title: Monitoring autophagic degradation of p62/SQSTM1.
abstract: The p62 protein, also called sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold protein that colocalizes with ubiquitinated protein aggregates in many neurodegenerative diseases and proteinopathies of the liver. The protein is able to polymerize via an N-terminal PB1 domain and can interact with ubiquitinated proteins via the C-terminal UBA domain. Also, p62/SQSTM1 binds directly to LC3 and GABARAP family proteins via a specific sequence motif. The protein is itself degraded by autophagy and may serve to link ubiquitinated proteins to the autophagic machinery to enable their degradation in the lysosome. Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced, p62 may be used as a marker to study autophagic flux. Here, we present several protocols for monitoring autophagy-mediated degradation of p62 using Western blots, pulse-chase measurement of p62 half-life, immunofluorescence and immuno-electron microscopy, as well as live cell imaging with a pH-sensitive mCherry-GFP double tag. We also present data on species-specificity and map the epitopes recognized by several commercially available anti-p62 antibodies.
authors.affiliation: Biochemistry Department, Institute of Medical Biology, University of Tromsø, Norway.
authors.forename: Geir
authors.initials: G
authors.lastname: Bjørkøy
authors.forename: Trond
authors.initials: T
authors.lastname: Lamark
authors.forename: Serhiy
authors.initials: S
authors.lastname: Pankiv
authors.forename: Aud
authors.initials: A
authors.lastname: Øvervatn
authors.forename: Andreas
authors.initials: A
authors.lastname: Brech
authors.forename: Terje
authors.initials: T
authors.lastname: Johansen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D049452:Green Fluorescent Proteins