Autophagosomes mature by the addition of membrane material from various intracellular sources and the attachment of peripheral proteins that remain bound through a covalent lipidation reaction. However, the origin and the mechanism of generation of the pre-autophagic membrane are poorly understood. We addressed this question with the development and analysis of a cell-free reaction that reproduces the lipidation of a major peripheral autophagosomal protein, LC3. A crude membrane fraction isolated from cells deficient in lipidation was mixed with cytosol harvested from normal cells that were untreated or subjected to a stress regimen known to induce autophagy. On addition of ATP, incubation of the mixture resulted in the formation of lipidated LC3. The reaction requires both membranes and cytosol, and was found to be stimulated 2- to 5-fold when the cytosol was taken from stress-induced cells. Autophagosome maturation requires a class III PI-3 kinase (VPS34 homolog); LC3 lipidation in our cell-free reaction is inhibited by inhibitors of this kinase, and by the addition of a peptide containing a PI3P-bnding sequence, the FYVE domain. Using cell fractionation techniques we have identified the ER-Golgi intermediate (ERGIC) compartment as the major site for lipidation of LC-3. This cell-free reaction may now be used to understand the molecular mechanism of autophagosome maturation.
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Ge, L, Melville, D., Zhang, M. and Schekman, R. (2013) The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis eLife DOI: http://dx.doi.org/10.7554/eLife.00947