Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants
Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants
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DOI：
10.1080/15548627.2017.1327940
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发表时间：
2017-01-01
期刊：
影响因子：
13.3
通讯作者：
Mizushima, Noboru
中科院分区：
文献类型：
--
作者：
Uematsu, Masaaki;Nishimura, Taki;Mizushima, Noboru
巨噬/自噬是细胞中主要的降解系统之一，它是由一种特殊的细胞器——自噬体介导的。自噬体与溶酶体融合前的纯化对自噬体的机制和生理研究都很重要。在这里，我们报告了一种简单的方法来积累未消化的自噬体。缺乏n端结构域（NTD）的自噬体Qa-SNARE STX17 （syntaxin 17）或n端标记的GFP-STX17的过表达导致自噬体的积累。在四环素应答启动子控制下表达GFP-STX17DNTD或全长GFP-STX17的HeLa细胞系，在强力霉素处理后积累了大量未消化的不含溶酶体标记物或早期自噬因子的自噬小体。使用该诱导细胞系，新生自噬体可以很容易地通过OptiPrep密度梯度离心和免疫沉淀纯化。这种新方法应该有助于进一步表征新生自噬体。
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-STX17DNTD 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.