Assays for Autophagy III: Observing Dynamic Protein Trafficking

Methods Mol Biol. 2021:2196:211-222. doi: 10.1007/978-1-0716-0868-5_16.

Abstract

Macroautophagy, by its very nature, is a protein trafficking process. Cargos are transported and processed. Atg proteins come and go. In this chapter, we present three assays to monitor these dynamic events: a non-radioactive pulse-chase labeling assay to monitor the transport of prApe1 and two fluorescent microscopy-based assays to assess the trafficking of Atg8 and Atg9.

Keywords: Ape1; Atg8; Atg9; Autophagy; Cvt pathway; Fluorescent microscopy; Intracellular trafficking; Pulse-chase; Yeast.

MeSH terms

  • Autophagosomes / metabolism
  • Autophagy*
  • Autophagy-Related Proteins / metabolism*
  • Microscopy, Fluorescence
  • Protein Transport
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Vesicular Transport Proteins / metabolism

Substances

  • Autophagy-Related Proteins
  • Saccharomyces cerevisiae Proteins
  • Vesicular Transport Proteins