Shun-ichi Yamashita

Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways

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.

Mitophagy in yeast: Molecular mechanisms and physiological role ☆

Mitochondria autophagy (mitophagy) is a process that selectively degrades mitochondria via autophagy. Recently, there has been significant progress in the understanding of mitophagy in yeast. Atg32, a mitochondrial outer membrane receptor, is indispensable for mitophagy. Phosphorylation of Atg32 is an initial cue for selective mitochondrial degradation. Atg32 expression and phosphorylation regulate the induction and efficiency of mitophagy. In addition to Atg32-related processes, recent studies have revealed that mitochondrial fission and the mitochondria-endoplasmic reticulum (ER) contact site may play important roles in mitophagy. Mitochondrial fission is required to regulate mitochondrial size. Mitochondria-ER contact is mediated by the ER-mitochondria encounter structure and is important to supply lipids from the ER for autophagosome biogenesis for mitophagy. Mitophagy is physiologically important for regulating the number of mitochondria, diminishing mitochondrial production of reactive oxygen species, and extending chronological lifespan under caloric restriction. These findings suggest that mitophagy contributes to maintain mitochondrial homeostasis. However, whether mitophagy selectively degrades damaged or dysfunctional mitochondria in yeast is unknown.

Mitochondrial division occurs concurrently with autophagosome formation but independently of Drp1 during mitophagy

It remains controversial whether Dnm1/Drp1-mediated mitochondrial division is essential for mitophagy. Yamashita et al. show that Dnm1/Drp1-independent mitochondrial division occurs after formation of isolation membranes and in cooperation with autophagosome formation during mitophagy.

Peroxisome homeostasis: Mechanisms of division and selective degradation of peroxisomes in mammals☆

Peroxisome number and quality are maintained by its biogenesis and turnover and are important for the homeostasis of peroxisomes. Peroxisomes are increased in number by division with dynamic morphological changes including elongation, constriction, and fission. In the course of peroxisomal division, peroxisomal morphogenesis is orchestrated by Pex11β, dynamin-like protein 1 (DLP1), and mitochondrial fission factor (Mff). Conversely, peroxisome number is reduced by its degradation. Peroxisomes are mainly degraded by pexophagy, a type of autophagy specific for peroxisomes. Upon pexophagy, an adaptor protein translocates on peroxisomal membrane and connects peroxisomes to autophagic machineries. Molecular mechanisms of pexophagy are well studied in yeast systems where several specific adaptor proteins are identified. Pexophagy in mammals also proceeds in a manner dependent on adaptor proteins. In this review, we address the recent progress in studies on peroxisome morphogenesis and pexophagy.

Role of Vac8 in formation of the vacuolar sequestering membrane during micropexophagy.

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.

Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy.

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy.

How autophagy eats large mitochondria: Autophagosome formation coupled with mitochondrial fragmentation

ABSTRACT Mitochondrial autophagy (mitophagy) is thought to be a multi-step pathway wherein mitochondria are first divided into small fragments, which are subsequently recognized by the phagophore. DNM1L (dynamin 1 like) plays a pivotal role in mitochondrial division; however, its role in mitophagy remains controversial. In our recent study, we examined the contribution of DNM1L to mitophagy and showed that mitophagy and mitochondrial division occur even in DNM1L-defective cells. Furthermore, time-lapse imaging of mitophagy showed that DNM1L-independent mitochondrial division occurs concomitantly with autophagosome formation. Upstream factors of autophagosome formation, i.e., RB1CC1/FIP200, ATG14, and WIPIs, are required for mitochondrial division, whereas ATG5 and ATG3 are dispensable. These results indicate that a portion of the tubular mitochondria is first recognized and then divided into small fragments by a phagophore-mediated event, independently of DNM1L. This autophagic process suggests that autophagy has the potential to degrade substrates larger than autophagosomes.

Detection of Hypoxia-Induced and Iron Depletion-Induced Mitophagy in Mammalian Cells

Mitochondrial quality and quantity are not only regulated by mitochondrial fusion and fission but also by mitochondria degradation. Mitophagy, an autophagy specific for damaged or unnecessary mitochondria, is believed to be an important pathway for mitochondrial homeostasis. To date, several stimuli are known to induce mitophagy. Some of these stimuli, however, including hypoxia, iron depletion, and nitrogen starvation, induce mild mitophagy, which is difficult to detect through decreased mitochondrial mass. Recently, we have clearly detected mitophagy induced under these conditions using mito-Keima as a reporter. In this chapter, we describe the protocols for induction and detection of hypoxia-induced and iron depletion-induced mitophagy using mito-Keima-expressed cells.

Detection of Iron Depletion- and Hypoxia-Induced Mitophagy in Mammalian Cells

Mitochondrial autophagy or mitophagy is a process that selectively degrades mitochondria via autophagy. It is believed that mitophagy degrades damaged or unnecessary mitochondria and is important for maintaining mitochondrial homeostasis. To date, it is known that several stimuli can induce mitophagy. However, some of these stimuli (including iron depletion, hypoxia, and nitrogen starvation) induce mild mitophagy, which is difficult to detect by measuring the decrease in mitochondrial mass. Recently, we have successfully detected mitophagy induced under these conditions using mito-Keima as a reporter. In this chapter, we describe the protocols for induction and detection of iron depletion- and hypoxia-induced mitophagy using the mito-Keima-expressing cells.

The PP2A-like Protein Phosphatase Ppg1 and the Far Complex Cooperatively Counteract CK2-Mediated Phosphorylation of Atg32 to Inhibit Mitophagy

Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151-200) of Atg32 caused the same phenotypes as in ppg1Δ cells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy.