Assessment of mammalian endosomal microautophagy

Gregory J. Krause, Ana Maria Cuervo

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Endosomal microautophagy (eMI) is a type of autophagy that allows for the selective uptake and degradation of cytosolic proteins in late endosome/multi-vesicular bodies (LE/MVB). This process starts with the recognition of a pentapeptide amino acid KFERQ-like targeting motif in the substrate protein by the hsc70 chaperone, which then enables binding and subsequent uptake of the protein into the LE/MVB compartment. The recognition of a KFERQ-like motif by hsc70 is the same initial step in chaperone-mediated autophagy (CMA), a form of selective autophagy that degrades the hsc70-targeted proteins in lysosomes in a LAMP-2A dependent manner. The shared step of substrate recognition by hsc70, originally identified for CMA, makes it now necessary to differentiate between the two pathways. Here, we detail biochemical and imaging-based methods to track eMI activity in vitro with isolated LE/MVBs and in cells in culture using fluorescent reporters and highlight approaches to distinguish whether a protein is a substrate of eMI or CMA.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc.
DOIs
StateAccepted/In press - 2020

Publication series

NameMethods in Cell Biology
ISSN (Print)0091-679X

Keywords

  • Autophagy
  • Chaperones
  • Late endosomes
  • Multi-vesicular bodies
  • Organelle isolation
  • Protein degradation
  • Protein targeting
  • Proteostasis

ASJC Scopus subject areas

  • Cell Biology

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