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

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

Author information

1
Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany.
2
Department of Cell Biology, University of Groningen, University Medical Center Groningen, Groningen, Netherlands.
3
Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany cu@uos.de.
4
Center of Cellular Nanoanalytics Osnabrück (CellNanOs), University of Osnabrück, Osnabrück, Germany.

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.

PMID:
30097515
PMCID:
PMC6168247
[Available on 2019-04-01]
DOI:
10.1083/jcb.201804039

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