GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis
GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis
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DOI：
10.4161/auto.7.12.18424
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发表时间：
2011-12-01
期刊：
影响因子：
13.3
通讯作者：
Krick, Roswitha
中科院分区：
文献类型：
--
作者：
Nair, Usha;Thumm, Michael;Krick, Roswitha
也许大型自噬最复杂的步骤是形成双膜自噬小体。大多数自噬相关(ATG)蛋白被认为参与了噬菌体的成核和扩张，和/或完成了这个隔室。监测过程的这一部分是困难的，通常涉及电子显微镜分析；然而，除非进行三维断层成像，否则即使是这种方法也不能轻易确定吞噬体是否完全封闭。因此，一种补充的方法是检查被隔离货物对外源添加的蛋白酶的可及性。这种类型的蛋白酶保护分析已被用于通过检测前体氨基肽酶I(PrApe1)的蛋白酶敏感性来监测细胞质到空泡靶向(CVT)小泡和自噬小体的形成。然而，对于确定在非选择性自噬过程中形成的自噬小体的状态，prApe1不是最好的标记蛋白。在这里，我们描述了一种检查自噬小体完成情况的替代方法，该方法使用GFP-Atg8作为蛋白酶保护的标记。
Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenausly added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.