Mitochondria in disease: changes in shapes and dynamics.

Mitochondrial structure often determines the function of these highly dynamic, multifunctional, eukaryotic organelles, which are essential for maintaining cellular health. The dynamic nature of mitochondria is apparent in descriptions of different mitochondrial shapes [e.g., donuts, megamitochondria (MGs), and nanotunnels] and crista dynamics. This review explores the significance of dynamic alterations in mitochondrial morphology and regulators of mitochondrial and cristae shape. We focus on studies across tissue types and also describe new microscopy techniques for detecting mitochondrial morphologies both in vivo and in vitro that can improve understanding of mitochondrial structure. We highlight the potential therapeutic benefits of regulating mitochondrial morphology and discuss prospective avenues to restore mitochondrial bioenergetics to manage diseases related to mitochondrial dysfunction.

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

Mitochondrial compartmentalization: emerging themes in structure and function.

Within cellular structures, compartmentalization is the concept of spatial segregation of macromolecules, metabolites, and biochemical pathways. Therefore, this concept bridges organellar structure and function. Mitochondria are morphologically complex, partitioned into several subcompartments by a topologically elaborate two-membrane system. They are also dynamically polymorphic, undergoing morphogenesis events with an extent and frequency that is only now being appreciated. Thus, mitochondrial compartmentalization is something that must be considered both spatially and temporally. Here, we review new developments in how mitochondrial structure is established and regulated, the factors that underpin the distribution of lipids and proteins, and how they spatially demarcate locations of myriad mitochondrial processes. Consistent with its pre-eminence, disturbed mitochondrial compartmentalization contributes to the dysfunction associated with heritable and aging-related diseases.

Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

Diverse mitochondrial abnormalities in a new cellular model of TAFFAZZIN deficiency are remediated by cardiolipin-interacting small molecules.

Barth syndrome (BTHS) is an X-linked disorder of mitochondrial phospholipid metabolism caused by pathogenic variants in TAFFAZIN, which results in abnormal cardiolipin (CL) content in the inner mitochondrial membrane. To identify unappreciated pathways of mitochondrial dysfunction in BTHS, we utilized an unbiased proteomics strategy and identified that complex I (CI) of the mitochondrial respiratory chain and the mitochondrial quality control protease presenilin-associated rhomboid-like protein (PARL) are altered in a new HEK293-based tafazzin-deficiency model. Follow-up studies confirmed decreased steady state levels of specific CI subunits and an assembly factor in the absence of tafazzin; this decrease is in part based on decreased transcription and results in reduced CI assembly and function. PARL, a rhomboid protease associated with the inner mitochondrial membrane with a role in the mitochondrial response to stress, such as mitochondrial membrane depolarization, is increased in tafazzin-deficient cells. The increased abundance of PARL correlates with augmented processing of a downstream target, phosphoglycerate mutase 5, at baseline and in response to mitochondrial depolarization. To clarify the relationship between abnormal CL content, CI levels, and increased PARL expression that occurs when tafazzin is missing, we used blue-native PAGE and gene expression analysis to determine that these defects are remediated by SS-31 and bromoenol lactone, pharmacologic agents that bind CL or inhibit CL deacylation, respectively. These findings have the potential to enhance our understanding of the cardiac pathology of BTHS, where defective mitochondrial quality control and CI dysfunction have well-recognized roles in the pathology of diverse forms of cardiac dysfunction.

Tafazzin deficiency impairs CoA-dependent oxidative metabolism in cardiac mitochondria.

Barth syndrome is a mitochondrial myopathy resulting from mutations in the tafazzin (TAZ) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear how tafazzin deficiency impacts cardiac mitochondrial metabolism. To address this question while avoiding confounding effects of cardiomyopathy on mitochondrial phenotype, we utilized Taz-shRNA knockdown (TazKD ) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human Barth syndrome but normal cardiac function into adulthood. Consistent with previous reports from other models, mitochondrial H2O2 emission and oxidative damage were greater in TazKD than in wild-type (WT) hearts, but there were no differences in oxidative phosphorylation coupling efficiency or membrane potential. Fatty acid and pyruvate oxidation capacities were 40-60% lower in TazKD mitochondria, but an up-regulation of glutamate oxidation supported respiration rates approximating those with pyruvate and palmitoylcarnitine in WT. Deficiencies in mitochondrial CoA and shifts in the cardiac acyl-CoA profile paralleled changes in fatty acid oxidation enzymes and acyl-CoA thioesterases, suggesting limitations of CoA availability or "trapping" in TazKD mitochondrial metabolism. Incubation of TazKD mitochondria with exogenous CoA partially rescued pyruvate and palmitoylcarnitine oxidation capacities, implicating dysregulation of CoA-dependent intermediary metabolism rather than respiratory chain defects in the bioenergetic impacts of tafazzin deficiency. These findings support links among cardiolipin abnormalities, respiratory supercomplex instability, and mitochondrial oxidant production and shed new light on the distinct metabolic consequences of tafazzin deficiency in the mammalian heart.

Systems Analysis of the 22q11.2 Microdeletion Syndrome Converges on a Mitochondrial Interactome Necessary for Synapse Function and Behavior.

Neurodevelopmental disorders offer insight into synaptic mechanisms. To unbiasedly uncover these mechanisms, we studied the 22q11.2 syndrome, a recurrent copy number variant, which is the highest schizophrenia genetic risk factor. We quantified the proteomes of 22q11.2 mutant human fibroblasts from both sexes and mouse brains carrying a 22q11.2-like defect, Df(16)A+/- Molecular ontologies defined mitochondrial compartments and pathways as some of top ranked categories. In particular, we identified perturbations in the SLC25A1-SLC25A4 mitochondrial transporter interactome as associated with the 22q11.2 genetic defect. Expression of SLC25A1-SLC25A4 interactome components was affected in neuronal cells from schizophrenia patients. Furthermore, hemideficiency of the Drosophila SLC25A1 or SLC25A4 orthologues, dSLC25A1-sea and dSLC25A4-sesB, affected synapse morphology, neurotransmission, plasticity, and sleep patterns. Our findings indicate that synapses are sensitive to partial loss of function of mitochondrial solute transporters. We propose that mitoproteomes regulate synapse development and function in normal and pathological conditions in a cell-specific manner.SIGNIFICANCE STATEMENT We address the central question of how to comprehensively define molecular mechanisms of the most prevalent and penetrant microdeletion associated with neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. This complex mutation reduces gene dosage of ∼63 genes in humans. We describe a disruption of the mitoproteome in 22q11.2 patients and brains of a 22q11.2 mouse model. In particular, we identify a network of inner mitochondrial membrane transporters as a hub required for synapse function. Our findings suggest that mitochondrial composition and function modulate the risk of neurodevelopmental disorders, such as schizophrenia.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

Defining functional classes of Barth syndrome mutation in humans.

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ.

Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION.

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3 First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high.

Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.

In yeast, phosphatidylglycerol (PG) is a minor phospholipid under standard conditions; it can be utilized for cardiolipin (CL) biosynthesis by CL synthase, Crd1p, or alternatively degraded by the phospholipase Pgc1p. The Saccharomyces cerevisiae deletion mutants crd1Δ and pgc1Δ both accumulate PG. Based on analyses of the phospholipid content of pgc1Δ and crd1Δ yeast, we revealed that in yeast mitochondria, two separate pools of PG are present, which differ in their fatty acid composition and accessibility for Pgc1p-catalyzed degradation. In contrast to CL-deficient crd1Δ yeast, the pgc1Δ mutant contains normal levels of CL. This makes the pgc1Δ strain a suitable model to study the effect of accumulation of PG per se. Using fluorescence microscopy, we show that accumulation of PG with normal levels of CL resulted in increased fragmentation of mitochondria, while in the absence of CL, accumulation of PG led to the formation of large mitochondrial sheets. We also show that pgc1Δ mitochondria exhibited increased respiration rates due to increased activity of cytochrome c oxidase. Taken together, our results indicate that not only a lack of anionic phospholipids, but also excess PG, or unbalanced ratios of anionic phospholipids in mitochondrial membranes, have harmful consequences on mitochondrial morphology and function.

The complexity of cardiolipin in health and disease.

Cardiolipin, the signature phospholipid of mitochondria, is a lipid dimer that is important for a diverse range of mitochondrial activities beyond the process of ATP production. Thus not surprisingly, derangements in cardiolipin metabolism are now appreciated to contribute to an assortment of pathological conditions. A comprehensive inventory of enzymes involved in cardiolipin biosynthesis and remodeling was just recently obtained. Post-biosynthesis, the acyl chain composition of cardiolipin is modified by up to three distinct remodeling enzymes that produce either a homogeneous tissue-specific mature form of cardiolipin or alternatively 'bad' cardiolipin that has been linked to mitochondrial dysfunction. In this review, we initially focus on the newly identified players in cardiolipin metabolism and then shift our attention to how changes in cardiolipin metabolism contribute to human disease.

Phosphatidylserine decarboxylase 1 autocatalysis and function does not require a mitochondrial-specific factor.

Phosphatidylethanolamine (PE) is a major cellular phospholipid that can be made by four separate pathways, one of which resides in the mitochondrion. The mitochondrial enzyme that generates PE is phosphatidylserine decarboxylase 1 (Psd1p). The pool of PE produced by Psd1p, which cannot be compensated for by the other cellular PE metabolic pathways, is important for numerous mitochondrial functions, including oxidative phosphorylation and mitochondrial dynamics and morphology, and is essential for murine development. To become catalytically active, Psd1p undergoes an autocatalytic processing step involving a conserved LGST motif that separates the enzyme into α and ß subunits that remain non-covalently attached and are anchored to the inner membrane by virtue of the membrane-embedded ß subunit. It was speculated that Psd1p autocatalysis requires a mitochondrial-specific factor and that for Psd1p to function in vivo, it had to be embedded with the correct topology in the mitochondrial inner membrane. However, the identity of the mitochondrial factor required for Psd1p autocatalysis has not been identified. With the goal of defining molecular requirements for Psd1p autocatalysis, we demonstrate that: 1) despite the conservation of the LGST motif from bacteria to humans, only the serine residue is absolutely required for Psd1p autocatalysis and function; 2) yeast Psd1p does not require its substrate phosphatidylserine for autocatalysis; and 3) contrary to a prior report, yeast Psd1p autocatalysis does not require mitochondrial-specific phospholipids, proteins, or co-factors, because Psd1p re-directed to the secretory pathway undergoes autocatalysis normally and is fully functional in vivo.

Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.

Seven functional classes of Barth syndrome mutation.

Patients with Barth syndrome (BTHS), a rare X-linked disease, suffer from skeletal and cardiomyopathy and bouts of cyclic neutropenia. The causative gene encodes tafazzin, a transacylase, which is the major determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, CL. In addition to numerous frame shift and splice-site mutations, 36 missense mutations have been associated with BTHS. Previously, we established a BTHS-mutant panel in the yeast Saccharomyces cerevisiae that successfully models 18/21 conserved pathogenic missense mutations and defined the loss-of-function mechanism associated with a subset of the mutant tafazzins. Here, we report the biochemical and cell biological characterization of the rest of the yeast BTHS-mutant panel and in so doing identify three additional modes of tafazzin dysfunction. The largest group of mutant tafazzins is catalytically null, two mutants encode hypomorphic alleles, and another two mutants are temperature sensitive. Additionally, we have expanded the defects associated with previously characterized matrix-mislocalized-mutant tafazzins to include the rapid degradation of aggregation-prone polypeptides that correctly localize to the mitochondrial IMS. In sum, our in-depth characterization of the yeast BTHS-mutant panel has identified seven functional classes of BTHS mutation.

Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver.

Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi-dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O2 consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.

The Taz1p transacylase is imported and sorted into the outer mitochondrial membrane via a membrane anchor domain.

Mutations in the mitochondrial transacylase tafazzin, Taz1p, in Saccharomyces cerevisiae cause Barth syndrome, a disease of defective cardiolipin remodeling. Taz1p is an interfacial membrane protein that localizes to both the outer and inner membranes, lining the intermembrane space. Pathogenic point mutations in Taz1p that alter import and membrane insertion result in accumulation of monolysocardiolipin. In this study, we used yeast as a model to investigate the biogenesis of Taz1p. We show that to achieve this unique topology in mitochondria, Taz1p follows a novel import pathway in which it crosses the outer membrane via the translocase of the outer membrane and then uses the Tim9p-Tim10p complex of the intermembrane space to insert into the mitochondrial outer membrane. Taz1p is then transported to membranes of an intermediate density to reach a location in the inner membrane. Moreover, a pathogenic mutation within the membrane anchor (V224R) alters Taz1p import so that it bypasses the Tim9p-Tim10p complex and interacts with the translocase of the inner membrane, TIM23, to reach the matrix. Critical targeting information for Taz1p resides in the membrane anchor and flanking sequences, which are often mutated in Barth syndrome patients. These studies suggest that altering the mitochondrial import pathway of Taz1p may be important in understanding the molecular basis of Barth syndrome.

Stem cell models of TAFAZZIN deficiency reveal novel tissue-specific pathologies in Barth Syndrome.

Barth syndrome (BTHS) is a rare mitochondrial disease caused by pathogenic variants in the gene TAFAZZIN, which leads to abnormal cardiolipin (CL) metabolism on the inner mitochondrial membrane. Although TAFAZZIN is ubiquitously expressed, BTHS involves a complex combination of tissue specific phenotypes including cardiomyopathy, neutropenia, skeletal myopathy, and growth delays, with a relatively minimal neurological burden. To understand both the developmental and functional effects of TAZ-deficiency in different tissues, we generated isogenic TAZ knockout (TAZ- KO) and WT cardiomyocytes (CMs) and neural progenitor cells (NPCs) from CRISPR-edited induced pluripotent stem cells (iPSCs). In TAZ-KO CMs we discovered evidence of dysregulated mitophagy including dysmorphic mitochondria and mitochondrial cristae, differential expression of key autophagy-associated genes, and an inability of TAZ-deficient CMs to properly initiate stress-induced mitophagy. In TAZ-deficient NPCs we identified novel phenotypes including a reduction in CIV abundance and CIV activity in the CIII2&CIV2 intermediate complex. Interestingly, while CL acyl chain manipulation was unable to alter mitophagy defects in TAZ-KO CMs, we found that linoleic acid or oleic acid supplementation was able to partially restore CIV abundance in TAZ-deficient NPCs. Taken together, our results have implications for understanding the tissue-specific pathology of BTHS and potential for tissue-specific therapeutic targeting. Moreover, our results highlight an emerging role for mitophagy in the cardiac pathophysiology of BTHS and reveal a potential neuron-specific bioenergetic phenotype.

Phosphatidylethanolamine biosynthesis in mitochondria: phosphatidylserine (PS) trafficking is independent of a PS decarboxylase and intermembrane space proteins UPS1P and UPS2P.

Phosphatidylethanolamine (PE) plays important roles for the structure and function of mitochondria and other intracellular organelles. In yeast, the majority of PE is produced from phosphatidylserine (PS) by a mitochondrion-located PS decarboxylase, Psd1p. Because PS is synthesized in the endoplasmic reticulum (ER), PS is transported from the ER to mitochondria and converted to PE. After its synthesis, a portion of PE moves back to the ER. Two mitochondrial proteins located in the intermembrane space, Ups1p and Ups2p, have been shown to regulate PE metabolism by controlling the export of PE. It remains to be determined where PS is decarboxylated in mitochondria and whether decarboxylation is coupled to trafficking of PS. Here, using fluorescent PS as a substrate in an in vitro assay for Psd1p-dependent PE production in isolated mitochondria, we show that PS is transferred from the mitochondrial outer membrane to the inner membrane independently of Psd1p, Ups1p, and Ups2p and decarboxylated to PE by Psd1p in the inner membrane. Interestingly, Ups1p is required for the maintenance of Psd1p and therefore PE production. Restoration of Psd1p levels rescued PE production defects in ups1Δ mitochondria. Our data provide novel mechanistic insight into PE biogenesis in mitochondria.

Role for two conserved intermembrane space proteins, Ups1p and Ups2p, [corrected] in intra-mitochondrial phospholipid trafficking.

Mitochondrial membranes maintain a specific phospholipid composition. Most phospholipids are synthesized in the endoplasmic reticulum (ER) and transported to mitochondria, but cardiolipin and phosphatidylethanolamine are produced in mitochondria. In the yeast Saccharomyces cerevisiae, phospholipid exchange between the ER and mitochondria relies on the ER-mitochondria encounter structure (ERMES) complex, which physically connects the ER and mitochondrial outer membrane. However, the proteins and mechanisms involved in phospholipid transport within mitochondria remain elusive. Here, we investigated the role of the conserved intermembrane space proteins, Ups1p and Ups2p, and an inner membrane protein, Mdm31p, in phospholipid metabolism. Our data show that loss of the ERMES complex, Ups1p, and Mdm31p causes similar defects in mitochondrial phospholipid metabolism, mitochondrial morphology, and cell growth. Defects in cells lacking the ERMES complex or Ups1p are suppressed by Mdm31p overexpression as well as additional loss of Ups2p, which antagonizes Ups1p. Combined loss of the ERMES complex and Ups1p exacerbates phospholipid defects. Finally, pulse-chase experiments using [(14)C]serine revealed that Ups1p and Ups2p antagonistically regulate conversion of phosphatidylethanolamine to phosphatidylcholine. Our results suggest that Ups proteins and Mdm31p play important roles in phospholipid biosynthesis in mitochondria. Ups proteins may function in phospholipid trafficking between the outer and inner mitochondrial membranes.