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How cellular cues alter the mitochondrial proteome and impact the composition of mitochondrial proteins remains poorly understood. In this issue of The EMBO Journal, Patron et al (2022) identify TMBIM5 as an important link between calcium homeostasis, proton motive force, and mitochondrial proteolysis, by which the organelle can modify its protein composition. The results may be crucial for our understanding of the plasticity of mitochondria.

Background: Left ventricular noncompaction (LVNC) is the third most common pediatric cardiomyopathy characterized by a thinned myocardium and prominent trabeculations. Next-generation genetic testing has led to a rapid increase in the number of genes reported to be associated with LVNC, but we still have little understanding of its pathogenesis. We sought to grade the strength of the gene-disease relationship for all genes reported to be associated with LVNC and identify molecular pathways that could be implicated. Methods: Following a systematic PubMed review, all genes identified with LVNC were graded using a validated, semi-quantitative system based on all published genetic and experimental evidence created by the Clinical Genome Resource (ClinGen). Genetic pathway analysis identified molecular processes and pathways associated with LVNC. Results: We identified 189 genes associated with LVNC: 11 (6%) were classified as definitive, 21 (11%) were classified as moderate, and 140 (74%) were classified as limited, but 17 (9%) were classified as no evidence. Of the 32 genes classified as definitive or moderate, the most common gene functions were sarcomere function (n=11; 34%), transcriptional/translational regulator (n=6; 19%), mitochondrial function (n=3; 9%), and cytoskeletal protein (n=3; 9%). Furthermore, 18 (56%) genes were implicated in noncardiac syndromic presentations. Lastly, 3 genetic pathways (cardiomyocyte differentiation via BMP receptors, factors promoting cardiogenesis in vertebrates, and Notch signaling) were found to be unique to LVNC and not overlap with pathways identified in dilated cardiomyopathy and hypertrophic cardiomyopathy. Conclusions: LVNC is a genetically heterogeneous cardiomyopathy. Distinct from dilated or hypertrophic cardiomyopathies, LVNC appears to arise from abnormal developmental processes.

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.

Mitochondrial cristae are extraordinarily crowded with proteins, which puts stress on the bilayer organization of lipids. We tested the hypothesis that the high concentration of proteins drives the tafazzin‐catalyzed remodeling of fatty acids in cardiolipin, thereby reducing bilayer stress in the membrane. Specifically, we tested whether protein crowding induces cardiolipin remodeling and whether the lack of cardiolipin remodeling prevents the membrane from accumulating proteins. In vitro, the incorporation of large amounts of proteins into liposomes altered the outcome of the remodeling reaction. In yeast, the concentration of proteins involved in oxidative phosphorylation (OXPHOS) correlated with the cardiolipin composition. Genetic ablation of either remodeling or biosynthesis of cardiolipin caused a substantial drop in the surface density of OXPHOS proteins in the inner membrane of the mouse heart and Drosophila flight muscle mitochondria. Our data suggest that OXPHOS protein crowding induces cardiolipin remodelling and that remodeled cardiolipin supports the high concentration of these proteins in the inner mitochondrial membrane.

Barth syndrome is a multisystem disorder caused by an abnormal metabolism of the mitochondrial lipid cardiolipin. In this review, we discuss physical properties, biosynthesis, membrane assembly, and function of cardiolipin. We hypothesize that cardiolipin reduces packing stress in the inner mitochondrial membrane, which arises as a result of protein crowding. According to this hypothesis, patients with Barth syndrome are unable to meet peak energy demands because they fail to concentrate the proteins of oxidative phosphorylation to a high surface density in the inner mitochondrial membrane. This article is protected by copyright. All rights reserved.

Pediatric heart failure remains poorly understood, distinct in many aspects from adult heart failure. Limited data point to roles of altered mitochondrial functioning and, in particular, changes in mitochondrial lipids, especially cardiolipin. Barth syndrome is a mitochondrial disorder caused by tafazzin mutations that lead to abnormal cardiolipin profiles. Patients are afflicted by cardiomyopathy, skeletal myopathy, neutropenia, and growth delay. A mouse model of Barth syndrome was developed a decade ago, which relies on a doxycycline-inducible short hairpin RNA to knock down expression of tafazzin mRNA (TAZKD). Our objective was to review published data from the TAZKD mouse to determine its contributions to our pathogenetic understanding of, and potential treatment strategies for, Barth syndrome. In regard to the clinical syndrome, the reported physiological, biochemical, and ultrastructural abnormalities of the mouse model mirror those in Barth patients. Using this model, the peroxisome proliferator-activated receptor pan-agonist bezafibrate has been suggested as potential therapy because it ameliorated the cardiomyopathy in TAZKD mice, while increasing mitochondrial biogenesis. A clinical trial is now underway to test bezafibrate in Barth syndrome patients. Thus the TAZKD mouse model of Barth syndrome has led to important insights into disease pathogenesis and therapeutic targets, which can potentially translate to pediatric heart failure.

The acrosome is a protease-rich organelle in sperm essential for fertilization but little is known about acrosome biogenesis. Ren et al. find that the mitochondrial lipid cardiolipin and some mitochondrial proteins translocate to the acrosome during spermatogenesis, suggesting that mitochondria directly contribute to the assembly of this sperm-specific organelle., Mitochondria contain cardiolipin (CL), an organelle-specific phospholipid that carries four fatty acids with a strong preference for unsaturated chains. Unsaturation is essential for the stability and for the function of mitochondrial CL. Surprisingly, we found tetrapalmitoyl-CL (TPCL), a fully saturated species, in the testes of humans and mice. TPCL was absent from other mouse tissues but was the most abundant CL species in testicular germ cells. Most intriguingly, TPCL was not localized in mitochondria but was in other cellular membranes even though mitochondrial CL was the substrate from which TPCL was synthesized. During spermiogenesis, TPCL became associated with the acrosome, a sperm-specific organelle, along with a subset of authentic mitochondrial proteins, including Ant4, Suox, and Spata18. Our data suggest that mitochondria-derived membranes are assembled into the acrosome, challenging the concept that this organelle is strictly derived from the Golgi apparatus and revealing a novel function of mitochondria.

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

The spatial dynamics of picophytoplankton were investigated by flow cytometry and high-throughput sequencing in Lake Hongze, a large river-connecting lake. Picophytoplankton were mainly composed of phycocyanin-rich picocyanobacteria (PCY) and photosynthetic picoeukaryotes (PPEs). Picocyanobacteria was the dominant picophytoplankton group since the contribution of PPEs to total picophytoplankton was only 11.78%. However, PPEs were highly diverse and were composed of Chlorophyta, Bacillariophyta and Chrysophyceae. Environmental factors showed spatial differences, particularly in total phosphorus (TP), suspended solids (SS) and chemical oxygen demand (CODMn), which showed relatively high concentrations around the river channel. The abundances of PPEs and PCY showed similar spatial patterns, which were relatively low in the river course since they were negatively related to SS and CODMn. The top 10 OTUs contributed 79.18% of the total PPEs reads and affiliated with main species in PPEs. CCA results showed that, PPEs community composition was significantly impacted by temperature and DO at sites away from river channel, while was only influenced by nitrite at sites around the river channel.

Tafazzin is the mitochondrial enzyme that catalyzes transacylation between a phospholipid and a lysophospholipid in remodeling. Mutations in tafazzin cause Barth syndrome, a potentially life-threatening disease with the major symptom of cardiomyopathy. In the tafazzin-deficient heart, cardiolipin (CL) acyl chains become abnormally heterogeneous unlike those in the normal heart with a single dominant linoleoyl species; tetralinoleoyl CL. In addition, the amount of CL decreases and monolysocardiolipin (MLCL) accumulates. Here we determined using high-resolution 31P NMR with cryoprobe technology the fundamental phospholipid composition, including the major but oxidation-labile plasmalogens, in the tafazzin-knockdown (TAZ-KD) mouse heart as a model of Barth syndrome. In addition to confirming less CL (6.4 ± 0.1→2.0 ± 0.4 mol% of total phospholipid) and accumulated MLCL (not detected→3.3 ± 0.5 mol%) in the TAZ-KD, we found a substantial reduction in plasmenylcholine (30.8 ± 2.8→18.1 ± 3.1 mol%), the most abundant phospholipid in control wild type. Quantitative Western blot revealed that while the level of peroxisomes, where early steps of plasmalogen synthesis take place, was normal in the TAZ-KD, expression of Far1 as a rate-determining enzyme in plasmalogen synthesis was dramatically upregulated by 8.3 (± 1.6)-fold to accelerate the synthesis in response to the reduced plasmalogen. We confirmed lyso-plasmenylcholine (/plasmenylcholine) is a substrate of purified tafazzin for transacylation with CL (/MLCL). Our results suggest that plasmenylcholine, abundant in linoleoyl species, is important in remodeling CL in the heart. Tafazzin deficiency thus has the major impact on the cardiac plasmenylcholine level, and thereby its functions.

Tafazzin is a mitochondrial enzyme that transfers fatty acids from phospholipids to lysophospholipids. Mutations in tafazzin cause abnormal molecular species of cardiolipin and the clinical phenotype of Barth syndrome. However, the mechanism by which tafazzin creates acyl specificity has been controversial. We have shown that the lipid phase state can produce acyl specificity in the tafazzin reaction, but others have reported that tafazzin itself carries enzymatic specificity. To resolve this issue, we replicated and expanded the controversial experiments, i.e. the transfer of different acyl groups from phosphatidylcholine to monolysocardiolipin by yeast tafazzin. Our data show that this reaction requires the presence of detergent and does not take place in liposomes but in mixed micelles. To separate thermodynamic (lipid-dependent) from kinetic (enzyme-dependent) parameters, we followed the accumulation of cardiolipin during the reaction from the initial state to the equilibrium state. The transacylation rates of different acyl groups varied over 2 orders of magnitude and correlated tightly with the concentration of cardiolipin in the equilibrium state (lipid-dependent parameter). In contrast, the rates by which different transacylations approached the equilibrium state were very similar (enzyme-dependent parameter). Furthermore, we found that tafazzin catalyzes the remodeling of cardiolipin by combinations of forward and reverse transacylations, essentially creating an equilibrium distribution of acyl groups. These data strongly support the idea that the acyl specificity of the tafazzin reaction results from the physical properties of lipids.

Lipid metabolism plays an important role in the regulation of cellular homeostasis. However, because it is difficult to measure the actual rates of synthesis and degradation of individual lipid species, lipid compositions are often used as a surrogate to evaluate lipid metabolism even though they provide only static snapshots of the lipodome. Here, we designed a simple method to determine the turnover rate of phospholipid and acylglycerol species based on the incorporation of (13)C(6)-glucose combined with LC-MS/MS. We labeled adult Drosophila melanogaster with (13)C(6)-glucose that incorporates into the entire lipidome, derived kinetic parameters from mass spectra, and studied effects of deletion of CG6718, the fly homolog of the calcium-independent phospholipase A2β, on lipid metabolism. Although (13)C(6)-glucose gave rise to a complex pattern of (13)C incorporation, we were able to identify discrete isotopomers in which (13)C atoms were confined to the glycerol group. With these isotopomers, we calculated turnover rate constants, half-life times, and fluxes of the glycerol backbone of multiple lipid species. To perform these calculations, we estimated the fraction of labeled molecules in glycerol-3-phosphate, the lipid precursor, by mass isotopomer distribution analysis of the spectra of phosphatidylglycerol. When we applied this method to D. melanogaster, we found a range of lipid half-lives from 2 to 200 days, demonstrated tissue-specific fluxes of individual lipid species, and identified a novel function of CG6718 in triacylglycerol metabolism. This method provides fluxomics-type data with significant potential to improve the understanding of complex lipid regulation in a variety of research models.

Objective To determine the prevalence of Barth syndrome in the pediatric population. Study design Data were collected from the Barth Syndrome Foundation Registry and relevant literature. With the advent of genetic testing and whole-exome sequencing, a multipronged Bayesian analysis was used to estimate the prevalence of Barth syndrome based on published data on the incidence and prevalence of cardiomyopathy and neutropenia, and the respective subpopulations of patients with Barth syndrome indicated in these publications. Results Based on 7 published studies of cardiomyopathy and 2 published studies of neutropenia, the estimated prevalence of Barth syndrome is approximately 1 case per million male population. This contrasts with 99 cases in the Barth Syndrome Foundation Registry, 58 of which indicate a US location, and only 230-250 cases known worldwide. Conclusions It appears that Barth syndrome is greatly underdiagnosed. There is a need for better education and awareness of this rare disease to move toward early diagnosis and treatment.

Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.

Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in subjects with Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin, whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but this association is compromised in subjects with Barth syndrome, leading cardiolipin to become unstable, which in turn causes the accumulation of monolyso-cardiolipin.

Tafazzin is a transacylase that affects cardiolipin fatty acid composition and mitochondrial function. Mutations in human tafazzin cause Barth syndrome yet the enzyme has mostly been characterized in yeast. To study tafazzin in higher organisms, we isolated mitochondria from Drosophila and mammalian cell cultures. Our data indicate that tafazzin binds to multiple protein complexes in these organisms, and that the interactions of tafazzin lack strong specificity. Very large tafazzin complexes could only be detected in the presence of cardiolipin, but smaller complexes remained intact even upon treatment with phospholipase A2. In mammalian cells, tafazzin had a half-life of only 3–6 h, which was much shorter than the half-life of other mitochondrial proteins. The data suggest that tafazzin is a transient resident of multiple protein complexes.

Background The PGC-1α/PPAR axis has been proposed as a potential therapeutic target for several metabolic disorders. The aim was to evaluate the efficacy of the pan-PPAR agonist, bezafibrate, in tafazzin knockdown mice (TazKD), a mouse model of Barth syndrome that exhibits age-dependent dilated cardiomyopathy with left ventricular (LV) dysfunction. Results The effect of bezafibrate on cardiac function was evaluated by echocardiography in TazKD mice with or without beta-adrenergic stress. Adrenergic stress by chronic isoproterenol infusion exacerbates the cardiac phenotype in TazKD mice, significantly depressing LV systolic function by 4.5 months of age. Bezafibrate intake over 2 months substantially ameliorates the development of LV systolic dysfunction in isoproterenol-stressed TazKD mice. Without beta-adrenergic stress, TazKD mice develop dilated cardiomyopathy by 7 months of age. Prolonged treatment with suprapharmacological dose of bezafibrate (0.5% in rodent diet) over a 4-month period effectively prevented LV dilation in mice isoproterenol treatment. Bezafibrate increased mitochondrial biogenesis, however also promoted oxidative stress in cardiomyocytes. Surprisingly, improvement of systolic function in bezafibrate-treated mice was accompanied with simultaneous reduction of cardiolipin content and increase of monolysocardiolipin levels in cardiac muscle. Conclusions Thus, we demonstrate that bezafibrate has a potent therapeutic effect on preventing cardiac dysfunction in a mouse model of Barth syndrome with obvious implications for treating the human disease. Additional studies are needed to assess the potential benefits of PPAR agonists in humans with Barth syndrome.

Considerable progress has recently been made in understanding the role of cardiolipin in mitochondria. In this brief review, we discuss new data that show how cardiolipin specifically contributes to the lateral organization of mitochondrial membranes. We argue that the function of cardiolipin has to be understood in the context of dynamic membrane assembly rather than static membrane structure, and we propose that remodeling of cardiolipin, i.e. the formation of uniformly substituted molecular species, may reduce the energy barrier of the assembly process.

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.

Barth Syndrome, with a major symptom of dilated cardiomyopathy, is caused by mutations in the enzyme tafazzin that catalyzes acylchain exchange between phospholipids and lysophospholipids. In a tafazzin-deficient heart, the acyl chain composition of cardiolipin, an essential component of cardiac lipids, becomes heterogeneous unlike in a normal heart with a single dominant species. In addition, cardiolipin decreases and monolysocardiolipin appears. In this work we have compared the lipid composition of hearts obtained from normal mice with that of hearts obtained from tafazzin knock-down mice. An extensive series of 31P NMR experiments using a cryoprobe on the biological materials with different solution conditions enabled us to determine detailed compositions of phospholipids in heart tissue. In addition to confirming that hearts obtained from tafazzin knock-down mice had less cardiolipin and detectable amounts of monolysocardiolipin, we found a decrease in plasmenylcholine. While the decrease in both cardiolipin and plasmenylcholine were large, both ∼35%, the decrease in plasmenylcholine in terms of changes in the total lipid content was drastic, ∼ 11% (32.4→21.5%); while the decrease in cardiolipin was only 2.3% (6.3→4.0%). In order to obtain an accurate and stable value for the amount of plasmalogen from highly resolved NMR resonances, it was required to have both a chelating agent EDTA and an antioxidant BHT present. Different tissues of the same mammalian species vary with regard to the relative amounts of the two principle forms of plasmalogen: plasmenylcholine and plasmenylethanolamine. Heart contains mostly plasmenylcholine, while lymphoblasts have principally plasmenylethanolamine. As a system directly related to the disease, we also studied human lymphoblasts derived from individuals with Barth Syndrome vs. normal controls. In this case we observed a marked decrease in the plasmenylethanolamine in mitochondria from individuals with Barth Syndrome.

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.

Barth syndrome is an X-linked disease presenting with cardiomyopathy and skeletal muscle weakness. It is caused by mutations in tafazzin, a putative acyl transferase that has been associated with altered metabolism of the mitochondrial phospholipid cardiolipin. To investigate the molecular basis of Barth syndrome, we created Drosophila melanogaster mutants, resulting from imprecise excision of a P element inserted upstream of the coding region of the tafazzin gene. Homozygous flies for that mutation were unable to express the full-length isoform of tafazzin, as documented by RNA and Western blot analysis, but two shorter tafazzin transcripts were still present, although the expression levels of their encoded proteins were too low to be detectable by Western blotting. The tafazzin mutation caused an 80% reduction of cardiolipin and a diversification of its molecular composition, similar to the changes seen in Barth patients. Other phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were not affected. Flies with the tafazzin mutation showed a reduced locomotor activity, measured in flying and climbing assays, and their indirect flight muscles displayed frequent mitochondrial abnormalities, mostly in the cristae membranes. Thus, tafazzin mutations in Drosophila generated a Barth-related phenotype, with the triad of abnormal cardiolipin, pathologic mitochondria, and motor weakness, suggesting causal links between these findings. We conclude that a lack of full-length tafazzin is responsible for the cardiolipin deficiency, which is integral to the disease mechanism, leading to mitochondrial myopathy.

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.

To elucidate the biogenetic pathways for the generation of lysosome-related organelles, we have chosen to study the Drosophila eye pigment granules because they are lysosome-related and the fruit fly provides the advantages of a genetic system in which many mutations affect eye color. Here, we report the molecular identification of two classic Drosophila eye-color genes required for pigment granule biogenesis, claret and lightoid ; the former encodes a protein containing seven repeats with sequence similarity to those that characterize regulator of chromosome condensation 1 (RCC1, a guanine nucleotide exchange factor for the small GTPase, Ran), and the latter encodes a rab GTPase, Rab-RP1. We demonstrate in transfected cells that Claret, through its RCC1-like domain, interacts preferentially with the nucleotide-free form of Rab-RP1, and this interaction involves Claret's first three RCC1-like repeats that are also critical for Claret's function in pigment granule biogenesis in transgenic rescue experiments. In addition, double-mutant analyses suggest that the gene products of claret and lightoid function in the same pathway, which is different from that of garnet and ruby (which encode the δ- and β-subunit of the tetrameric adaptor protein 3 complex, respectively). Taken together, our results suggest that Claret functions as a guanine nucleotide exchange factor for Lightoid/Rab-RP1 in an adaptor protein 3-independent vesicular trafficking pathway of pigment granule biogenesis.

The terminally differentiated umbrella cells of bladder epithelium contain unique cytoplasmic organelles, the fusiform vesicles, which deliver preassembled crystalline arrays of uroplakin proteins to the apical cell surface of urothelial umbrella cells. We have investigated the possible role of Rab proteins in this delivery process, and found Rab27b to be expressed at an extraordinary high level (0.1% of total protein) in urothelium, whereas Rab27b levels were greatly reduced (to

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

Since it has been recognized that mitochondria are crucial not only for energy metabolism but also for other cellular functions, there has been a growing interest in cardiolipin, the specific phospholipid of mitochondrial membranes. Indeed, cardiolipin is a universal component of mitochondria in all eukaryotes. It has a unique dimeric structure comprised of two phosphatidic acid residues linked by a glycerol bridge, which gives rise to unique physicochemical properties. Cardiolipin plays an important role in the structural organization and the function of mitochondrial membranes. In this article, we review the literature on cardiolipin biology, focusing on the most important discoveries of the past decade. Specifically, we describe the formation, the migration, and the degradation of cardiolipin and we discuss how cardiolipin affects mitochondrial function. We also give an overview of the various phenotypes of cardiolipin deficiency in different organisms.

We have identified and cloned the cDNA for a 912-aa protein, rab11BP, that interacts with the GTP-containing active form of rab11, a GTP-binding protein that plays a critical role in receptor recycling. Although rab11BP is primarily cytosolic, a significant fraction colocalizes with rab11 in endosomal membranes of both the sorting and recycling subcompartments. In vitro binding of rab11 to native rab11BP requires partial denaturation of the latter to expose an internal binding site located between residues 334 and 504 that is apparently masked by the C-terminal portion of the protein, which includes six repeats known as WD40 domains. Within the cell, rab11BP must undergo a conformational change in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins formed abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not affect transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1–504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant negative rab11 mutant protein that does not bind GTP. Strikingly, the inhibition caused by the truncated rab11BP was prevented completely when the cells also expressed a C-terminally deleted, nonprenylatable form of rab11 that, by itself, has no effect on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent on the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP.

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913–924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37°C of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37°C or 16°C. We show that incubation at 16°C blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16°C. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16°C is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli.

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

Background Barth syndrome is a rare, multisystem disorder caused by mutations in tafazzin that lead to cardiolipin deficiency and mitochondrial abnormalities. Patients most commonly develop an early‐onset cardiomyopathy in infancy or fetal life. Methods and Results Knockdown of tafazzin (TAZKD) in a mouse model was induced from the start of gestation via a doxycycline‐inducible shRNA transgenic approach. All liveborn TAZKD mice died within the neonatal period, and in vivo echocardiography revealed prenatal loss of TAZKD embryos at E12.5‐14.5. TAZKD E13.5 embryos and newborn mice demonstrated significant tafazzin knockdown, and mass spectrometry analysis of hearts revealed abnormal cardiolipin profiles typical of Barth syndrome. Electron microscopy of TAZKD hearts demonstrated ultrastructural abnormalities in mitochondria at both E13.5 and newborn stages. Newborn TAZKD mice exhibited a significant reduction in total mitochondrial area, smaller size of individual mitochondria, reduced cristae density, and disruption of the normal parallel orientation between mitochondria and sarcomeres. Echocardiography of E13.5 and newborn TAZKD mice showed good systolic function, but early diastolic dysfunction was evident from an abnormal flow pattern in the dorsal aorta. Strikingly, histology of E13.5 and newborn TAZKD hearts showed myocardial thinning, hypertrabeculation and noncompaction, and defective ventricular septation. Altered cellular proliferation occurring within a narrow developmental window accompanied the myocardial hypertrabeculation‐noncompaction. Conclusions In this murine model, tafazzin deficiency leads to a unique developmental cardiomyopathy characterized by ventricular myocardial hypertrabeculation‐noncompaction and early lethality. A central role of cardiolipin and mitochondrial functioning is strongly implicated in cardiomyocyte differentiation and myocardial patterning required for heart development. ( J Am Heart Assoc . 2012;1:e000455 doi: 10.1161/JAHA.111.000455.)

Cardiolipin is a mitochondrial phospholipid with a unique composition and distribution of acyl groups. The cardiolipin composition depends on tafazzin, a phospholipid-lysophospholipid transacylase, although the enzyme itself lacks acyl specificity. We incubated isolated tafazzin with various mixtures of phospholipids and lysophospholipids, characterized the lipid phase state by 31P-NMR, and measured newly formed molecular species by mass spectrometry. Significant transacylation activity was observed only in non-bilayer lipid aggregates, in which lipids had a low packing order. The lipid phase state profoundly affected the substrate specificity of the tafazzin reaction. In particular, tetralinoleoyl-cardiolipin, a prototype molecular species, formed only under conditions that favor the inverted hexagonal phase. In isolated mitochondria, less than 2 percent of lipids participated in transacylations, suggesting that tafazzin acts only on privileged lipid domains. We propose that tafazzin reacts with non-bilayer lipids in mitochondria and that acyl specificity arises from spontaneous self-organization of these domains.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ran genes encode a family of well-conserved small nuclear GTPases (Ras-related nuclear proteins), whose function is implicated in both normal cell cycle progression and the transport of RNA and proteins between the nucleus and the cytoplasm. Previous studies of Ran proteins have utilized cell-free systems, yeasts, and cultured mammalian cells. We have now characterized patterns of Ran gene expression in the mouse. Serum starvation suppressed Ran gene transcription in mouse 3T3 cells. Ran mRNA reappeared in cells within 3 h after refeeding. A single Ran mRNA species was detected at low levels in most somatic tissues of the adult mouse. In testis, this Ran mRNA was abundant, as were other larger transcripts. Analysis of testis-derived Ran cDNA clones revealed the presence of two transcripts, one specifying an amino acid sequence identical to that of human Ran/TC4 and one specifying an amino acid sequence 94% identical. Northern blotting and reverse transcriptase-PCR assays with oligonucleotide probes and primers specific for each transcript demonstrated that the isoform identical to Ran/TC4 was expressed in both somatic tissues and testis, while the variant form was transcribed only in testis. The existence of tissue-specific Ran isoforms may help to rationalize the diverse roles suggested for Ran by previous biochemical studies.

Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex.

Cardiolipin is a dimeric phospholipid with a characteristic acyl composition that is generated by fatty acid remodeling after de novo synthesis. Several enzymes have been proposed to participate in acyl remodeling of cardiolipin. In order to compare the effect of these enzymes, we determined the pattern of cardiolipin molecular species in Drosophila strains with specific enzyme deletions, using MALDI-TOF mass spectrometry with internal standards. We established the linear range of the method for cardiolipin quantification, determined the relative signal intensities of several cardiolipin standards, and demonstrated satisfying signal-to-noise ratios in cardiolipin spectra from a single fly. Our data demonstrate changes in the cardiolipin composition during the Drosophila life cycle. Comparison of cardiolipin spectra, using vector algebra, showed that inactivation of tafazzin had a large effect on the molecular composition of cardiolipin, inactivation of calcium-independent phospholipase A2 had a small effect, whereas inactivation of acyl-CoA:lysocardiolipin-acyltransferase and of the trifunctional enzyme did not affect the cardiolipin composition.

F1F0 ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane.

The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Delta5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Delta5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin.