Accumulation of undegraded autophagosomes by expression of dominant-negative syntaxin 17 mutants (Uematsu et al., Autophagy)
2017.07.27 Recent Publications
Uematsu M, Nishimura T, Sakamaki Y, Yamamoto H, Mizushima N.
Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants
Autophagy, 2017 July 27 DOI: 10.1080/15548627.2017.1327940
Macroautophagy/autophagy, which is one of the main degradation systems in the cell, is mediated by a specialized organelle, the autophagosome. Purification of autophagosomes before fusion with lysosomes is important for both mechanistic and physiological studies of the autophagosome. Here, we report a simple method to accumulate undigested autophagosomes. Overexpression of the autophagosomal Qa-SNARE STX17 (syntaxin 17) lacking the N-terminal domain (NTD) or N-terminally tagged GFP-STX17 causes accumulation of autophagosomes. A HeLa cell line, which expresses GFP-STX17ΔNTD or full-length GFP-STX17 under the control of the tetracycline-responsive promoter, accumulates a large number of undigested autophagosomes devoid of lysosomal markers or early autophagy factors upon treatment with doxycycline. Using this inducible cell line, nascent autophagosomes can be easily purified by OptiPrep density-gradient centrifugation and immunoprecipitation. This novel method should be useful for further characterization of nascent autophagosomes.