

Methods Enzymol. 2019;619:337-366. doi: 10.1016/bs.mie.2018.12.022. Epub 2019 Mar 7.


Almost 70 years after the discovery of the lysosome, and about four decades following the unraveling of ubiquitin as a specific "mark of death," the field of protein turnover-the numerous processes it regulates, the pathologies resulting from its dysregulation, and the drugs that have been developed to target them-is still growing exponentially. Accordingly, the need for new technologies and methods is ever growing. One interesting question in the field is the mechanism(s) by which the "predators become prey". We have reported recently that the 26S proteasome, the catalytic arm of the ubiquitin system, is degraded by the autophagy-lysosome machinery, in a process requiring specific ubiquitination of the proteasome, and subsequent recognition by the shuttle protein p62/SQSTM1. Studying the modification(s), recognition sites, engulfment, and breakdown of the 26S proteasome via such "proteaphagy" has required the use of microscopy, subcellular fractionation, 'classical biochemistry', and proteomics. In this chapter, we present the essentials of these protocols, with emphasis on the refinements we have introduced in order for them to better suit the particular study of proteaphagy.

Keywords: Autophagosome/autolysosome purification; Autophagy; Fluorescent microscopy; Proteasome; Protein degradation; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagosomes / metabolism*
  • Autophagosomes / ultrastructure
  • Autophagy
  • Fluorescent Antibody Technique / methods
  • HeLa Cells
  • Humans
  • Lysosomes / metabolism
  • Lysosomes / ultrastructure
  • Microscopy, Fluorescence / methods*
  • Proteasome Endopeptidase Complex / analysis
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteolysis
  • Ubiquitination


  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease