A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles

J Cell Biol. 2018 Oct 1;217(10):3670-3682. doi: 10.1083/jcb.201804039. Epub 2018 Aug 10.

Abstract

Autophagy is a catabolic pathway that delivers intracellular material to the mammalian lysosomes or the yeast and plant vacuoles. The final step in this process is the fusion of autophagosomes with vacuoles, which requires SNARE proteins, the homotypic vacuole fusion and protein sorting tethering complex, the RAB7-like Ypt7 GTPase, and its guanine nucleotide exchange factor, Mon1-Ccz1. Where these different components are located and function during fusion, however, remains to be fully understood. Here, we present a novel in vitro assay to monitor fusion of intact and functional autophagosomes with vacuoles. This process requires ATP, physiological temperature, and the entire fusion machinery to tether and fuse autophagosomes with vacuoles. Importantly, we uncover Ykt6 as the autophagosomal SNARE. Our assay and findings thus provide the tools to dissect autophagosome completion and fusion in a test tube.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagosomes / metabolism*
  • Biological Assay*
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism
  • Membrane Fusion / physiology*
  • R-SNARE Proteins / genetics
  • R-SNARE Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Vacuoles / genetics
  • Vacuoles / metabolism*
  • Vesicular Transport Proteins / genetics
  • Vesicular Transport Proteins / metabolism

Substances

  • Ccz1 protein, S cerevisiae
  • Guanine Nucleotide Exchange Factors
  • Mon1 protein, S cerevisiae
  • R-SNARE Proteins
  • Saccharomyces cerevisiae Proteins
  • Vesicular Transport Proteins
  • YKT6 protein, S cerevisiae