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Cell-free reconstitution of autophagy to dissect molecular mechanisms

Periodic Reporting for period 3 - AutoClean (Cell-free reconstitution of autophagy to dissect molecular mechanisms)

Reporting period: 2021-06-01 to 2022-11-30

Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy. However, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase and the molecular events triggered, will enable us to provide a definitive mechanistic understanding of autophagy. To achieve this goal, we aim at reconstituting different steps of autophagy in vitro, to then dissect the underlying events in detail. As autophagy function is pivotal to prevent diseases such as cancer and neurodegeneration, understanding the molecular events of autophagy will also help to address its potential in therapeutics.
We have devised a novel in vitro reconstitution assay, allowing us to recapitulate autophagosome-vacuole fusion in vitro. This system allowed us to dissect the players involved and the place and time of action. We found the SNARE protein Ykt6 acting on the autophagosome, directing it to the vacuole to allow fusion. Further work addressed the regulation of this autophagosome-vacuole fusion event. We found that the Atg1 kinase directly phosphorylates Ykt6, thereby preventing premature fusion. This again highlights the central role of the Atg1 kinase in regulating different steps in autophagy. We have now alos reconstituted this step in mammalian cells and analyzed the conservation of the process.
We have also set up a synthetic in vivo system to dissect the molecular events of autophagosome formation. Using this system we found that the vacuolar protein Vac8 acts as a coordinator of this process, and that the initiation and progression of autophagosome formation is regulated by avidity.
AutoClean has established novel in vitro approaches and synthetic in vivo approaches in yeast and mammalian cells, to dissect molecular events underlying autophagy function. These approaches are unique in that different steps of autophagy can be addressed at the molecular level, which is often impossible in live cells. These novel assays will allow the future study of further molecular events in autophagy and provide a detailed molecular understanding of the autophagy pathway. In the long term we expect that the AutoClean molecular analysis will contribute to our understanding of autophagy dysfunction in diseases, and provide insight into its potential in therapeutics.
Yeast cell in which cargo ("waste", in blue) is packaged by an autophagosomal membrane (red).