Yao Zhiyuan, Liu Xu, Klionsky Daniel J
Department of Molecular, Cellular and Developmental Biology, Life Sciences Institute, University of Michigan, Room 6036, 210 Washtenaw Avenue, Ann Arbor, MI, 48109-2216, USA.
Methods Mol Biol. 2018;1759:85-93. doi: 10.1007/7651_2017_12.
Mitophagy, a selective type of macroautophagy (hereafter referred to as autophagy), specifically mediates the vacuole/lysosome-dependent degradation of damaged or surplus mitochondria. Because this process regulates the number and quality of mitochondria, it is vital for proper cellular homeostasis. Mitophagy also plays critical roles in the clearance of paternal mitochondria in C. elegans embryos, in erythroid cell maturation, and in the prevention of neurodegenerative disease and cancer. In order to study the molecular mechanism and regulation of mitophagy, sensitive assays are necessary to quantitatively measure mitophagy activity. In the budding yeast, Saccharomyces cerevisiae, a "mitoPho8Δ60" assay was developed to study mitophagy. In this assay, Pho8, a vacuolar phosphatase protein, is genetically engineered to be targeted to mitochondria. When mitophagy is induced, the phosphatase protein, along with mitochondria, is conveyed to the vacuole, where its C-terminal propeptide is removed and the phosphatase activity becomes activated; under growing conditions only a background level of delivery occurs. For this reason, the enzymatic activity of mitoPho8Δ60 is correlated with the amount of mitochondria delivered to the vacuole. Thus, this assay serves as a very convenient tool to quantitatively monitor mitophagy activity in yeast.
线粒体自噬是一种选择性的巨自噬（以下简称自噬），专门介导受损或多余线粒体的液泡/溶酶体依赖性降解。由于这个过程调节线粒体的数量和质量，对维持细胞内环境稳定至关重要。线粒体自噬在秀丽隐杆线虫胚胎中父系线粒体的清除、红细胞成熟以及预防神经退行性疾病和癌症方面也起着关键作用。为了研究线粒体自噬的分子机制和调控，需要灵敏的检测方法来定量测量线粒体自噬活性。在芽殖酵母酿酒酵母中，开发了一种“mitoPho8Δ60”检测方法来研究线粒体自噬。在该检测方法中，液泡磷酸酶蛋白Pho8经过基因工程改造后靶向线粒体。当诱导线粒体自噬时，磷酸酶蛋白与线粒体一起被转运到液泡中，在那里其C末端前肽被切除，磷酸酶活性被激活；在正常生长条件下，只有背景水平的转运发生。因此，mitoPho8Δ60的酶活性与转运到液泡中的线粒体数量相关。因此，该检测方法是定量监测酵母中线粒体自噬活性的非常方便的工具。
