A Genomic Screen for Yeast Mutants Defective in Selective Mitochondria Autophagy

Mitophagy is the process of selective mitochondrial degradation via autophagy, which has an important role in mitochondrial quality control. Very little is known, however, about the molecular mechanism of mitophagy. A genome-wide yeast mutant screen for mitophagy-defective strains identified 32 mutants with a block in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We further characterized one of these mutants, ylr356wΔ that corresponds to a gene whose function has not been identified. YLR356W is a mitophagy-specific gene that was not required for other types of selective autophagy or macroautophagy. The deletion of YLR356W partially inhibited mitophagy during starvation, whereas there was an almost complete inhibition at post-log phase. Accordingly, we have named this gene ATG33. The new mutants identified in this analysis will provide a useful foundation for researchers interested in the study of mitochondrial homeostasis and quality control.     

Atg24-assisted mitophagy in the foot cells is necessary for proper asexual differentiation in Magnaporthe oryzae

Macroautophagy-mediated glycogen catabolism is required for asexual differentiation in the blast fungus, Magnaporthe oryzae. However, the function(s) of selective subtypes of autophagy has not been studied therein. Here, we report that mitophagy, selective autophagic delivery of mitochondria to the vacuoles for degradation, occurs during early stages of Magnaporthe conidiation. Specifically, mitophagy was evident in the foot cells while being undetectable in aerial hyphae and/or conidiophores. We show that loss of MoAtg24, a sorting nexin related to yeast Snx4, disrupts mitophagy and consequently leads to highly reduced conidiation, suggesting that mitophagy in the foot cells plays an important role during asexual development in Magnaporthe. Ectopic expression of yeast ScATG32 partially suppressed the conidiation initiation defects associated with MoATG24 deletion. MoAtg24 was neither required for pexophagy nor for macroautophagy, or for MoAtg8 localization per se, but directly associated with and likely recruited mitochondria to the autophagic structures during mitophagy. Lastly, MoAtg24 was also required for oxidative stress response in Magnaporthe.     

MAPKs regulate mitophagy in Saccharomyces cerevisiae

The autophagy-dependent selective degradation of mitochondria (mitophagy) plays an important role in removing excessive, damaged and dysfunctional mitochondria to maintain a proper cellular homeostasis. Relative to its significance in cell physiology, very little is known about the molecular machinery and regulatory mechanism of mitophagy in mammalian cells or yeast. We found that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is involved in both mitophagy and pexophagy (the selective degradation of peroxisomes through autophagy), whereas Hog1 functions specifically in mitophagy.     

Peroxisomal protein PEX13 functions in selective autophagy

PEX13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. Mutations in PEX13 and other peroxin proteins are associated with Zellweger syndrome spectrum (ZSS) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. The lack of functional peroxisomes in ZSS patients is widely accepted as the underlying cause of disease; however, our understanding of disease pathogenesis is still incomplete. Here, we demonstrate that PEX13 is required for selective autophagy of Sindbis virus (virophagy) and of damaged mitochondria (mitophagy) and that disease‐associated PEX13 mutants I326T and W313G are defective in mitophagy. The mitophagy function of PEX13 is shared with another peroxin family member PEX3, but not with two other peroxins, PEX14 and PEX19, which are required for general autophagy. Together, our results demonstrate that PEX13 is required for selective autophagy, and suggest that dysregulation of PEX13‐mediated mitophagy may contribute to ZSS pathogenesis.     

Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A₁, Atg7 knockdown or in atg5^?/? MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.     

PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury

Damaged or dysfunctional mitochondria are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Therefore, timely removal of these organelles is critical to cellular homeostasis and viability. Mitophagy is the mechanism of selective degradation of mitochondria via autophagy. The significance of mitophagy in kidney diseases, including ischemic acute kidney injury (AKI), has yet to be established, and the involved pathway of mitophagy remains poorly understood. Here, we show that mitophagy is induced in renal proximal tubular cells in both in vitro and in vivo models of ischemic AKI. Mitophagy under these conditions is abrogated by Pink1 and Park2 deficiency, supporting a critical role of the PINK1-PARK2 pathway in tubular cell mitophagy. Moreover, ischemic AKI is aggravated in pink1 andpark2 single- as well as double-knockout mice. Mechanistically, Pink1 and Park2 deficiency enhances mitochondrial damage, reactive oxygen species production, and inflammatory response. Taken together, these results indicate that PINK1-PARK2-mediated mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function during AKI.     

Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy

Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity.     

Monitoring mitophagy in yeast: The Om45-GFP processing assay

Macroautophagy (hereafter autophagy) is a ubiquitous degradative process in eukaryotic cells.¹ Mitochondria autophagy (mitophagy) is a type of selective autophagy that degrades mitochondria selectively.² Mitophagy is thought to play an important role for maintaining the quality of these organelles by eliminating damaged mitochondria, and it is involved in cellular differentiation, whereas dysfunctional mitophagy is related with neurodegenerative diseases;^3-5 however, the mechanism of mitophagy is poorly understood. To facilitate the analysis of mitophagy, we recently established a simple method to monitor mitophagy in yeast, the Om45-GFP processing assay.⁶ Om45-GFP is a mitochondrial outer membrane protein. Following the uptake of mitochondria into the vacuole, Om45-GFP is degraded, releasing the intact form of GFP, which is detected by immunoblotting. Therefore, the amount of free GFP reflects the level of mitophagy.     

Aup1p, a yeast mitochondrial protein phosphatase homolog, is required for efficient stationary phase mitophagy and cell survival

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.     

The selective autophagy receptor SQSTM1/p62 improves lifespan and proteostasis in an evolutionarily conserved manner

ABSTRACT

The degradation of specific cargos such as ubiquitinated protein aggregates and dysfunctional mitochondria via macroautophagy/autophagy is facilitated by SQSTM1/p62, the first described selective autophagy receptor in metazoans. While the general process of autophagy plays crucial roles during aging, it remains unclear whether and how selective autophagy mediates effects on longevity and health. Two recent studies in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster observed gene expression changes of the respective SQSTM1 orthologs in response to environmental stressors or age and showed that overexpression of SQSTM1 is sufficient to extend lifespan and improve proteostasis and mitochondrial function in an autophagy-dependent manner in these model organisms. These findings show that increased expression of the selective autophagy receptor SQSTM1 is sufficient to induce aggrephagy in C. elegans, and mitophagy in Drosophila, and demonstrate an evolutionarily conserved role for SQSTM1 in lifespan determination.     

Phospholipid methylation controls Atg32‐mediated mitophagy and Atg8 recycling

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy‐inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria‐anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy‐incompetent lipid conjugate of the autophagy‐related ubiquitin‐like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3‐null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32‐mediated mitophagy.     

In vivo imaging reveals mitophagy independence in the maintenance of axonal mitochondria during normal aging

Mitophagy is thought to be a critical mitochondrial quality control mechanism in neurons and has been extensively studied in neurological disorders such as Parkinson's disease. However, little is known about how mitochondria are maintained in the lengthy neuronal axons in the context of physiological aging. Here, we utilized the unique Drosophila wing nerve model and in vivo imaging to rigorously profile changes in axonal mitochondria during aging. We revealed that mitochondria became fragmented and accumulated in aged axons. However, lack of Pink1 or Parkin did not lead to the accumulation of axonal mitochondria or axonal degeneration. Further, unlike in in vitro cultured neurons, we found that mitophagy rarely occurred in intact axons in vivo, even in aged animals. Furthermore, blocking overall mitophagy by knockdown of the core autophagy genes Atg12 or Atg17 had little effect on the turnover of axonal mitochondria or axonal integrity, suggesting that mitophagy is not required for axonal maintenance; this is regardless of whether the mitophagy is PINK1‐Parkin dependent or independent. In contrast, downregulation of mitochondrial fission–fusion genes caused age‐dependent axonal degeneration. Moreover, Opa1 expression in the fly head was significantly decreased with age, which may underlie the accumulation of fragmented mitochondria in aged axons. Finally, we showed that adult‐onset, neuronal downregulation of the fission–fusion, but not mitophagy genes, dramatically accelerated features of aging. We propose that axonal mitochondria are maintained independently of mitophagy and that mitophagy‐independent mechanisms such as fission–fusion may be central to the maintenance of axonal mitochondria and neural integrity during normal aging.     

Mitophagy: The Life-or-Death Dichotomy Includes Yeast

The degradation and recycling of mitochondria is an important household chore in eukaryotic cells. It is thought that mitochondrial autophagy, or mitophagy, is the major route by which mitochondria are degraded. In this view, the cell would selectively induce mitophagy to expunge malfunctioning mitochondria, thus ridding the cell of troublesome sources of reactive oxygen species, apoptosis-inducing factors, or unnecessary metabolic burden. This standard view of mitophagy, in addition to some experimental reports, points to a pro-survival role of mitophagy. However, there is also a significant amount of evidence that suggests a pro-death role of this process, some of it coming from studies in yeast. Aup1 is a protein phosphatase homolog that shows a genetic interaction with the Atg1 protein kinase, localizes to mitochondria, and is required for mitophagy under stationary phase conditions in lactate medium. In contrast with previous yeast studies on mitophagy, deletion of AUP1 results in decreased viability under mitophagy-inducing conditions, suggesting a pro-survival role under physiologically relevant conditions. Thus, the Janus-faced nature of mitophagy is conserved between yeast and mammalian systems.

Addendum to:

Aup1p, a Yeast Mitochondrial Protein Phosphatase Homolog, is Required for Efficient Stationary Phase Mitophagy and Cell Survival

R. Tal, G. Winter, N. Ecker, D.J. Klionsky and H. Abeliovich

J Biol Chem 2006; 282: 5617-24     

Zinc prevents mitochondrial superoxide generation by inducing mitophagy in the setting of hypoxia/reoxygenation in cardiac cells

Zinc plays a role in autophagy and protects cardiac cells from ischemia/reperfusion injury. This study aimed to test if zinc can induce mitophagy leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation (H/R) in cardiac cells. H9c2 cells were subjected to 4?h hypoxia followed by 2?h reoxygenation. Under normoxic conditions, treatments of cells with ZnCl₂ increased both the LC3-II/LC3-I ratio and GFP-LC3 puncta, implying that zinc induces autophagy. Further experiments showed that endogenous zinc is required for the autophagy induced by starvation and rapamycin. Zinc down-regulated TOM20, TIM23, and COX4 both in normoxic cells and the cells subjected to H/R, indicating that zinc can trigger mitophagy. Zinc increased ERK activity and Beclin1 expression, and zinc-induced mitophagy was inhibited by PD98059 and Beclin1 siRNA during reoxygenation. Zinc-induced Beclin1 expression was reversed by PD98059, implying that zinc promotes Beclin1 expression via ERK. In addition, zinc failed to induce mitophagy in cells transfected with PINK1 siRNA and stabilized PINK1 in mitochondria. Moreover, zinc-induced PINK1 stabilization was inhibited by PD98059. Finally, zinc prevented mitochondrial superoxide generation and dissipation of mitochondrial membrane potential (ΔΨ_m) at reoxygenation, which was blocked by both the Beclin1 and PINK1 siRNAs, suggesting that zinc prevents mitochondrial oxidative stress through mitophagy. In summary, zinc induces mitophagy through PINK1 and Beclin1 via ERK leading to the prevention of mitochondrial superoxide generation in the setting of H/R. Clearance of damaged mitochondria may account for the cardioprotective effect of zinc on H/R injury.     

MicroRNA-137 Is a Novel Hypoxia-responsive MicroRNA That Inhibits Mitophagy via Regulation of Two Mitophagy Receptors FUNDC1 and NIX

Mitophagy receptors mediate the selective recognition and targeting of damaged mitochondria by autophagosomes. The mechanism for the regulation of these receptors remains unknown. Here, we demonstrated that a novel hypoxia-responsive microRNA, microRNA-137 (miR-137), markedly inhibits mitochondrial degradation by autophagy without affecting global autophagy. miR-137 targets the expression of two mitophagy receptors NIX and FUNDC1. Impaired mitophagy in response to hypoxia caused by miR-137 is reversed by re-expression of FUNDC1 and NIX expression vectors lacking the miR-137 recognition sites at their 3′ UTR. Conversely, miR-137 also suppresses the mitophagy induced by fundc1 (CDS+3′UTR) but not fundc1 (CDS) overexpression. Finally, we found that miR-137 inhibits mitophagy by reducing the expression of the mitophagy receptor thereby leads to inadequate interaction between mitophagy receptor and LC3. Our results demonstrated the regulatory role of miRNA to mitophagy receptors and revealed a novel link between miR-137 and mitophagy.     

Mitochondrial fission facilitates mitophagy in Saccharomyces cerevisiae

As a highly dynamic organelle, mitochondria undergo constitutive fusion and fission as well as biogenesis and degradation. Mitophagy, selective mitochondrial degradation through autophagy, is a conserved cellular process used for the elimination of excessive and damaged mitochondria in eukaryotes. Despite the significance of mitophagy in cellular physiology and pathophysiologies, the underlying mechanism of this process is far from clear. In this report, we studied the role of mitochondrial fission during mitophagy, and uncover a direct link between the fission complex and mitophagy machinery in Saccharomyces cerevisiae.     

Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson's disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner. p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1-mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy.     

Backbone and side chain resonance assignments for a structured domain within Atg32

Autophagy is a catabolic cellular process that targets cytosolic material, including mitochondria, to the vacuole or lysosomes for degradation. The selective degradation of mitochondria by autophagy is termed mitophagy. Dysfunctional mitophagy, which leads to the accumulation of damaged mitochondria, has been implicated in Parkinson’s disease, cancer, cardiac disease and metabolic disease. In Saccharomyces cerevisiae, mitophagy is initiated by the autophagy receptor Atg32, an outer mitochondrial membrane protein. A lack of structural information for Atg32 has hindered our understanding of the molecular mechanisms of mitophagy initiation. To gain new structural insight into Atg32, we have identified the location of a structured domain within the cytosolic region of Atg32 and completed the backbone and side chain resonance assignments for this domain.     

Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. Accumulating evidence has also shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination through mitochondrial E3 ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics, and mitophagy. Here we showed that MUL1, a mitochondria-localized E3 ligase, regulates selenite-induced mitophagy in an ATG5 and ULK1-dependent manner. ULK1 partially translocated to mitochondria after selenite treatment and interacted with MUL1. We also demonstrated that ULK1 is a novel substrate of MUL1. These results suggest the association of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent.