Michael J. Baker

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

OPA1 processing by YEM1L and OMA1 is dispensable for mitochondrial fusion and instead drives mitochondrial fragmentation, which is crucial for mitochondrial integrity and quality control.

Quality Control of Mitochondrial Proteostasis

A decline in mitochondrial activity has been associated with aging and is a hallmark of many neurological diseases. Surveillance mechanisms acting at the molecular, organellar, and cellular level monitor mitochondrial integrity and ensure the maintenance of mitochondrial proteostasis. Here we will review the central role of mitochondrial chaperones and proteases, the cytosolic ubiquitin-proteasome system, and the mitochondrial unfolded response in this interconnected quality control network, highlighting the dual function of some proteases in protein quality control within the organelle and for the regulation of mitochondrial fusion and mitophagy.

Imbalanced OPA1 processing and mitochondrial fragmentation cause heart failure in mice

A change of heart (mitochondria) Mitochondria provide an essential source of energy to drive cellular processes and are particularly important in heart muscle cells (see the Perspective by Gottlieb and Bernstein). After birth, the availability of oxygen and nutrients to organs and tissues changes. This invokes changes in metabolism. Gong et al. studied the developmental transitions in mouse heart mitochondria soon after birth. Mitochondria were replaced wholesale via mitophagy in cardiomyocytes over the first 3 weeks after birth. Preventing this turnover by interfering with parkin-mediated mitophagy specifically in cardiomyocytes prevented the normal metabolic transition and caused heart failure. Thus, the heart has coopted a quality-control pathway to facilitate a major developmental transition after birth. Wai et al. examined the role of mitochondrial fission and fusion in mouse cardiomyocytes. Disruption of these processes led to “middle-aged” death from a form of dilated cardiomyopathy. Mice destined to develop cardiomyopathy were protected by feeding with a high-fat diet, which altered cardiac metabolism. Science, this issue p. 10.1126/science.aad2459, p. 10.1126/science.aad0116; see also p. 1162 Mitochondrial fragmentation in cardiomyocytes causes heart failure in mice and can be rescued by metabolic intervention. [Also see Perspective by Gottlieb and Bernstein] INTRODUCTION Mitochondria are essential organelles whose form and function are inextricably linked. Balanced fusion and fission events shape mitochondria to meet metabolic demands and to ensure removal of damaged organelles. A fragmentation of the mitochondrial network occurs in response to cellular stress and is observed in a wide variety of disease conditions, including heart failure, neurodegenerative disorders, cancer, and obesity. However, the physiological relevance of stress-induced mitochondrial fragmentation remains unclear. RATIONALE Proteolytic processing of the dynamin-like guanosine triphosphatase (GTPase) OPA1 in the inner membrane of mitochondria is emerging as a critical regulatory step to balance mitochondrial fusion and fission. Two mitochondrial proteases, OMA1 and the AAA protease YME1L, cleave OPA1 from long (L-OPA1) to short (S-OPA1) forms. L-OPA1 is required for mitochondrial fusion, but S-OPA1 is not, although accumulation of S-OPA1 in excess accelerates fission. In cultured mammalian cells, stress conditions activate OMA1, which cleaves L-OPA1 and inhibits mitochondrial fusion resulting in mitochondrial fragmentation. In this study, we generated conditional mouse models for both YME1L and OMA1 and examined the role of OPA1 processing and mitochondrial fragmentation in the heart, a metabolically demanding organ that depends critically on mitochondrial functions. RESULTS Deletion of Yme1l in cardiomyocytes did not grossly affect mitochondrial respiration but induced the proteolytic cleavage of OPA1 by the stress-activated peptidase OMA1 and drove fragmentation of mitochondria in vivo. These mice suffered from dilated cardiomyopathy characterized by well-established features of heart failure that include necrotic cell death, fibrosis and ventricular remodelling, and a metabolic switch away from fatty acid oxidation and toward glucose use. We discovered that additional deletion of Oma1 in cardiomyocytes prevented OPA1 processing altogether and restored normal mitochondrial morphology and cardiac health. On the other hand, mice lacking YME1L in both skeletal muscle and cardiomyocytes exhibited normal cardiac function and life span despite mitochondrial fragmentation in cardiomyocytes. Imbalanced OPA1 processing in skeletal muscle, which is an insulin signaling tissue, induced systemic glucose intolerance and prevented cardiac glucose overload and cardiomyopathy. We observed a similar effect on cardiac metabolism upon feeding mice lacking Yme1l in cardiomyocytes a high-fat diet, which preserved heart function despite mitochondrial fragmentation. CONCLUSION Our work highlights the importance of balanced fusion and fission of mitochondria for cardiac function and unravels an intriguing link between mitochondrial dynamics and cardiac metabolism in the adult heart in vivo. Mitochondrial fusion mediated by L-OPA1 preserves cardiac function, whereas its stress-induced processing by OMA1 and mitochondrial fragmentation triggers dilated cardiomyopathy and heart failure. In contrast to previous genetic models of the mitochondrial fusion machinery, mice lacking Yme1l in cardiomyocytes do not show pleiotropic respiratory deficiencies and thus provide a tool to directly assess the physiological importance of mitochondrial dynamics. Preventing mitochondrial fragmentation by deleting Oma1 protects against cell death and heart failure. The identification of OMA1 as a critical regulator of mitochondrial morphology and cardiomyocyte survival holds promise for translational applications in cardiovascular medicine. Mitochondrial fragmentation induces a metabolic switch from fatty acid to glucose utilization in the heart. It turns out that reversing this switch and restoring normal cardiac metabolism is sufficient to preserve heart function despite mitochondrial fragmentation. These findings raise the intriguing possibility that the switch in fuel usage that occurs in the failing adult heart may, in fact, be maladaptive and could contribute to the pathogenesis of heart failure. Critical role of balanced mitochondrial fusion and fission for cardiac metabolism and heart function. Induced processing of the dynamin-like GTPase OPA1 in the inner membrane by the stress-activated peptidase OMA1 leads to mitochondrial fragmentation, cardiomyopathy, and heart failure, which is characterized by a switch in fuel utilization. Heart function can be preserved by reversing this metabolic switch without suppressing mitochondrial fragmentation. Mitochondrial morphology is shaped by fusion and division of their membranes. Here, we found that adult myocardial function depends on balanced mitochondrial fusion and fission, maintained by processing of the dynamin-like guanosine triphosphatase OPA1 by the mitochondrial peptidases YME1L and OMA1. Cardiac-specific ablation of Yme1l in mice activated OMA1 and accelerated OPA1 proteolysis, which triggered mitochondrial fragmentation and altered cardiac metabolism. This caused dilated cardiomyopathy and heart failure. Cardiac function and mitochondrial morphology were rescued by Oma1 deletion, which prevented OPA1 cleavage. Feeding mice a high-fat diet or ablating Yme1l in skeletal muscle restored cardiac metabolism and preserved heart function without suppressing mitochondrial fragmentation. Thus, unprocessed OPA1 is sufficient to maintain heart function, OMA1 is a critical regulator of cardiomyocyte survival, and mitochondrial morphology and cardiac metabolism are intimately linked.

Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin‐like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1‐mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress‐sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1‐mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.

DNAJC19, a Mitochondrial Cochaperone Associated with Cardiomyopathy, Forms a Complex with Prohibitins to Regulate Cardiolipin Remodeling

Prohibitins form large protein and lipid scaffolds in the inner membrane of mitochondria that are required for mitochondrial morphogenesis, neuronal survival, and normal lifespan. Here, we have defined the interactome of PHB2 in mitochondria and identified DNAJC19, mutated in dilated cardiomyopathy with ataxia, as binding partner of PHB complexes. We observed impaired cell growth, defective cristae morphogenesis, and similar transcriptional responses in the absence of either DNAJC19 or PHB2. The loss of PHB/DNAJC19 complexes affects cardiolipin acylation and leads to the accumulation of cardiolipin species with altered acyl chains. Similar defects occur in cells lacking the transacylase tafazzin, which is mutated in Barth syndrome. Our experiments suggest that PHB/DNAJC19 membrane domains regulate cardiolipin remodeling by tafazzin and explain similar clinical symptoms in two inherited cardiomyopathies by an impaired cardiolipin metabolism in mitochondrial membranes.

Proteolytic control of mitochondrial function and morphogenesis.

Mitochondrial proteostasis depends on a hierarchical system of tightly controlled quality surveillance mechanisms. Proteases within mitochondria take center stage in this network. They eliminate misfolded and damaged proteins and ensure the biogenesis and morphogenesis of mitochondria by processing or degrading short-lived regulatory proteins. Mitochondrial gene expression, the mitochondrial phospholipid metabolism and the fusion of mitochondrial membranes are under proteolytic control. Furthermore, in response to stress and mitochondrial dysfunction, proteolysis inhibits fusion and facilitates mitophagy and apoptosis. Defining these versatile activities of mitochondrial proteases will be pivotal for understanding the pathogenesis of various neurodegenerative disorders associated with defective mitochondria-associated proteolysis. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

The regulation of mitochondrial DNA copy number in glioblastoma cells

As stem cells undergo differentiation, mitochondrial DNA (mtDNA) copy number is strictly regulated in order that specialized cells can generate appropriate levels of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to undertake their specific functions. It is not understood whether tumor-initiating cells regulate their mtDNA in a similar manner or whether mtDNA is essential for tumorigenesis. We show that human neural stem cells (hNSCs) increased their mtDNA content during differentiation in a process that was mediated by a synergistic relationship between the nuclear and mitochondrial genomes and results in increased respiratory capacity. Differentiating multipotent glioblastoma cells failed to match the expansion in mtDNA copy number, patterns of gene expression and increased respiratory capacity observed in hNSCs. Partial depletion of glioblastoma cell mtDNA rescued mtDNA replication events and enhanced cell differentiation. However, prolonged depletion resulted in impaired mtDNA replication, reduced proliferation and induced the expression of early developmental and pro-survival markers including POU class 5 homeobox 1 (OCT4) and sonic hedgehog (SHH). The transfer of glioblastoma cells depleted to varying degrees of their mtDNA content into immunocompromised mice resulted in tumors requiring significantly longer to form compared with non-depleted cells. The number of tumors formed and the time to tumor formation was relative to the degree of mtDNA depletion. The tumors derived from mtDNA depleted glioblastoma cells recovered their mtDNA copy number as part of the tumor formation process. These outcomes demonstrate the importance of mtDNA to the initiation and maintenance of tumorigenesis in glioblastoma multiforme.

Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import

The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.

Impaired folding of the mitochondrial small TIM chaperones induces clearance by the i-AAA protease.

The intermembrane space of mitochondria contains a dedicated chaperone network-the small translocase of the inner membrane (TIM) family-for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX(3)C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1).

Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms. DOI: http://dx.doi.org/10.7554/eLife.17463.001