Cardiolipin Synthesis Research Articles

Moraxella catarrhalis Expresses a Cardiolipin Synthase That Impacts Adherence to Human Epithelial Cells

The major phospholipid constituents of Moraxella catarrhalis membranes are phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin (CL). However, very little is known regarding the synthesis and function of these phospholipids in M. catarrhalis. In this study, we discovered that M. catarrhalis expresses a cardiolipin synthase (CLS), termed MclS, that is responsible for the synthesis of CL within the bacterium. The nucleotide sequence of mclS is highly conserved among M. catarrhalis isolates and is predicted to encode a protein with significant amino acid similarity to the recently characterized YmdC/ClsC protein of Escherichia coli. Isogenic mclS mutant strains were generated in M. catarrhalis isolates O35E, O12E, and McGHS1 and contained no observable levels of CL. Site-directed mutagenesis of a highly conserved HKD motif of MclS also resulted in a CL-deficient strain. Moraxella catarrhalis, which depends on adherence to epithelial cells for colonization of the human host, displays significantly reduced levels of adherence to HEp-2 and A549 cell lines in the mclS mutant strains compared to wild-type bacteria. The reduction in adherence appears to be attributed to the absence of CL. These findings mark the first instance in which a CLS has been related to a virulence-associated trait.

Reduced Cardiolipin Synthesis Protects Against the Development of Obesity and Insulin Resistance

s / Can J Diabetes 37 (2013) S13eS84 S63 Methods: Eight healthy men (21.4 1.3 year, 21.5 0.3 kg/m2) underwent a 2-stage (2 3h) pancreatic clamp, with octreotide suppression and basal glucagon and hGH replacement. Glycemia was clamped at w5.5 mmol/L and branched-chain amino acids w350 mmol/L. Insulin was infused to maintain postabsorptive then raised to postprandial levels. Glucose turnover was measured using 3-3H-glucose. Vastus lateralis muscle biopsies were taken before and at 30 and 120 min during hyperinsulinemia. Primary skeletal myoblasts were isolated, cultured and differentiated into myotubes. Results: Hyperinsulinemia at 30 min (589.2 37.0 pmol/L) was maintained (150 to 180 min, 551.2 23.2 pmol/L). Glucose disposal increased from 2.73 0.08 mg/kg/min at postabsorptive to 9.54 0.62 120 min (P<0.00001) hyperinsulinemia. In biopsies, AktSer473 phosphorylation was maximal within 30 min and sustained at 120 min hyperinsulinemia. Grb10Ser476 phosphorylation was not different at 30 min (2.36 0.91 vs. 2.14 0.68; n1⁄44), but doubled by 120 min (4.16 1.14; P1⁄40.067). siRNA-mediated silencing reduced Grb10 protein abundance by 54-94%. The insulin receptor abundance was 2.5-fold higher in Grb10-depleted myotubes. Knockdown of Grb10 increased both basal and insulin-stimulated phosphorylation of IRS1Tyr632 and AktThr308. Acute inhibition of mTORC1 by rapamycin eliminated basal and insulin-stimulated Grb10Ser476 phosphorylation, while enhancing insulin-stimulated phosphorylation of AktThr308. Conclusions: Grb10 negatively regulates insulin signaling in part by modulating its receptor abundance. Insulin-stimulated Grb10 phosphorylation by mTORC1 is a negative feedback mechanism of PI3K/Akt signaling in human skeletal muscle.

The Importance of Cardiolipin Synthase for Mitochondrial Ultrastructure, Respiratory Function, Plant Development, and Stress Responses in Arabidopsis

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

Mitochondrial lipid trafficking

Mitochondria are dynamic organelles surrounded by two membranes with a defined lipid composition. The majority of lipids are synthesized in the endoplasmic reticulum (ER) and transported to the mitochondria, but the synthesis of cardiolipin and phosphatidylethanolamine in the inner membrane of mitochondria highlights their general importance for cellular lipid metabolism. Extensive exchange of lipids and their precursors occurs between the ER and mitochondria as well as between mitochondrial membranes. The recent identification of membrane-tethering complexes and lipid-transfer proteins in mitochondria now provides the first insight into the mechanisms of these transport processes, which are of fundamental importance for mitochondrial activities and cell homeostasis. Here, we summarize the current understanding of lipid trafficking at the mitochondria and discuss emerging models for the mechanisms of lipid transfer.

Mitochondrial Cardiolipin Is Required for Nlrp3 Inflammasome Activation

Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic linezolid as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically themitochondrial lipid cardiolipin. Cardiolipin bound toNlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.

TRIAP1/PRELI Complexes Prevent Apoptosis by Mediating Intramitochondrial Transport of Phosphatidic Acid

Cardiolipin (CL), a mitochondria-specific glycerophospholipid, is required for diverse mitochondrial processes and orchestrates the function of various death-inducing proteins during apoptosis. Here, we identify a complex of the p53-regulated protein TRIAP1 (p53CSV) and PRELI in the mitochondrial intermembrane space (IMS), which ensures the accumulation of CL in mitochondria. TRIAP1/PRELI complexes exert lipid transfer activity in vitro and supply phosphatidic acid (PA) for CL synthesis in the inner membrane. Loss of TRIAP1 or PRELI impairs the accumulation of CL, facilitates the release of cytochrome c, and renders cells vulnerable to apoptosis upon intrinsic and extrinsic stimulation. Survival of TRIAP1- and PRELI-deficient cells is conferred by an excess of exogenously provided phosphatidylglycerol. Our results reveal a p53-dependent cell-survival pathway and highlight the importance of the CL content of mitochondrial membranes in apoptosis.

Unveiling the last missing link of the cardiolipin synthetic pathway in mitochondria

Mitochondria produce cellular energy ATP by using electrochemical energy stored as a proton gradient across the mitochondrial inner membrane (IM). To generate the proton gradient, the respiratory-chain complexes in the IM pump up protons from the matrix to the inter-membrane space by coupling it to the electron transport through the respiratory chain. The efficient electron flow relies on the respiratory-chain super-complex formation, which is facilitated by the mitochondrial signature phospholipid cardiolipin (CL) [1]. CL directly interacts with the respiratory-chain complexes, mitochondrial carrier proteins including ADP/ATP carrier (AAC) etc. and stabilizes the super complexes consisting of Complexes I, III and IV in mammals and Complexes III and IV with AAC in yeast [1]. In addition, it is known that the assembly and maintenance of the TIM23 complex, a mitochondrial protein translocator in the IM, depends on the presence of CL [2]. Although the electron transport is essential for energy production in mitochondria, it could also generate reactive oxygen species (ROS), which are the potential cause of the oxidative damage of DNA and proteins as well as phospholipids in mitochondria. Accumulation of oxidative damage is thought to lead to mitochondrial and cellular dysfunction associated with aging [3]. CL is one of the main targets of ROS in mitochondria due to its IM location close to the sites of ROS production and its feature containing high-levels of unsaturated fatty acids [3]. Therefore CL could be a key molecule in aging process. CL is synthesized through several steps of modifications of phosphatidic acid (PA) in the IM and is remodeled by deacylation and re-acylation that generate mature CL with unsaturated fatty acids [1]. Despite its physiological importance, it was not clear how CL is synthesized in mitochondria. In particular, an enzyme responsible for the first step of the CL synthesis, i.e. generation of an important intermediate phospholipid, CDP-diacylglycerol (CDP-DAG), in mitochondria was not identified. In the issue of Cell Metabolism (Volume 17, Issue 5, 709-718, 7 May 2013), we reported identification of Tam41 as a mitochondrial CDP-DAG synthase and the cellular system to compensate the loss of Tam41. Tam41 was originally identified as a protein important for assembly and maintenance of the TIM23 complex in the IM [4]. However, a subsequent study showed that loss of Tam41 leads to significant decrease in CL and accumulation of PA, the precursor phospholipid of CDP-DAG [5]. Besides, respiratory-chain super complexes tend to be destabilized in cells lacking Tam41, which indicates that the role of Tam41 is not limited to the TIM23 complex but to the protein complexes in the IM in general [5]. We thus reasoned that Tam41 could be a mitochondrial CDP-DAG synthase and asked whether Tam41 directly catalyzes the CDP-DAG formation. To this end, we purified Tam41 and its loss-of-function mutants from yeast cells and measured their CDP-DAG synthase activities in vitro. We observed generation of CDP-DAG upon incubation of wild-type Tam41, but not the Tam41 mutants, with fluorescence-labeled PA and CTP. These results demonstrate that Tam41 is a mitochondrial CDP-DAG synthase, which had been elusive previously (Figure ​(Figure11). Figure 1 Roles of Tam41 and Art5 in yeast hospholipid biosynthetic pathways Our previous study identified Art5 as a multi-copy suppressor of tam41Δ cells [6]. Art5 is a member of the arrestin-related trafficking adaptor (ART) protein family and is known to facilitate degradation of inositol transporter in plasma membrane in response to exogenous inositol [7]. Indeed, we found that growth defects of tam41Δ cells were restored in inositol-depleted media (Figure ​(Figure1).1). Surprisingly, despite the restoration of the growth defects of tam41Δ cells by overexpressing Art5, the low level of CL hardly changed while the CDP-DAG, PA, and PE (phophatidylethanolamine) levels increased and the PI (phosphatidylinositol) level decreased. Hence CL itself is not critical for cell growth, but overall alteration of the phospholipid composition is capable of compensation of the loss of Tam41. This observation also points to the fact that yeast cells have a backup system to tolerate the loss of Tam41. In relation to this, Connerth et al. showed that defects in cell growths and decrease in the CL level caused by the loss of Tam41 can be largely restored by the simultaneous loss of a PA transfer protein Ups1 [8]. This suggests the presence of an alternative CL synthetic pathway, which may be activated in the absence of both Tam41 and Ups1. These findings may offer hints about possible treatments of diseases and protection against aging processes associated with mitochondrial dysfunction arising from the compromised CL synthesis or CL oxidation.

Tam41 Is a CDP-Diacylglycerol Synthase Required for Cardiolipin Biosynthesis in Mitochondria

CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.

Phosphatidylglycerol homeostasis in glycerol-phosphate auxotrophs of Staphylococcus aureus

BackgroundThe balanced synthesis of membrane phospholipids, fatty acids and cell wall constituents is a vital facet of bacterial physiology, but there is little known about the biochemical control points that coordinate these activities in Gram-positive bacteria. In Escherichia coli, the glycerol-phosphate acyltransferase (PlsB) plays a key role in coordinating fatty acid and phospholipid synthesis, but pathogens like Staphylococcus aureus have a different acyltransferase (PlsY), and the headgroup of the major membrane phospholipid, phosphatidylglycerol (PtdGro), is used as a precursor for lipoteichoic acid synthesis.ResultsThe PlsY acyltransferase in S. aureus was switched off by depriving strain PDJ28 (ΔgpsA) of the required glycerol supplement. Removal of glycerol from the growth medium led to the rapid cessation of phospholipid synthesis. However, the continued utilization of the headgroup caused a reduction in PtdGro coupled with the accumulation of CDP-diacylglycerol and phosphatidic acid. PtdGro was further decreased by its stimulated conversion to cardiolipin. Although acyl-acyl carrier protein (ACP) and malonyl-CoA accumulated, fatty acid synthesis continued at a reduced level leading to the intracellular accumulation of unusually long-chain free fatty acids.ConclusionsThe cessation of new phospholipid synthesis led to an imbalance in membrane compositional homeostasis. PtdGro biosynthesis was not coupled to headgroup turnover leading to the accumulation of pathway intermediates. The synthesis of cardiolipin significantly increased revealing a stress response to liberate glycerol-phosphate for PtdGro synthesis. Acyl-ACP accumulation correlated with a decrease in fatty acid synthesis; however, the coupling was not tight leading to the accumulation of intracellular fatty acids.

The effects of chronic muscle use and disuse on cardiolipin metabolism

Cardiolipin (CL) is a phospholipid that maintains the integrity of mitochondrial membranes. We previously demonstrated that CL content increases with chronic muscle use, and decreases with denervation-induced disuse. To investigate the underlying mechanisms, we measured the mRNA expression of 1) CL synthesis enzymes cardiolipin synthase (CLS) and CTP:PA-cytidylyltransferase-1 (CDS-1); 2) remodeling enzymes tafazzin and acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1); and 3) outer membrane CL enzymes, mitochondrial phospholipase D and phospholipid scramblase 3 (Plscr3), during chronic contractile activity (CCA)-induced mitochondrial biogenesis and denervation. With CCA, CDS-1 expression increased by 128%, parelleling CL levels. Surprisingly, denervation also led to large increases in CDS-1 and CLS, despite a decrease in mitochondria, possibly due to a compensatory mechanism to restore lost CL. ALCAT1 decreased by 32% with CCA, but increased by 290% following denervation, indicating that both CCA and denervation alter CL remodeling. CCA and denervation also elicited 60-90% increases in Plscr3, likely to facilitate CL movement to the outer membrane. The expression of these genes was not affected by aging, but changes due to CCA and denervation were attenuated compared with young animals. The absence of PPARγ coactivator-1α in knockout animals led to a decrease in CDS-1 and an increase in ALCAT1 mRNA levels, implicating PGC-1α in regulating both CL synthesis and remodeling. These data suggest that chronic muscle use and disuse modify the expression of mRNAs encoding CL metabolism enzymes. Our data also illustrate, for the first time, that PPARγ coactivator-1α regulates the CL metabolism pathway in muscle.

Ablation of ALCAT1 Mitigates Hypertrophic Cardiomyopathy through Effects on Oxidative Stress and Mitophagy

Oxidative stress causes mitochondrial dysfunction and heart failure through unknown mechanisms. Cardiolipin (CL), a mitochondrial membrane phospholipid required for oxidative phosphorylation, plays a pivotal role in cardiac function. The onset of age-related heart diseases is characterized by aberrant CL acyl composition that is highly sensitive to oxidative damage, leading to CL peroxidation and mitochondrial dysfunction. Here we report a key role of ALCAT1, a lysocardiolipin acyltransferase that catalyzes the synthesis of CL with a high peroxidation index, in mitochondrial dysfunction associated with hypertrophic cardiomyopathy. We show that ALCAT1 expression was potently upregulated by the onset of hyperthyroid cardiomyopathy, leading to oxidative stress and mitochondrial dysfunction. Accordingly, overexpression of ALCAT1 in H9c2 cardiac cells caused severe oxidative stress, lipid peroxidation, and mitochondrial DNA (mtDNA) depletion. Conversely, ablation of ALCAT1 prevented the onset of T4-induced cardiomyopathy and cardiac dysfunction. ALCAT1 deficiency also mitigated oxidative stress, insulin resistance, and mitochondrial dysfunction by improving mitochondrial quality control through upregulation of PINK1, a mitochondrial GTPase required for mitochondrial autophagy. Together, these findings implicate a key role of ALCAT1 as the missing link between oxidative stress and mitochondrial dysfunction in the etiology of age-related heart diseases.

Diagnosis of Barth syndrome using a novel LC‐MS/MS method for leukocyte cardiolipin analysis

Barth syndrome (BTHS) is an X-linked disorder characterised by cardiomyopathy, skeletal myopathy, growth retardation, neutropenia and 3-methylglutaconic aciduria. It is caused by mutations in the TAZ gene which codes for tafazzin, a protein with acyl transferase activity involved in synthesis of cardiolipin. Monolysocardiolipin (MLCL) is an intermediate in this process. Diagnosis of BTHS is difficult, as clinical and biochemical features are variable and numerous TAZ mutations have been described. These factors, together with lack of a straightforward diagnostic test are thought to have contributed to under-diagnosis of the condition. A novel method for cardiolipin analysis by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is reported which is less complicated and faster than previously described methods and uses a readily available sample type. The equipment, reagents and expertise required are found in most clinical laboratories performing metabolic investigations. Leukocytes were prepared from whole blood, phospholipids extracted and tetralinoleyl cardiolipin (CL4) and MLCL analysed by UPLC-MS/MS. Reference values were derived from analysis of 76 control and 23 BTHS samples as follows: CL4 in controls >132 (95% CI 100-169), BTHS <30.2 (21.3-40.4) pmol/mg protein; MLCL/CL4 ratio in controls <0.006 (0.004-0.009) and >2.52 (1.51-4.22) in BTHS patients. We describe an improved method for CL4 and MLCL/CL4 analysis which can be incorporated into the routine work of a clinical biochemistry laboratory. It shows 100% sensitivity and specificity for BTHS, making it a suitable diagnostic test.

Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates

Depending on growth phase and culture conditions, cardiolipin (CL) makes up 5-15% of the phospholipids in Escherichia coli with the remainder being primarily phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In E. coli, the cls and ybhO genes (renamed clsA and clsB, respectively) each encode a CL synthase (Cls) that catalyzes the condensation of two PG molecules to form CL and glycerol. However, a ΔclsAB mutant still makes CL in the stationary phase, indicating the existence of additional Cls. We identified a third Cls encoded by ymdC (renamed clsC). ClsC has sequence homology with ClsA and ClsB, which all belong to the phospholipase D superfamily. The ΔclsABC mutant lacks detectible CL regardless of growth phase or growth conditions. CL can be restored to near wild-type levels in stationary phase in the triple mutant by expressing either clsA or clsB. Expression of clsC alone resulted in a low level of CL in the stationary phase, which increased to near wild-type levels by coexpression of its neighboring gene, ymdB. CL synthesis by all Cls is increased with increasing medium osmolarity during logarithmic growth and in stationary phase. However, only ClsA contributes detectible levels of CL at low osmolarity during logarithmic growth. Mutation of the putative catalytic motif of ClsC prevents CL formation. Unlike eukaryotic Cls (that use PG and CDP-diacylglycerol as substrates) or ClsA, the combined YmdB-ClsC used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.

Possible Roles of the Mitochondria in Sulfur Dioxide Production by Lager Yeast

Sulfur dioxide is one of the key lager yeast metabolites that brewers seek to control, as it has multiple effects on the quality of lager beers. It has been widely reported that yeast cells, when fermented in high glucose worts, are susceptible to producing elevated sulfur dioxide, yet the exact mechanisms are not fully understood. It is known that when yeast cells utilize glucose as a primary carbon source to fuel yeast growth, this negatively affects mitochondrial development, specifically in its unique membrane lipid, cardiolipin. In this study, we show the results of incubation experiments aimed at altering cardiolipin formation and mitochondrial development with stimulators of cardiolipin synthesis (L-thyroxine and glycerol) and an inhibitor (inositol). These results demonstrated significantly lower sulfite levels (P < 0.05), adjusted for ethanol production, under conditions promoting cardiolipin formation. In addition, fermentations with respiratory deficient mutants also revealed significantly lower efficiency in the conversion of sulfate, as measured by the ratio of the amount of sulfite produced to the amount of sulfate taken up, as compared to the parental strain (P < 0.05). These findings suggest that this overlooked organelle may have a vital role in sulfite production, which we speculate is related to a key prosthetic group in the enzyme complex, sulfite reductase. This group, namely the [4Fe-4S] iron-sulfur cluster, provides the catalytic site in sulfite reductase electrons to reduce sulfur dioxide to sulfide. It is postulated that limitations in mitochondrial ATP levels affect [4Fe-4S] cluster formation or export of the cluster for cytosolic apoprotein modification.

Cardiac Mitochondrial Phenotype of the taz shRNA Mouse Model of Human Barth Syndrome

Barth syndrome (BTHS) is an X‐linked cardioskeletal myopathy that results from a mutation in the Tafazzin (taz) gene. Tafazzin is a phospholipid transacylase required for the optimal synthesis of cardiolipin (CL), a mitochondrial phospholipid that provides functional support to several mitochondrial proteins. In this study, the cardiac mitochondrial phenotype of a new mouse model of BTHS (taz shRNA; TAZ) was characterized. Cardiac mitochondria were isolated from male TAZ and wild type (WT) mice at 3–4 months of age for assessment of respiratory parameters, H2O2 production and Ca2+ tolerance. Mitochondria from TAZ mice had 50–60% lower rates of state 3 and state 4 respiration, with little change in respiratory efficiency vs. WT. Response to increasing levels of ADP was also severely blunted in TAZ, indicating impaired respiratory control. Absolute levels of H2O2 production were similar in TAZ and WT but were higher in TAZ when normalized for electron flow rate. Ca2+‐induced swelling was impaired in TAZ, as was sensitivity to cyclosporin A, indicating resistance to mitochondrial permeability transition. These findings demonstrate that taz deficiency impairs several aspects of mitochondrial function and provide the basis for further inquiry into the precise roles of taz and cardiolipin metabolism in mitochondrial (patho)physiology. Funding: AHA and Barth Syndrome Foundation

An essential bacterial-type cardiolipin synthase mediates cardiolipin formation in a eukaryote

Cardiolipin is important for bacterial and mitochondrial stability and function. The final step in cardiolipin biosynthesis is catalyzed by cardiolipin synthase and differs mechanistically between prokaryotes and eukaryotes. To study the importance of cardiolipin synthesis for mitochondrial integrity, membrane protein complex formation, and cell proliferation in the human and animal pathogenic protozoan parasite, Trypanosoma brucei, we generated conditional cardiolipin synthase-knockout parasites. We found that cardiolipin formation in T. brucei procyclic forms is catalyzed by a bacterial-type cardiolipin synthase, providing experimental evidence for a prokaryotic-type cardiolipin synthase in a eukaryotic organism. Ablation of enzyme expression resulted in inhibition of de novo cardiolipin synthesis, reduction in cellular cardiolipin levels, alterations in mitochondrial morphology and function, and parasite death in culture. By using immunofluorescence microscopy and blue-native gel electrophoresis, cardiolipin synthase was shown to colocalize with inner mitochondrial membrane proteins and to be part of a large protein complex. During depletion of cardiolipin synthase, the levels of cytochrome oxidase subunit IV and cytochrome c1, reflecting mitochondrial respiratory complexes IV and III, respectively, decreased progressively.

Cardiolipin synthase is required for Streptomyces coelicolor morphogenesis

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.

Phosphatidylglycerol-containing ER-transport vesicles built and restore outer mitochondrial membrane and deliver nuclear DNA translation products to generate cardiolipin in the inner mitochondrial membrane

Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.

Trans-10, cis-12 conjugated linoleic acid decreases de novo lipid synthesis in human adipocytes

Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid metabolism. The lipid-lowering effects of 10,12 CLA were unique, as other trans FA did not reduce TG content to the same extent. Using low levels of [14C]-CLA isomers, it was shown that both isomers were readily incorporated into acylglycerols and phospholipids, albeit at lower levels than [14C]-oleic or [14C]-linoleic acids. When using [14C]-acetic acid and [14C]-pyruvic acid as substrates, 30 μM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3–24 h. Treatment with 30 μM 10,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within 7–12 h, respectively. The mRNA levels of liver X receptor (LXR)α and sterol regulatory element binding protein (SREBP)-1c, transcription factors that regulate SCD-1, were decreased by 10,12 CLA within 5 h. These data suggest that the isomer-specific decrease in de novo lipid synthesis by 10,12 CLA is due, in part, to the rapid repression of lipogenic transcription factors that regulate MUFA synthesis, suggesting an anti-obesity mechanism unique to this trans FA.

Structural and functional analysis of PTPMT1, a phosphatase required for cardiolipin synthesis

PTPMT1 (PTP localized to the Mitochondrion 1) is a member of the protein tyrosine phosphatase superfamily that is localized exclusively to the mitochondrion. We recently reported that PTPMT1 dephosphorylates phosphatidylglycerol phosphate, an essential intermediate of cardiolipin biosynthesis. To gain further insights into the molecular basis of PTPMT1 function, we determined the crystal structures of the phosphatase domain of PTPMT1. PTPMT1 exhibits a canonical protein tyrosine phosphatase domain fold, resembling many dual-specificity phosphatases such as phosphatase and tensin homolog and vaccinia H1-related phosphatase. We also determined the structure of the catalytically inactive phosphatase in complex with a surrogate substrate, phosphatidylinositol 5-phosphate, which sheds light on the substrate recognition and specificity of PTPMT1. Comparison of the apo and substrate-bound structures of PTPMT1 suggests that it undergoes significant conformational change during catalysis, and we further demonstrated that an evolutionarily conserved EEYE loop is important for its activity.