Journal
JOURNAL OF CELL BIOLOGY
Volume 217, Issue 10, Pages 3670-3682Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.201804039
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Funding
- ALW Open Program [822.02.014]
- Deutsche Forschungsgemeinschaft/Netherlands Organisation for Scientific Research cooperation [DN82-303]
- Swiss National Science Foundation Sinergia [CRS II3_154421]
- Marie Sklodowska-Curie Cofund [713660]
- ZonMW Vici [016.130.606]
- Deutsche Forschungsgemeinschaft grant [UN111/7-3]
- Sonderforschungsbereich 944 [P11]
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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.
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