Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When autophagy selects mitochondria as a cargo, Atg32 is bound by Atg11. It is shown that the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediates the Atg11–Atg32 interaction and mitophagy.

Background:The physiological importance of mitophagy in yeast has been largely unexplored. Results: Mitochondrial DNA deletion frequently occurs in mitophagy-deficient cells during nitrogen starvation because of overproduction of the reactive oxygen species from unregulated mitochondria. Conclusion: Mitophagy prevents excess reactive oxygen species production and mitochondrial DNA mutation. Significance: Our findings provide insight into mitophagy-related disorders such as Parkinson disease.

HM1.24/Bst2/CD317 is a protein highly expressed in multiple myeloma cells and has unique topology with two membrane anchor domains, an NH 2 -terminal transmembrane domain and a glycosylphosphatidylinositol attached to the COOH terminus. We show here that human HM1.24 is localized not only on the cell surface but also in the trans-Golgi network and/or recycling endosomes, where it resides in detergent-resistant microdomains, lipid rafts. In contrast to other glycosylphosphatidylinositol-anchored proteins, HM1.24 was internalized from lipid rafts on the cell surface by clathrin-mediated endocytosis. Interestingly, a non-canonical tyrosine-based motif, which contains two tyrosine residues, Tyr-6 and Tyr-8, present in the NH 2 -terminal cytoplasmic tail, was essential for endocytosis through interaction with an ␣-adaptin, but not 2-subunit, of the AP-2 complex. Indeed, an appendage domain of ␣-adaptin was identified as a protein interacting with the cytoplasmic tail of HM1.24. Furthermore, overexpression of the appendage domain of ␣-adaptin in cells depleted of ␣-adaptin could rescue the clathrin-mediated endocytosis of HM1.24 but not of the transferrin receptor. Taken together, our findings suggest that clathrin-dependent endocytosis of human HM1.24 from the cell surface lipid rafts is mediated by direct interaction with ␣-adaptin.

Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy-related; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; ER, endoplasmic reticulum; FUNDC1, FUN14 Domain Containing 1; LC3, microtubule-associated protein 1 light chain 3; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; MEK, MAPK-ERK kinase; MKK, MAP kinase kinase; PE, phosphatidylethanolamine; PINK1, PTEN-induced putative kinase protein 1; PTEN, phosphatase and tensin homolog; siRNA, short interfering RNA; SQSTM1, sequestosome 1; Tet, tetracycline.In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.