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.     

Casein kinase 2 is essential for mitophagy

Mitophagy is a process that selectively degrades mitochondria. When mitophagy is induced in yeast, the mitochondrial outer membrane protein Atg32 is phosphorylated, interacts with the adaptor protein Atg11 and is recruited into the vacuole with mitochondria. We screened kinase‐deleted yeast strains and found that CK2 is essential for Atg32 phosphorylation, Atg32–Atg11 interaction and mitophagy. Inhibition of CK2 specifically blocks mitophagy, but not macroautophagy, pexophagy or the Cvt pathway. In vitro, CK2 phosphorylates Atg32 at serine 114 and serine 119. We conclude that CK2 regulates mitophagy by directly phosphorylating Atg32.     

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.     

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.     

Phosphorylation of Serine 114 on Atg32 mediates mitophagy

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When mitophagy is induced in yeast, mitochondrial residential protein Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and it is recruited into the vacuole along with mitochondria. The Atg11-Atg32 interaction is believed to be the initial molecular step in which the autophagic machinery recognizes mitochondria as a cargo, although how this interaction is mediated is poorly understood. Therefore, we studied the Atg11-Atg32 interaction in detail. We found that the C-terminus region of Atg11, which included the fourth coiled-coil domain, interacted with the N-terminus region of Atg32 (residues 100-120). When mitophagy was induced, Ser-114 and Ser-119 on Atg32 were phosphorylated, and then the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediated the Atg11-Atg32 interaction and mitophagy. These findings suggest that cells can regulate the amount of mitochondria, or select specific mitochondria (damaged or aged) that are degraded by mitophagy, by controlling the activity and/or localization of the kinase that phosphorylates Atg32. We also found that Hog1 and Pbs2, which are involved in the osmoregulatory signal transduction cascade, are related to Atg32 phosphorylation and mitophagy.     

Protein N-terminal Acetylation by the NatA Complex Is Critical for Selective Mitochondrial Degradation

Mitophagy is an evolutionarily conserved autophagy pathway that selectively degrades mitochondria. Although it is well established that this degradation system contributes to mitochondrial quality and quantity control, mechanisms underlying mitophagy remain largely unknown. Here, we report that protein N-terminal acetyltransferase A (NatA), an enzymatic complex composed of the catalytic subunit Ard1 and the adaptor subunit Nat1, is crucial for mitophagy in yeast. NatA is associated with the ribosome via Nat1 and acetylates the second amino acid residues of nascent polypeptides. Mitophagy, but not bulk autophagy, is strongly suppressed in cells lacking Ard1, Nat1, or both proteins. In addition, loss of NatA enzymatic activity causes impairment of mitochondrial degradation, suggesting that protein N-terminal acetylation by NatA is important for mitophagy. Ard1 and Nat1 mutants exhibited defects in induction of Atg32, a protein essential for mitophagy, and formation of mitochondria-specific autophagosomes. Notably, overexpression of Atg32 partially recovered mitophagy in NatA-null cells, implying that this acetyltransferase participates in mitophagy at least in part via Atg32 induction. Together, our data implicate NatA-mediated protein modification as an early regulatory step crucial for efficient mitophagy.     

Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy

Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.     

Receptor-mediated mitophagy in yeast and mammalian systems

Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.     

Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.     

A pseudo-receiver domain in Atg32 is required for mitophagy

Mitochondria are targeted for degradation by mitophagy, a selective form of autophagy. In Saccharomyces cerevisiae, mitophagy is dependent on the autophagy receptor, Atg32, an outer mitochondrial membrane protein. Once activated, Atg32 recruits the autophagy machinery to mitochondria, facilitating mitochondrial capture in phagophores, the precursors to autophagosomes. However, the mechanism of Atg32 activation remains poorly understood. To investigate this crucial step in mitophagy regulation, we examined the structure of Atg32. We have identified a structured domain in Atg32 that is essential for the initiation of mitophagy, as it is required for the proteolysis of the C-terminal domain of Atg32 and the subsequent recruitment of Atg11. The solution structure of this domain was determined by NMR spectroscopy, revealing that Atg32 contains a previously undescribed pseudo-receiver (PsR) domain. Our data suggests that the PsR domain of Atg32 regulates Atg32 activation and the initiation of mitophagy.

Abbreviations:AIM: Atg8-interacting motif; GFP: green fluorescent protein; LIR: LC3-interacting region; NMR: nuclear magnetic resonance; NOESY: nuclear Overhauser effect spectroscopy; PDB: protein data bank; PsR: pseudo-receiver; RMSD: root-mean-square deviation     

BCL2L13 is a mammalian homolog of the yeast mitophagy receptor Atg32

Although Atg32 is essential for mitophagy in yeast, no mammalian homolog has been identified. Here, we demonstrate that BCL2L13 (BCL2-like 13 [apoptosis facilitator]) is a functional mammalian homolog of Atg32. First, we hypothesized that a mammalian mitophagy receptor will share certain molecular features with Atg32. Using the molecular profile of Atg32 as a search tool, we screened public databases for novel Atg32 functional homologs and identified BCL2L13. BCL2L13 induces mitochondrial fragmentation and mitophagy in HEK293 cells. In BCL2L13, the BH domains are important for fragmentation, whereas the WXXI motif, an LC3 interacting region, is needed for mitophagy. BCL2L13 induces mitochondrial fragmentation and mitophagy even in the absence of DNM1L/Drp1 and PARK2/Parkin, respectively. BCL2L13 is indispensable for mitochondrial damage-induced fragmentation and mitophagy. Furthermore, BCL2L13 induces mitophagy in Atg32-deficient yeast. Induction and/or phosphorylation of BCL2L13 may regulate its activity. Our findings thus open a new chapter in mitophagy research.     

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.     

A landmark protein essential for mitophagy

Degradation of mitochondria is a fundamental process conserved from yeast to humans that utilizes the machinery of autophagy. In contrast to starvation-induced, nonselective autophagy responsible for nutrient recycling, selective autophagy, which involves particular cues and receptors required for induction and cargo recognition, respectively, mediates mitochondria-specific breakdown. Although numerous studies highlight that mitochondria autophagy (mitophagy) contributes to homeostatic control of mitochondria, the molecular mechanisms underlying this selective clearance process are poorly understood. Using a genome-wide visual screen, we identified Atg32, a protein essential for mitophagy in budding yeast. During respiratory growth, Atg32 is highly expressed, likely in response to oxidative stress, and anchored on the surface of mitochondria. We also demonstrate that Atg32 interacts with Atg8 and Atg11, proteins critical for recognition of cargo receptors. Notably, Atg32 contains WXXI/L/V, a conserved motif that serves as a binding site for the Atg8 family members. Our recent findings suggest that Atg32 is a transmembrane receptor that directs autophagosome formation to mitochondria.     

Coupling mitogenesis and mitophagy for longevity

Maintenance of mitochondrial function and energy homeostasis requires both generation of newly synthesized and elimination of dysfunctional mitochondria. Impaired mitochondrial function and excessive mitochondrial content are major characteristics of aging and several human pathophysiological conditions, highlighting the pivotal role of the coordination between mitochondrial biogenesis and mitophagy. However, the cellular and molecular underpinnings of mitochondrial mass homeostasis remain obscure. In our recent study, we demonstrate that DCT-1, the Caenorhabditis elegans homolog of mammalian BNIP3 and BNIP3L/NIX, is a key mediator of mitophagy promoting longevity under stress. DCT-1 acts downstream of the PINK-1-PDR-1/Parkin pathway and is ubiquitinated upon mitophagy-inducing conditions to mediate the removal of damaged mitochondria. Accumulation of damaged mitochondria triggers SKN-1 activation, which initiates a bipartite retrograde signaling pathway stimulating the coordinated induction of both mitochondrial biogenesis and mitophagy genes. Taken together, our results unravel a homeostatic feedback loop that allows cells to adjust their mitochondrial population in response to environmental and intracellular cues. Age-dependent decline of mitophagy both inhibits removal of dysfunctional or superfluous mitochondria and impairs mitochondrial biogenesis resulting in progressive mitochondrial accretion and consequently, deterioration of cell function.     

Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina

Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.     

Mitochondrial Dysfunction in Age‐Related Metabolic Disorders

Aging is a natural biological process in living organisms characterized by receding bioenergetics. Mitochondria are crucial for cellular bioenergetics and thus an important contributor to age‐related energetics deterioration. In addition, mitochondria play a major role in calcium signaling, redox homeostasis, and thermogenesis making this organelle a major cellular component that dictates the fate of a cell. To maintain its quantity and quality, mitochondria undergo multiple processes such as fission, fusion, and mitophagy to eliminate or replace damaged mitochondria. While this bioenergetics machinery is properly protected, the functional decline associated with age and age‐related metabolic diseases is mostly a result of failure in such protective mechanisms. In addition, metabolic by‐products like reactive oxygen species also aid in this destructive pathway. Mitochondrial dysfunction has always been thought to be associated with diseases. Moreover, studies in recent years have pointed out that aging contributes to the decay of mitochondrial health by promoting imbalances in key mitochondrial‐regulated pathways. Hence, it is crucial to understand the nexus of mitochondrial dysfunction in age‐related diseases. This review focuses on various aspects of basic mitochondrial biology and its status in aging and age‐related metabolic diseases.     

Evidence that a mitochondrial death spiral underlies antagonistic pleiotropy

The antagonistic pleiotropy (AP) theory posits that aging occurs because alleles that are detrimental in older organisms are beneficial to growth early in life and thus are maintained in populations. Although genes of the insulin signaling pathway likely participate in AP, the insulin‐regulated cellular correlates of AP have not been identified. The mitochondrial quality control process called mitochondrial autophagy (mitophagy), which is inhibited by insulin signaling, might represent a cellular correlate of AP. In this view, rapidly growing cells are limited by ATP production; these cells thus actively inhibit mitophagy to maximize mitochondrial ATP production and compete successfully for scarce nutrients. This process maximizes early growth and reproduction, but by permitting the persistence of damaged mitochondria with mitochondrial DNA mutations, becomes detrimental in the longer term. I suggest that as mitochondrial ATP output drops, cells respond by further inhibiting mitophagy, leading to a further decrease in ATP output in a classic death spiral. I suggest that this increasing ATP deficit is communicated by progressive increases in mitochondrial ROS generation, which signals inhibition of mitophagy via ROS‐dependent activation of insulin signaling. This hypothesis clarifies a role for ROS in aging, explains why insulin signaling inhibits autophagy, and why cells become progressively more oxidized during aging with increased levels of insulin signaling and decreased levels of autophagy. I suggest that the mitochondrial death spiral is not an error in cell physiology but rather a rational approach to the problem of enabling successful growth and reproduction in a competitive world of scarce nutrients.     

Mitophagy: mechanisms, pathophysiological roles, and analysis

Abstract Mitochondria are essential organelles that regulate cellular energy homeostasis and cell death. The removal of damaged mitochondria through autophagy, a process called mitophagy, is thus critical for maintaining proper cellular functions. Indeed, mitophagy has been recently proposed to play critical roles in terminal differentiation of red blood cells, paternal mitochondrial degradation, neurodegenerative diseases, and ischemia or drug-induced tissue injury. Removal of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Recent progress in mitophagy studies reveals that mitochondrial priming is mediated either by the Pink1-Parkin signaling pathway or the mitophagic receptors Nix and Bnip3. In this review, we summarize our current knowledge on the mechanisms of mitophagy. We also discuss the pathophysiological roles of mitophagy and current assays used to monitor mitophagy.     

Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons

Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many nonneuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial, leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψ(m)-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with nonneuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring-like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.