The enzymatic function of tafazzin

Tafazzin is a putative enzyme that is involved in cardiolipin metabolism, it may carry mutations responsible for Barth syndrome. To identify the biochemical reaction catalyzed by tafazzin, we expressed the full-length isoform of Drosophila melanogaster tafazzin in a baculovirus-Sf9 insect cell system. Tafazzin expression induced a new enzymatic function in Sf9 cell mitochondria, namely 1-palmitoyl-2-[14C]linoleoyl-phosphatidylcholine:monolysocardiolipin linoleoyltransferase. We also found evidence for the reverse reaction, because tafazzin expression caused transfer of acyl groups from phospholipids to 1-[14C]palmitoyl-2-lyso-phosphatidylcholine. An affinity-purified tafazzin construct, tagged with the maltose-binding protein, catalyzed both forward and reverse transacylations between cardiolipin and phosphatidylcholine, but was unable to utilize CoA or acyl-CoA as substrates. Whereas tafazzin supported transacylations between various phospholipid-lysophospholipid pairs, it showed the highest rate for the phosphatidylcholine-cardiolipin transacylation. Transacylation activities were about 10-fold higher for linoleoyl groups than for oleoyl groups, and they were negligible for arachidonoyl groups. The data show that Drosophila tafazzin is a CoA-independent, acyl-specific phospholipid transacylase with substrate preference for cardiolipin and phosphatidylcholine.     

The Mitochondrial Quality Control Protein Yme1 Is Necessary to Prevent Defective Mitophagy in a Yeast Model of Barth Syndrome

The Saccharomyces cerevisiae TAZ1 gene is an orthologue of human TAZ; both encode the protein tafazzin. Tafazzin is a transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids. Mutations in human TAZ cause Barth syndrome, a fatal childhood cardiomyopathy biochemically characterized by reduced cardiolipin mass and increased monolysocardiolipin levels. To uncover cellular processes that require tafazzin to maintain cell health, we performed a synthetic genetic array screen using taz1Δ yeast cells to identify genes whose deletion aggravated its fitness. The synthetic genetic array screen uncovered several mitochondrial cellular processes that require tafazzin. Focusing on the i-AAA protease Yme1, a mitochondrial quality control protein that degrades misfolded proteins, we determined that in cells lacking both Yme1 and Taz1 function, there were substantive mitochondrial ultrastructural defects, ineffective superoxide scavenging, and a severe defect in mitophagy. We identify an important role for the mitochondrial protease Yme1 in the ability of cells that lack tafazzin function to maintain mitochondrial structural integrity and mitochondrial quality control and to undergo mitophagy.     

Cardiolipin remodeling and the function of tafazzin

Cardiolipin, the specific phospholipid of mitochondria, is involved in the biogenesis, the dynamics, and the supramolecular organization of mitochondrial membranes. Cardiolipin acquires a characteristic composition of fatty acids by post-synthetic remodeling, a process that is crucial for cardiolipin homeostasis and function. The remodeling of cardiolipin depends on the activity of tafazzin, a non-specific phospholipid–lysophospholipid transacylase. This review article discusses recent findings that suggest a novel function of tafazzin in mitochondrial membranes. By shuffling fatty acids between molecular species, tafazzin transforms the lipid composition and by doing so supports changes in the membrane conformation, specifically the generation of membrane curvature. Tafazzin activity is critical for the differentiation of cardiomyocytes, in which the characteristic cristae-rich morphology of cardiac mitochondria evolves. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Cardiolipin remodeling by TAZ/tafazzin is selectively required for the initiation of mitophagy

Tafazzin (TAZ) is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of TAZ cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. Here we investigated the role of TAZ in regulating mitochondrial function and mitophagy. Using primary mouse embryonic fibroblasts (MEFs) with doxycycline-inducible knockdown of Taz, we showed that TAZ deficiency in MEFs caused defective mitophagosome biogenesis, but not other autophagic processes. Consistent with a key role of mitophagy in mitochondria quality control, TAZ deficiency in MEFs also led to impaired oxidative phosphorylation and severe oxidative stress. Together, these findings provide key insights on mitochondrial dysfunction in Barth syndrome, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for this lethal condition.     

Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria

Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation.     

Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.     

Barth syndrome, a human disorder of cardiolipin metabolism

Barth syndrome is an X-linked recessive disease caused by mutations in the tafazzin gene. Patients have reduced concentration and altered composition of cardiolipin, the specific mitochondrial phospholipid, and they have variable clinical findings, often including heart failure, myopathy, neutropenia, and growth retardation. This article provides an overview of the molecular basis of Barth syndrome. It is argued that tafazzin, a phospholipid acyltransferase, is involved in acyl-specific remodeling of cardiolipin, which promotes structural uniformity and molecular symmetry among the cardiolipin molecular species. Inhibition of this pathway leads to changes in mitochondrial architecture and function.     

The enigmatic role of tafazzin in cardiolipin metabolism

The mitochondrial phospholipid cardiolipin plays an important role in cellular metabolism as exemplified by its involvement in mitochondrial energy production and apoptosis. Following its biosynthesis, cardiolipin is actively remodeled to achieve its final acyl composition. An important cardiolipin remodeling enzyme is tafazzin, of which several mRNA splice variants exist. Mutations in the tafazzin gene cause the X-linked recessive disorder Barth syndrome. In addition to providing an overview of the current knowledge in literature about tafazzin, we present novel experimental data and use this to discuss the functional role of the different tafazzin variants in cardiolipin metabolism in relation to Barth syndrome. We developed and performed specific quantitative PCR analyses of different tafazzin mRNA splice variants in 16 human tissues and correlated this with the tissue cardiolipin profile. In BTHS fibroblasts we showed that mutations in the tafazzin gene affected both the level and distribution of tafazzin mRNA variants. Transient expression of selected human tafazzin variants in BTHS fibroblasts showed for the first time in a human cell system that tafazzin lacking exon5 indeed functions in cardiolipin remodeling.     

Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients

Mutations in the human TAZ gene are associated with Barth Syndrome, an often fatal X-linked disorder that presents with cardiomyopathy and neutropenia. The TAZ gene encodes Tafazzin, a putative phospholipid acyltranferase that is involved in the remodeling of cardiolipin, a phospholipid unique to the inner mitochondrial membrane. It has been shown that the disruption of the Tafazzin gene in yeast (Taz1) affects the assembly and stability of respiratory chain Complex IV and its supercomplex forms. However, the implications of these results for Barth Syndrome are restricted due to the additional presence of Complex I in humans that forms a supercomplex with Complexes III and IV. Here, we investigated the effects of Tafazzin, and hence cardiolipin deficiency in lymphoblasts from patients with Barth Syndrome, using blue-native polyacrylamide gel electrophoresis. Digitonin extraction revealed a more labile Complex I/III(2)/IV supercomplex in mitochondria from Barth Syndrome cells, with Complex IV dissociating more readily from the supercomplex. The interaction between Complexes I and III was also less stable, with decreased levels of the Complex I/III(2) supercomplex. Reduction of Complex I holoenzyme levels was observed also in the Barth Syndrome patients, with a corresponding decrease in steady-state subunit levels. We propose that the loss of mature cardiolipin species in Barth Syndrome results in unstable respiratory chain supercomplexes, thereby affecting Complex I biogenesis, respiratory activities and subsequent pathology.     

Barth syndrome mutations that cause tafazzin complex lability

Deficits in mitochondrial function result in many human diseases. The X-linked disease Barth syndrome (BTHS) is caused by mutations in the tafazzin gene TAZ1. Its product, Taz1p, participates in the metabolism of cardiolipin, the signature phospholipid of mitochondria. In this paper, a yeast BTHS mutant tafazzin panel is established, and 18 of the 21 tested BTHS missense mutations cannot functionally replace endogenous tafazzin. Four BTHS mutant tafazzins expressed at low levels are degraded by the intermembrane space AAA (i-AAA) protease, suggesting misfolding of the mutant polypeptides. Paradoxically, each of these mutant tafazzins assembles in normal protein complexes. Furthermore, in the absence of the i-AAA protease, increased expression and assembly of two of the BTHS mutants improve their function. However, the BTHS mutant complexes are extremely unstable and accumulate as insoluble aggregates when disassembled in the absence of the i-AAA protease. Thus, the loss of function for these BTHS mutants results from the inherent instability of the mutant tafazzin complexes.     

The human TAZ gene complements mitochondrial dysfunction in the yeast taz1Delta mutant. Implications for Barth syndrome

Barth syndrome is a genetic disorder that is caused by different mutations in the TAZ gene G4.5. The yeast gene TAZ1 is highly homologous to human TAZ, and the taz1Delta mutant has phospholipid defects similar to those observed in Barth syndrome cells, including aberrant cardiolipin species and decreased cardiolipin levels. Subcellular fractionation studies revealed that Taz1p is localized exclusively in mitochondria, which supports the theory that tafazzins are involved in cardiolipin remodeling. Because cardiolipin plays an important role in respiratory function, we measured the energy transformation and osmotic properties of isolated mitochondria from the taz1Delta mutant. Energy coupling in taz1Delta mitochondria was dependent on the rate of oxidative phosphorylation, as coupling was diminished when NADH was used as a respiratory substrate but was unaffected when ethanol was the substrate. Membrane stability was compromised in taz1Delta mitochondria exposed to increased temperature and hypotonic conditions. Mitochondria from taz1Delta also displayed decreased swelling in response to ATP, which induces the yeast mitochondrial unspecific channel, and to alamethicin, a membrane-disrupting agent. Coupling was measured in taz1Delta cells containing different splice variants of the human TAZ gene. Only the variant that restores wild type cardiolipin synthesis (lacking exon 5) restored coupling in hypotonic conditions and at elevated temperature. These findings may shed light on the mitochondrial deficiencies observed in Barth syndrome.     

The role of mitochondrial cardiolipin in heart function and its implication in cardiac disease

Mitochondria play an essential role in the energy metabolism of the heart. Many of the essential functions are associated with mitochondrial membranes and oxidative phosphorylation driven by the respiratory chain. Mitochondrial membranes are unique in the cell as they contain the phospholipid cardiolipin. The important role of cardiolipin in cardiovascular health is highlighted by several cardiac diseases, in which cardiolipin plays a fundamental role. Barth syndrome, Sengers syndrome, and Dilated cardiomyopathy with ataxia (DCMA) are genetic disorders, which affect cardiolipin biosynthesis. Other cardiovascular diseases including ischemia/reperfusion injury and heart failure are also associated with changes in the cardiolipin pool. Here, we summarize molecular functions of cardiolipin in mitochondrial biogenesis and morphology. We highlight the role of cardiolipin for the respiratory chain, metabolite carriers, and mitochondrial metabolism and describe links to apoptosis and mitochondria specific autophagy (mitophagy) with possible implications in cardiac disease.     

Cardiac Metabolic Pathways Affected in the Mouse Model of Barth Syndrome

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F₀F₁-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.     

Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions,and cellular proliferation

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.     

Cell biology of cardiac mitochondrial phospholipids.

Phospholipids are important structural and functional components of all biological membranes and define the compartmentation of organelles. Mitochondrial phospholipids comprise a significant proportion of the entire phospholipid content of most eukaroytic cells. In the heart, a tissue rich in mitochondria, the mitochondrial phospholipids provide for diverse roles in the regulation of various mitochondrial processes including apoptosis, electron transport, and mitochondrial lipid and protein import. It is well documented that alteration in the content and fatty acid composition of phospholipids within the heart is linked to alterations in myocardial electrical activity. In addition, reduction in the specific mitochondrial phospholipid cardiolipin is an underlying biochemical cause of Barth Syndrome, a rare and often fatal X-linked genetic disease that is associated with cardiomyopathy. Thus, maintenance of both the content and molecular composition of phospholipids synthesized within the mitochondria is essential for normal cardiac function. This review will focus on the function and regulation of the biosynthesis and resynthesis of mitochondrial phospholipids in the mammalian heart.     

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.     

Cardiolipin remodeling in Barth syndrome and other hereditary cardiomyopathies

Mitochondria play a prominent role in cardiac energy metabolism, and their function is critically dependent on the integrity of mitochondrial membranes. Disorders characterized by mitochondrial dysfunction are commonly associated with cardiac disease. The mitochondrial phospholipid cardiolipin directly interacts with a number of essential protein complexes in the mitochondrial membranes including the respiratory chain, mitochondrial metabolite carriers, and proteins critical for mitochondrial morphology. Barth syndrome is an X-linked disorder caused by an inherited defect in the biogenesis of the mitochondrial phospholipid cardiolipin. How cardiolipin deficiency impacts on mitochondrial function and how mitochondrial dysfunction causes cardiomyopathy has been intensively studied in cellular and animal models of Barth syndrome. These findings may also have implications for the molecular mechanisms underlying other inherited disorders associated with defects in cardiolipin, such as Sengers syndrome and dilated cardiomyopathy with ataxia (DCMA).     

Cellular functions of cardiolipin in yeast

Cardiolipin (CL), the signature lipid of mitochondria, plays a critical role in mitochondrial function and biogenesis. The availability of yeast mutants blocked in CL synthesis has facilitated studies of the biological role of this lipid. Perturbation of CL synthesis leads to growth defects not only during respiratory growth but also under conditions in which respiration is not essential. CL was shown to play a role in mitochondrial protein import, cell wall biogenesis, aging and apoptosis, ceramide synthesis, and translation of electron transport chain components. The genetic disorder Barth syndrome (BTHS) is caused by mutations in the tafazzin gene resulting in decreased total CL levels, accumulation of monolysocardiolipin (MLCL), and decreased unsaturated fatty acyl species of CL. The variation in clinical presentation of BTHS indicates that other physiological factors play a significant role in modifying the phenotype resulting from tafazzin deficiency. Elucidating the functions of CL is expected to shed light on the role of this important lipid in BTHS and other disorders of mitochondrial dysfunction.     

Promotion of plasmalogen biosynthesis reverse lipid changes in a Barth Syndrome cell model

In Barth syndrome (BTHS) mutations in tafazzin leads to changes in both the quantities and the molecular species of cardiolipin (CL), which are the hallmarks of BTHS. Contrary to the well-established alterations in CL associated with BTHS; recently a marked decrease in the plasmalogen levels in Barth specimens has been identified. To restore the plasmalogen levels, the present study reports the effect of promotion of plasmalogen biosynthesis on the lipidome of lymphoblasts derived from Barth patients as well as on cell viability, mitochondria biogenesis, and mitochondrial membrane potential. High resolution ³¹P NMR phospholipidomic analysis showed an increase in the levels of plasmenylethanolamine (the major plasmalogen in lymphoblasts), which reached values comparable to the control and a compensatory decrease in the levels of its diacyl-PE counterpart. Importantly, ³¹P NMR showed a significant increase in the levels of CL, while not altering the levels of monolysocardiolipin. Mass spectrometry measurements showed that the promotion of plasmalogen biosynthesis did not change the molecular species profile of targeted phospholipids. In addition, promotion of plasmalogen biosynthesis did not impact on cellular viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential. Overall, the results showed the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and highlight the potential beneficial effect of a diet supplemented with plasmalogen precursors to BTHS patients.