Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Sevilla, Sevilla, Spain.
Methods Mol Biol. 2022;2447:205-220. doi: 10.1007/978-1-0716-2079-3_17.
Deciphering the molecular mechanisms underlying the regulation of the ATG4 protease is essential to understand the regulation of ATG8 lipidation, a key step in the biogenesis of the autophagosome and hence in autophagy progression. Here, we describe two complementary approaches to monitor ATG4 proteolytic activity in the model green alga Chlamydomonas reinhardtii: an in vitro assay using recombinant ATG4 and recombinant ATG8 as substrate, and a cell-free assay using soluble total protein extract from Chlamydomonas and recombinant Chlamydomonas ATG8 as substrate. Both assays are followed by non-reducing SDS-PAGE and immuno-blot analysis. Given the high evolutionary conservation of the ATG8 maturation process, these assays have also been validated to monitor ATG4 activity in yeast using Chlamydomonas ATG8 as substrate.
解析 ATG4 蛋白酶调控的分子机制对于理解 ATG8 脂质化的调控至关重要，因为 ATG8 脂质化是自噬体生物发生的关键步骤，也是自噬体进程的关键步骤。在这里，我们描述了两种互补的方法来监测模型绿藻衣藻中的 ATG4 蛋白水解活性：一种使用重组 ATG4 和重组 ATG8 作为底物的体外测定法，以及一种使用可溶性总蛋白提取物和重组衣藻 ATG8 作为底物的无细胞测定法。这两种测定法都通过非还原 SDS-PAGE 和免疫印迹分析进行。鉴于 ATG8 成熟过程的高度进化保守性，这些测定法也已经通过使用衣藻 ATG8 作为底物在酵母中监测 ATG4 活性得到了验证。
