Musical chairs during mitophagy

Mitophagy, or the autophagic degradation of mitochondria, is thought to be important in mitochondrial quality control, and hence in cellular physiology. Defects in mitophagy correlate with late onset pathologies and aging. Here, we discuss recent results that shed light on the interrelationship between mitophagy and mitochondrial dynamics, based on proteomic analyses of protein dynamics in wild-type and mutant cells. These studies show that different mitochondrial matrix proteins undergo mitophagy at different rates, and that the rate differences are affected by mitochondrial dynamics. These results are consistent with models in which phase separation within the mitochondrial matrix leads to unequal segregation of proteins during mitochondrial fission. Repeated fusion and fission cycles may thus lead to “distillation” of components that are destined for degradation.     

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.     

Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy

Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.     

A new role for ATM: regulating mitochondrial function and mitophagy

The various pathologies in ataxia telangiectasia (A-T) patients including T-cell lymphomagenesis have been attributed to defects in the DNA damage response pathway because ATM, the gene mutated in this disease, is a key mediator of this process. Analysis of Atm-deficient thymocytes in mice reveals that the absence of this gene results in altered mitochondrial homeostasis, a phenomenon that appears to result from abnormal mitophagy engagement. Interestingly, allelic loss of the autophagic gene Becn1 delays tumorigenesis in Atm-null mice presumably by reversing the mitochondrial abnormalities and not by improving the DNA damage response (DDR) pathway. Thus, ATM plays a critical role in modulating mitochondrial homeostasis perhaps by regulating mitophagy.     

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.     

Autophagy across the eukaryotes: Is S. cerevisiae the odd one out?

Autophagy is conserved throughout the eukaryotes and for many years, work in Saccharomyces cerevisiae has been at the forefront of autophagy research. However as our knowledge of the autophagic machinery has increased, differences between S. cerevisiae and mammalian cells have become apparent. Recent work in other organisms, such as the amoeba Dictyostelium discoideum, indicate an autophagic pathway much more similar to mammalian cells than S. cerevisiae, despite its earlier evolutionary divergence. S. cerevisiae therefore appear to have significantly specialized, and the autophagic pathway in mammals is much more ancient than previously appreciated, which has implications for how we interpret data from organisms throughout the eukaryotic tree.     

Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage

Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.     

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.     

Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine‐tune hypoxic mitophagy

Mitophagy is an essential process for mitochondrial quality control and turnover. It is activated by two distinct pathways, one dependent on ubiquitin and the other dependent on receptors including FUNDC1. It is not clear whether these pathways coordinate to mediate mitophagy in response to stresses, or how mitophagy receptors sense stress signals to activate mitophagy. We find that the mitochondrial E3 ligase MARCH5, but not Parkin, plays a role in regulating hypoxia‐induced mitophagy by ubiquitylating and degrading FUNDC1. MARCH5 directly interacts with FUNDC1 to mediate its ubiquitylation at lysine 119 for subsequent degradation. Degradation of FUNDC1 by MARCH5 expression desensitizes mitochondria to hypoxia‐induced mitophagy, whereas knockdown of endogenous MARCH5 significantly inhibits FUNDC1 degradation and enhances mitochondrial sensitivity toward mitophagy‐inducing stresses. Our findings reveal a feedback regulatory mechanism to control the protein levels of a mitochondrial receptor to fine‐tune mitochondrial quality.     

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.     

Deubiquitinating enzymes regulate PARK2-mediated mitophagy

The selective degradation of mitochondria by the process of autophagy, termed mitophagy, is one of the major mechanisms of mitochondrial quality control. The best-studied mitophagy pathway is the one mediated by PINK1 and PARK2/Parkin. From recent studies it has become clear that ubiquitin-ligation plays a pivotal role and most of the focus has been on the role of ubiquitination of mitochondrial proteins in mitophagy. Even though ubiquitination is a reversible process, very little is known about the role of deubiquitinating enzymes (DUBs) in mitophagy. Here, we report that 2 mitochondrial DUBs, USP30 and USP35, regulate PARK2-mediated mitophagy. We show that USP30 and USP35 can delay PARK2-mediated mitophagy using a quantitative mitophagy assay. Furthermore, we show that USP30 delays mitophagy by delaying PARK2 recruitment to the mitochondria during mitophagy. USP35 does not delay PARK2 recruitment, suggesting that it regulates mitophagy through an alternative mechanism. Interestingly, USP35 only associates with polarized mitochondria, and rapidly translocates to the cytosol during CCCP-induced mitophagy. It is clear that PARK2-mediated mitophagy is regulated at many steps in this important quality control pathway. Taken together, these findings demonstrate an important role of mitochondrial-associated DUBs in mitophagy. Because defects in mitochondria quality control are implicated in many neurodegenerative disorders, our study provides clear rationales for the design and development of drugs for the therapeutic treatment of neurodegenerative diseases such as Parkinson and Alzheimer diseases.     

Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy

Mitochondrial fragmentation due to imbalanced fission and fusion of mitochondria is a prerequisite for mitophagy, however, the exact “coupling” of mitochondrial dynamics and mitophagy remains unclear. We have previously identified that FUNDC1 recruits MAP1LC3B/LC3B (LC3) through its LC3-interacting region (LIR) motif to initiate mitophagy in mammalian cells. Here, we show that FUNDC1 interacts with both DNM1L/DRP1 and OPA1 to coordinate mitochondrial fission or fusion and mitophagy. OPA1 interacted with FUNDC1 via its Lys70 (K70) residue, and mutation of K70 to Ala (A), but not to Arg (R), abolished the interaction and promoted mitochondrial fission and mitophagy. Mitochondrial stress such as selenite or FCCP treatment caused the disassembly of the FUNDC1-OPA1 complex while enhancing DNM1L recruitment to the mitochondria. Furthermore, we observed that dephosphorylation of FUNDC1 under stress conditions promotes the dissociation of FUNDC1 from OPA1 and association with DNM1L. Our data suggest that FUNDC1 regulates both mitochondrial fission or fusion and mitophagy and mediates the “coupling” across the double membrane for mitochondrial dynamics and quality control.     

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP 7 and IP 8; and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP 4 and IP 5. We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.     

Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE

The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Δ cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein–protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7–amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor–dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein–specific sorting signal in a t-SNARE protein, and suggest that AP-3–dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3–dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.     

BECN1 is involved in the initiation of mitophagy

The autophagy protein BECN1/Beclin 1 is known to play a central role in autophagosome formation and maturation. The results presented here demonstrate that BECN1 interacts with the Parkinson disease-related protein PARK2. This interaction does not require PARK2 translocation to mitochondria and occurs mostly in cytosol. However, our results suggest that BECN1 is involved in PARK2 translocation to mitochondria because loss of BECN1 inhibits CCCP- or PINK1 overexpression-induced PARK2 translocation. Our results also demonstrate that the observed PARK2-BECN1 interaction is functionally important. Measurements of the level of MFN2 (mitofusin 2), a PARK2 substrate, demonstrate that depletion of BECN1 prevents PARK2 translocation-induced MFN2 ubiquitination and loss. BECN1 depletion also rescues the MFN2 loss-induced suppression of mitochondrial fusion. In sum, our results demonstrate that BECN1 interacts with PARK2 and regulates PARK2 translocation to mitochondria as well as PARK2-induced mitophagy prior to autophagosome formation.     

Thyroid hormone induction of mitochondrial activity is coupled to mitophagy via ROS-AMPK-ULK1 signaling

Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T₃) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T₃ is coupled to oxidative phosphorylation and ROS production. We show that T₃ induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) mediated phosphorylation of PRKAA1/AMPK (5′ AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T₃-treated cells impairs both mitophagy as well as OXPHOS without affecting T₃ induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T₃-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.     

A cell-free assay allows reconstitution of Vps33p-dependent transport to the yeast vacuole/lysosome.

We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). Yeast spheroplasts are lysed by extrusion through polycarbonate filters. After differential centrifugation, a 125,000-g pellet is enriched for radiolabeled proCPY and is used as "donor" membranes. A 15,000-g pellet, harvested from nonradiolabeled cells and enriched for vacuoles, is used as "acceptor" membranes. When these membranes are incubated together with ATP and cytosolic extracts, approximately 50% of the radiolabeled proCPY is processed to mature CPY. Maturation was inhibited by dilution of donor and acceptor membranes during incubation, showed a 15-min lag period, and was temperature sensitive. Efficient proCPY maturation was possible when donor membranes were from a yeast strain deleted for the PEP4 gene (which encodes the principal CPY processing enzyme, proteinase A) and acceptor membranes from a PEP4 yeast strain, indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the VPS33 gene was less efficient at driving transport. Moreover, antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a VPS gene product.     

Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy

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