Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase 1

CDP diacylglycerol synthase (CDS) catalyses the conversion of phosphatidic acid (PA) to CDP-diacylglycerol, an essential intermediate in the synthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol (PI). CDS activity has been identified in mitochondria and endoplasmic reticulum of mammalian cells apparently encoded by two highly-related genes, CDS1 and CDS2. Cardiolipin is exclusively synthesised in mitochondria and recent studies in cardiomyocytes suggest that the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α and β) serve as transcriptional regulators of mitochondrial biogenesis and up-regulate the transcription of the CDS1 gene. Here we have examined whether CDS1 is responsible for the mitochondrial CDS activity. We report that differentiation of H9c2 cells with retinoic acid towards cardiomyocytes is accompanied by increased expression of mitochondrial proteins, oxygen consumption, and expression of the PA/PI binding protein, PITPNC1, and CDS1 immunoreactivity. Both CDS1 immunoreactivity and CDS activity were found in mitochondria of H9c2 cells as well as in rat heart, liver and brain mitochondria. However, the CDS1 immunoreactivity was traced to a peripheral p55 cross-reactive mitochondrial protein and the mitochondrial CDS activity was due to a peripheral mitochondrial protein, TAMM41, not an integral membrane protein as expected for CDS1. TAMM41 is the mammalian equivalent of the recently identified yeast protein, Tam41. Knockdown of TAMM41 resulted in decreased mitochondrial CDS activity, decreased cardiolipin levels and a decrease in oxygen consumption. We conclude that the CDS activity present in mitochondria is mainly due to TAMM41, which is required for normal mitochondrial function.

Related papers:

title: Mammalian cardiolipin biosynthesis.
abstract: Cardiolipin is a major phospholipid in mitochondria and is involved in the generation of cellular energy in the form of ATP. In mammalian and eukaryotic cells it is synthesized via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol phosphate pathway. This brief review will describe some of the more recent studies on mammalian cardiolipin biosynthesis and provide an overview of regulation of cardiolipin biosynthesis. In addition, the important role that this key phospholipid plays in disease processes including heart failure, diabetes, thyroid hormone disease and the genetic disease Barth Syndrome will be discussed.

title: Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle.
abstract: Two populations of mitochondria were observed upon ultrastructural examination of cardiac muscle tissue, one located directly beneath the sarcolemma (subsarcolemmal mitochondria) and another between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria were released by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by nagarse digestion of the remaining tissue. These results were supported by electron microscopy of Polytron-treated heart tissue showing rupture and loss of sarcolemma with release of the underlying mitochondria but with retention of intact mitochondria between the myofibrils. Electron microscopy of the isolated mitochondria indicated that both mitochondrial types maintained their structural integrity throughout the isolation procedure. Specific activities of succinate dehydrogenase and citrate synthase were higher in the interfibrillar mitochondria as compared to the subsarcolemmal mitochondria, while those of carnitine palmitoyltransferase and alpha-glycerophosphate dehydrogenase were nearly the same in both. Interfibrillar mitochondria oxidized all substrates tested approximately 1.5 times faster than did the subsarcolemmal mitochondria. Thus the two mitochondrial types differed not only in their respective locations in the cell, but also in certain biochemical properties.

title: Morphological dynamics of mitochondria--a special emphasis on cardiac muscle cells.
abstract: Mitochondria play a critical role in cellular energy metabolism, Ca(2+) homeostasis, reactive oxygen species generation, apoptosis, aging, and development. Many recent publications have shown that a continuous balance of fusion and fission of these organelles is important in maintaining their proper function. Therefore, there is a steep correlation between the form and function of mitochondria. Many major proteins involved in mitochondrial fusion and fission have been identified in different cell types, including heart. However, the functional role of mitochondrial dynamics in the heart remains, for the most part, unexplored. In this review we will cover the recent field of mitochondrial dynamics and its physiological and pathological implications, with a particular emphasis on the experimental and theoretical basis of mitochondrial dynamics in the heart.

title: Transcriptional integration of mitochondrial biogenesis
abstract: Gene regulatory factors encoded by the nuclear genome are essential for mitochondrial biogenesis and function. Some of these factors act exclusively within the mitochondria to regulate the control of mitochondrial transcription, translation, and other functions. Others govern the expression of nuclear genes required for mitochondrial metabolism and organelle biogenesis. The peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family of transcriptional coactivators play a major role in transducing and integrating physiological signals governing metabolism, differentiation, and cell growth to the transcriptional machinery controlling mitochondrial functional capacity. Thus, the PGC-1 coactivators serve as a central component of the transcriptional regulatory circuitry that coordinately controls the energy-generating functions of mitochondria in accordance with the metabolic demands imposed by changing physiological conditions, senescence, and disease.

title: Transcriptional control of mitochondrial biogenesis and function.
abstract: Mitochondria play central roles in energy homeostasis, metabolism, signaling, and apoptosis. Accordingly, the abundance, morphology, and functional properties of mitochondria are finely tuned to meet cell-specific energetic, metabolic, and signaling demands. This tuning is largely achieved at the level of transcriptional regulation. A highly interconnected network of transcription factors regulates a broad set of nuclear genes encoding mitochondrial proteins, including those that control replication and transcription of the mitochondrial genome. The same transcriptional network senses cues relaying cellular energy status, nutrient availability, and the physiological state of the organism and enables short- and long-term adaptive responses, resulting in adjustments to mitochondrial function and mitochondrial biogenesis. Mitochondrial dysfunction is associated with many human diseases. Characterization of the transcriptional mechanisms that regulate mitochondrial biogenesis and function can offer insights into possible therapeutic interventions aimed at modulating mitochondrial function.

title: A Role for Peroxisome Proliferator-activated Receptor γ Coactivator 1 (PGC-1) in the Regulation of Cardiac Mitochondrial Phospholipid Biosynthesis*♦
abstract: Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) α and β are transcriptional regulators of mitochondrial metabolism.  Loss of PGC-1 coactivators in mouse heart causes a defect in phospholipid biosynthesis resulting in mitochondrial ultrastructural abnormalities.  PGC-1 coactivators coordinately regulate mitochondrial metabolism and structure.  The mitochondrial phenotype of PGC-1-deficient mice resembles that of humans with genetic defects in mitochondrial phospholipid biosynthesis (Barth syndrome). The energy demands of the adult mammalian heart are met largely by ATP generated via oxidation of fatty acids in a high capacity mitochondrial system. Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)-α and -β serve as inducible transcriptional coregulators of genes involved in mitochondrial biogenesis and metabolism. Whether PGC-1 plays a role in the regulation of mitochondrial structure is unknown. In this study, mice with combined deficiency of PGC-1α and PGC-1β (PGC-1αβ−/−) in adult heart were analyzed. PGC-1αβ−/− hearts exhibited a distinctive mitochondrial cristae-stacking abnormality suggestive of a phospholipid abnormality as has been described in humans with genetic defects in cardiolipin (CL) synthesis (Barth syndrome). A subset of molecular species, containing n-3 polyunsaturated species in the CL, phosphatidylcholine, and phosphatidylethanolamine profiles, was reduced in PGC-1αβ-deficient hearts. Gene expression profiling of PGC-1αβ−/− hearts revealed reduced expression of the gene encoding CDP-diacylglycerol synthase 1 (Cds1), an enzyme that catalyzes the proximal step in CL biosynthesis. Cds1 gene promoter-reporter cotransfection experiments and chromatin immunoprecipitation studies demonstrated that PGC-1α coregulates estrogen-related receptors to activate the transcription of the Cds1 gene. We conclude that the PGC-1/estrogen-related receptor axis coordinately regulates metabolic and membrane structural programs relevant to the maintenance of high capacity mitochondrial function in heart.

title: Isolation and characterization of murine Cds (CDP-diacylglycerol synthase) 1 and 2.
abstract: Phototransduction in Drosophila is a phosphoinositide-mediated signalling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signalling. cds mutants develop light-induced retinal degeneration. We report here the isolation and characterization of two murine genes encoding this enzyme, Cds1 and Cds2. The genes encode proteins that are 73% identical and 92% similar but exhibit very different expression patterns. Cds1 shows a very restricted expression pattern but is expressed in the inner segments of the photoreceptors whilst Cds2 shows a ubiquitous pattern of expression. Using fluorescent in situ hybridization we have mapped Cds1 and Cds2 to chromosomes 5E3 and 2G1 respectively. These are regions of synteny with the corresponding human gene localization (4q21 and 20p13). Transient transfection experiments with epitope tagged proteins have also demonstrated that both are associated with the endoplasmic reticulum.

title: Isolation and chromosomal localization of two human CDP-diacylglycerol synthase (CDS) genes.
abstract: Phototransduction in Drosophila is a phosphoinositide-mediated signaling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signaling. cds mutants develop light-induced retinal degeneration. As part of a search for novel genes that may be involved in eye disease in human, using Drosophila phototransduction genes as a model system, two human CDP-diacylglycerol synthase genes (CDS1 and CDS2) were cloned and sequenced. Radiation hybrid panel mapping and fluorescence in situ hybridization were used to localize the genes to chromosomes 4q21 and 20p13. As yet, no known retinal diseases map to either of these regions.

title: Extraplastidial cytidinediphosphate diacylglycerol synthase activity is required for vegetative development in Arabidopsis thaliana.
abstract: Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the activation of phosphatidic acid to cytidinediphosphate (CDP)-diacylglycerol, a central intermediate in glycerolipid biosynthesis in prokaryotic and eukaryotic organisms. Cytidinediphosphate-diacylglycerol is the precursor to phosphatidylinositol, phosphatidylglycerol (PG) and cardiolipin of eukaryotic phospholipids that are essential for various cellular functions. Isoforms of CDS are located in plastids, mitochondria and the endomembrane system of plants and are encoded by five genes in Arabidopsis. Two genes have previously been shown to code for the plastidial isoforms which are indispensable for the biosynthesis of plastidial PG, and thus biogenesis and function of thylakoid membranes. Here we have focused on the extraplastidial CDS isoforms, encoded by CDS1 and CDS2 which are constitutively expressed contrary to CDS3. We provide evidence that these closely related CDS genes code for membrane proteins located in the endoplasmic reticulum and possess very similar enzymatic properties. Development and analysis of Arabidopsis mutants lacking either one or both CDS1 and CDS2 genes clearly shows that these two genes have redundant functions. As reflected in the seedling lethal phenotype of the cds1cds2 double mutant, plant cells require at least one catalytically active microsomal CDS isoform for cell division and expansion. According to the altered glycerolipid composition of the double mutant in comparison with wild-type seedlings, it is likely that the drastic decrease in the level of phosphatidylinositol and the increase in phosphatidic acid cause defects in cell division and expansion.

title: Two Closely Related Genes of Arabidopsis Encode Plastidial Cytidinediphosphate Diacylglycerol Synthases Essential for Photoautotrophic Growth1[C]
abstract: Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth.

title: The CDS1 Gene Encoding CDP-diacylglycerol Synthase In Saccharomyces cerevisiae Is Essential for Cell Growth (*)
abstract: An open reading frame (CDS1) residing on chromosome II of Saccharomyces cerevisiae encodes a hydrophobic protein with a predicted molecular mass of 51,789 Da, which exhibits 29 and 37% amino acid sequence identities with CDP-diacylglycerol synthases reported from Escherichia coli and Drosophila, respectively. Induction of expression of a GAL1 promoter-driven CDS1 gene on a multicopy plasmid in a cds1 null mutant background resulted in synthase activity 10 times that of wild-type cells and an elevation in the apparent initial rate of synthesis of phosphatidylinositol relative to phosphatidylserine. Without induction, activity was reduced to 10% of wild-type levels, which was sufficient to support growth but resulted in an inositol excretion phenotype, and had an opposite effect on the above phospholipid synthesis. Null cds1 mutants were incapable of spore germination or vegetative growth and could not be complemented under uninduced conditions with a GAL1 promoter-driven CDS1 gene on a low copy plasmid. Therefore, the essential CDS1 gene encodes the majority, if not all, of the synthase activity. The lack of consensus RNA splice sites derived from the genomic CDS1 sequence predicts that the multiple subcellular locations for synthase activities do not arise through RNA processing events.

title: Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli.
abstract: The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase. We now report the construction of plasmids which carry cds. Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene. This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase. The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570. Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon. We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase. This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., ). Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E. coli genetic map.

title: Regulation of PLC-mediated signalling in vivo by CDP-diacylglycerol synthase
abstract: CDP-diacylglycerol synthase (CDS) is an enzyme required for the regeneration of the signalling molecule phosphatidylinositol-4,5-bisphosphate (PtdlnsP2) from phosphatidic acid. A photo-receptor cell-specific isoform of CDS from Drosophila is a key regulator of phototransduction, a G-protein-coupled signalling cascade mediated by phospholipase C. cds mutants cannot sustain a light-activated current as a result of depletion of PtdlnsP2. Overexpression of CDS increases the amplitude of the light response, demonstrating that availability of PtdlnsP2 is a determinant in the gain of this pathway. cds mutants undergo light-dependent retinal degeneration which can be suppressed by a mutation in phospholipase C. Thus, enzymes involved in PtdlnsP2 metabolism regulate phosphoinositide-mediated signalling cascades in vivo.

title: CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis.
abstract: Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis.

title: Gene Cloning and Characterization of CDP-diacylglycerol Synthase from Rat Brain*
abstract: A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this rat CDS was 53 kDa, and putative primary structure included several possible membrane- spanning regions. At the amino acid sequence level, rat CDS shared 55.5%, 31.7%, and 20.9% identity with already known Drosophila, Saccharomyces cerevisiae, and Escherichia coli CDS, respectively. This rat CDS preferred 1-stearoyl-2-arachidonoyl phosphatidic acid as a substrate, and its activity was strongly inhibited by phosphatidylglycerol 4,5-bisphosphate. By immunoblotting analysis of COS cells overexpressed with the epitope-tagged for rat CDS, a 60-kDa band was detected. By epitope-tag immunocytochemistry, the CDS protein was mainly localized in close association with the membrane of the endoplasmic reticulum of the transfected cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, the pineal body, and the inner segment of photoreceptor cells. Additionally, very intense expression was detected in postmitotic spermatocytes and spermatids.

title: Structure and mechanism of an intramembrane liponucleotide synthetase central for phospholipid biosynthesis
abstract: Phospholipids are elemental building-block molecules for biological membranes. Biosynthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine requires a central liponucleotide intermediate named cytidine-diphosphate diacylglycerol (CDP-DAG). The CDP-DAG synthetase (Cds) is an integral membrane enzyme catalysing the formation of CDP-DAG, an essential step for phosphoinositide recycling during signal transduction. Here we report the structure of the Cds from Thermotoga maritima (TmCdsA) at 3.4 Å resolution. TmCdsA forms a homodimer and each monomer contains nine transmembrane helices arranged into a novel fold with three domains. An unusual funnel-shaped cavity penetrates half way into the membrane, allowing the enzyme to simultaneously accept hydrophilic substrate (cytidine 5′-triphosphate (CTP)/deoxy-CTP) from cytoplasm and hydrophobic substrate (phosphatidic acid) from membrane. Located at the bottom of the cavity, a Mg2+-K+ hetero-di-metal centre coordinated by an Asp-Asp dyad serves as the cofactor of TmCdsA. The results suggest a two-metal-ion catalytic mechanism for the Cds-mediated synthesis of CDP-DAG at the membrane–cytoplasm interface.

title: A highly dynamic ER-derived phosphatidylinositol-synthesizing organelle supplies phosphoinositides to cellular membranes.
abstract: Polyphosphoinositides are lipid signaling molecules generated from phosphatidylinositol (PtdIns) with critical roles in vesicular trafficking and signaling. It is poorly understood where PtdIns is located within cells and how it moves around between membranes. Here we identify a hitherto-unrecognized highly mobile membrane compartment as the site of PtdIns synthesis and a likely source of PtdIns of all membranes. We show that the PtdIns-synthesizing enzyme PIS associates with a rapidly moving compartment of ER origin that makes ample contacts with other membranes. In contrast, CDP-diacylglycerol synthases that provide PIS with its substrate reside in the tubular ER. Expression of a PtdIns-specific bacterial PLC generates diacylglycerol also in rapidly moving cytoplasmic objects. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.

title: Characterization of CDPdiacylglycerol hydrolase in mitochondrial and microsomal fractions from rat lung.
abstract: CDPdiacylglycerol pyrophosphatase (E.C. 3.6.1.26) activity has been examined in rat lung mitochondrial and microsomal fractions. While the mitochondrial hydrolase exhibited a broad pH optimum from pH 6-8, the microsomal activity decreased rapidly above pH 6.5. Apparent Km values of 36.2 and 23.6 microM and Vmax values of 311 and 197 pmol.min-1.mg protein-1 were observed for the mitochondrial and microsomal preparations, respectively. Addition of parachloromercuriphenylsulphonic acid led to a marked inhibition of the microsomal fraction but slightly stimulated the mitochondrial activity at low concentrations. Mercuric ions were inhibitory with both fractions. Although biosynthetic reactions utilizing CDPdiacylglycerol require divalent cations, addition of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, and Cu2+ all inhibited the catabolic CDPdiacylglycerol hydrolase activity in both fractions. EDTA and EGTA also produced an inhibitory effect, especially with the mitochondrial fraction. Although addition of either adenine or cytidine nucleotides led to a decrease in activity with both fractions, the marked susceptibility to AMP previously reported for this enzyme in Escherichia coli membranes, guinea pig brain lysosomes, and pig liver mitochondria was not observed. These results indicate that rat lung mitochondria and microsomes contain specific CDPdiacylglycerol hydrolase activities, which could influence the rate of formation of phosphatidylinositol and phosphatidylglycerol for pulmonary surfactant.

title: CDP‐diacylglycerol synthesis in rat liver mitochondria
abstract: CDP‐diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane‐associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP‐diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent‐soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP‐diacylglycerol synthase activity could be completely separated by anion‐exchange column chromatography. The mitochondrial and microsomal CDP‐diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.

title: Comparative studies of CDP-diacylglycerol synthase in rat liver mitochondria and microsomes.
abstract: CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. Contamination by microsomal membranes cannot account for the CDP-diacylglycerol synthesis found in the mitochondria. Phosphatidic acid from egg lecithin was the best substrate for the synthesis of CDP-diacylglycerol in both subcellular fractions. Concentration curves for CTP and Mg2+ differed for the two subcellular fractions. Microsomal CDP-diacylglycerol synthase was specifically stimulated by the nucleotide GTP; this stimulatory effect by GTP was not observed in the mitochondrial fraction. By comparison, the microsomal enzyme was more sensitive towards sulfhydryl inhibitors than the mitochondrial enzyme. The enzymes could be solubilized from the membrane fractions using 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the detergent-soluble activity could be partially restored by addition of phospholipids. Based on the differences in properties, it was concluded that there are two distinct enzyme localizations for CDP-diacylglycerol synthesis in mitochondria and microsomes from rat liver.

title: Cloning of CDP‐Diacylglycerol Synthase from a Human Neuronal Cell Line
abstract: A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP‐diacylglycerol, catalyzed by CDP‐diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP‐diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP‐diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP‐diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.

title: Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure
abstract: Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

title: Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria.
abstract: 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.

title: Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function
abstract: Cds1 is an evolutionarily conserved CDP-DG synthase. Fission yeast are used to demonstrate that cells deficient in its function exhibit markedly increased triacylglycerol content and assemble unusual ER-associated lipid droplets that recruit the triacylglycerol synthesis machinery and grow by expansion.

title: The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis
abstract: The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.

title: 14-3-3 protein and ATRAP bind to the soluble class IIB phosphatidylinositol transfer protein RdgBβ at distinct sites.
abstract: PITPs (phosphatidylinositol transfer proteins) are characterized by the presence of the PITP domain whose biochemical properties of binding and transferring PI (phosphatidylinositol) are well studied. Despite their wide-spread expression in both unicellular and multicellular organisms, they remain functionally uncharacterized. An emerging theme is that individual PITPs play highly specific roles in either membrane trafficking or signal transduction. To identify specific roles for PITPs, identification of interacting molecules would shed light on their molecular function. In the present paper, we describe binding partners for the class IIB PITP RdgBβ (retinal degeneration type Bβ). RdgBβ is a soluble PITP but is unique in that it contains a region of disorder at its C-terminus following its defining N-terminal PITP domain. The C-terminus of RdgBβ is phosphorylated at two serine residues, Ser274 and Ser299, which form a docking site for 14-3-3 proteins. Binding to 14-3-3 proteins protects RdgBβ from degradation that occurs at the proteasome after ubiquitination. In addition to binding 14-3-3, the PITP domain of RdgBβ interacts with the Ang II (angiotensin II)-associated protein ATRAP (Ang II receptor-associated protein). ATRAP is also an interacting partner for the AT1R (Ang II type 1 receptor). We present a model whereby RdgBβ functions by being recruited to the membrane by ATRAP and release of 14-3-3 from the C-terminus allows the disordered region to bind a second membrane to create a membrane bridge for lipid transfer, possibly under the control of Ang II.

title: The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein).
abstract: PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser(274) and Ser(299)) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.

title: Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid*
abstract: Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ) promotes metastatic colonization and angiogenesis in humans.  We demonstrate that RdgBβ is a phosphatidic acid (PA)- and phosphatidylinositol-binding protein and binds PA derived from the phospholipase D pathway.  RdgBβ is the first lipid-binding protein identified that can bind and transfer PA.  PA bound to RdgBβ is a likely effector downstream of phospholipase D. Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.

title: A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells
abstract: Metastatic progression of cancer is a complex and clinically daunting process. We previously identified a set of human microRNAs (miRNAs) that robustly suppress breast cancer metastasis to lung and bone and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of non-coding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers, non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK—novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 (IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network’s individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.

title: Phosphorylation of a Distinct Structural Form of Phosphatidylinositol Transfer Protein α at Ser166 by Protein Kinase C Disrupts Receptor-mediated Phospholipase C Signaling by Inhibiting Delivery of Phosphatidylinositol to Membranes*
abstract: Phosphatidylinositol transfer protein α (PITPα) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) β and γ signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPα at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165–172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPα. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPα from rat brain or in vitro phosphorylated PITPα demonstrated that phosphorylated PITPα is structurally distinct from the non-phosphorylated form. Phosphorylated PITPα was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPα structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.

title: Phosphatidylinositol- and phosphatidylcholine-transfer activity of PITPβ is essential for COPI-mediated retrograde transport from the Golgi to the endoplasmic reticulum
abstract: Vesicles formed by the COPI complex function in retrograde transport from the Golgi to the endoplasmic reticulum (ER). Phosphatidylinositol transfer protein β (PITPβ), an essential protein that possesses phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) lipid transfer activity is known to localise to the Golgi and ER but its role in these membrane systems is not clear. To examine the function of PITPβ at the Golgi-ER interface, RNA interference (RNAi) was used to knockdown PITPβ protein expression in HeLa cells. Depletion of PITPβ leads to a decrease in PtdIns(4)P levels, compaction of the Golgi complex and protection from brefeldin-A-mediated dispersal to the ER. Using specific transport assays, we show that anterograde traffic is unaffected but that KDEL-receptor-dependent retrograde traffic is inhibited. This phenotype can be rescued by expression of wild-type PITPβ but not by mutants defective in docking, PtdIns transfer and PtdCho transfer. These data demonstrate that the PtdIns and PtdCho exchange activity of PITPβ is essential for COPI-mediated retrograde transport from the Golgi to the ER.

title: Isolation and incubation conditions to study heart mitochondrial protein synthesis.
abstract: Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt.  21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

title: Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells
abstract: Many cellular processes require the proper cooperation between mitochondria and the endoplasmic reticulum (ER). Several recent works show that their functional interactions rely on dynamic structural contacts between both organelles. Such contacts, called mitochondria-associated membranes (MAMs), are crucial for the synthesis and intracellular transport of phospholipids, as well as for intracellular Ca2+ signaling and for the determination of mitochondrial structure. Although several techniques are available to isolate mitochondria, only few are specifically tuned to the isolation of MAM, containing unique regions of ER membranes attached to the outer mitochondrial membrane and mitochondria without contamination from other organelles (i.e., pure mitochondria). Here we provide optimized protocols to isolate these fractions from tissues and cells. These procedures require 4–5 h and can be easily modified and adapted to different tissues and cell types.

title: Modulation of L-type Calcium Channel Expression during Retinoic Acid-induced Differentiation of H9C2 Cardiac Cells*
abstract: The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca2+ channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (α1S) and cardiac (α1C) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nm all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca2+ channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca2+ channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (α1C) L-type Ca2+ channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca2+ channels in mature cardiac cells.

title: Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts.
abstract: H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.

title: Metabolic Remodeling During H9c2 Myoblast Differentiation: Relevance for In Vitro Toxicity Studies
abstract: H9c2 cells, derived from the ventricular part of an E13 BDIX rat heart, possess a proliferative and relatively undifferentiated phenotype but can be readily directed to differentiate under reduced serum conditions originating cells presenting muscle features. Skeletal or cardiac phenotypes can be originated depending on whether or not serum reduction is accompanied by a daily treatment with all-trans-retinoic acid. In the present study, we aimed to characterize and compare the metabolic profile of H9c2 cells at various differentiation states, correlating the differences between different populations with muscle-specific development. We determined that H9c2 myoblasts remodel their metabolism upon differentiation, with undifferentiated cells more reliant on glycolysis, as demonstrated by higher lactate production rates. Differentiated cells adopted a more oxidative metabolism with better coupling between the glycolytic and oxidative pathways, which is indicative of a metabolic evolvement toward a higher energetic efficiency state. Our findings emphasize the metabolic differences between differentiated and undifferentiated H9c2 cells and raise caution on how to adequately select the H9c2 differentiation state that will act as the better model for the design of experimental studies.

title: Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype
abstract: H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays.

title: ZEB1-responsive genes in non-small cell lung cancer.
abstract: The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-β plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease.

title: CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum
abstract: Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.

title: MAM (mitochondria-associated membranes) in mammalian cells: lipids and beyond.
abstract: One mechanism by which communication between the endoplasmic reticulum (ER) and mitochondria is achieved is by close juxtaposition between these organelles via mitochondria-associated membranes (MAM). The MAM consist of a region of the ER that is enriched in several lipid biosynthetic enzyme activities and becomes reversibly tethered to mitochondria. Specific proteins are localized, sometimes transiently, in the MAM. Several of these proteins have been implicated in tethering the MAM to mitochondria. In mammalian cells, formation of these contact sites between MAM and mitochondria appears to be required for key cellular events including the transport of calcium from the ER to mitochondria, the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, the formation of autophagosomes, regulation of the morphology, dynamics and functions of mitochondria, and cell survival. This review focuses on the functions proposed for MAM in mediating these events in mammalian cells. In light of the apparent involvement of MAM in multiple fundamental cellular processes, recent studies indicate that impaired contact between MAM and mitochondria might underlie the pathology of several human neurodegenerative diseases, including Alzheimer's disease. Moreover, MAM has been implicated in modulating glucose homeostasis and insulin resistance, as well as in some viral infections.

title: Characterization of Mmp37p, a Saccharomyces cerevisiae mitochondrial matrix protein with a role in mitochondrial protein import.
abstract: Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

title: Metabolism and function of mitochondrial cardiolipin.
abstract: 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.

title: Antibody anarchy: A call to order
abstract: Antibodies used in research often give murky results. Broader awareness and advanced technologies promise clarity.

title: Reproducibility: Standardize antibodies used in research
abstract: To save millions of dollars and dramatically improve reproducibility, protein-binding reagents must be defined by their sequences and produced as recombinant proteins, say Andrew Bradbury, Andreas Pluckthun and 110 co-signatories.

title: PITPNC1 Recruits RAB1B to the Golgi Network to Drive Malignant Secretion.
abstract: Enhanced secretion of tumorigenic effector proteins is a feature of malignant cells. The molecular mechanisms underlying this feature are poorly defined. We identify PITPNC1 as a gene amplified in a large fraction of human breast cancer and overexpressed in metastatic breast, melanoma, and colon cancers. Biochemical, molecular, and cell-biological studies reveal that PITPNC1 promotes malignant secretion by binding Golgi-resident PI4P and localizing RAB1B to the Golgi. RAB1B localization to the Golgi allows for the recruitment of GOLPH3, which facilitates Golgi extension and enhanced vesicular release. PITPNC1-mediated vesicular release drives metastasis by increasing the secretion of pro-invasive and pro-angiogenic mediators HTRA1, MMP1, FAM3C, PDGFA, and ADAM10. We establish PITPNC1 as a PI4P-binding protein that enhances vesicular secretion capacity in malignancy.

title: H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation.
abstract: Dysfunction of cardiac energy metabolism plays a critical role in many cardiac diseases, including heart failure, myocardial infarction and ischemia-reperfusion injury and organ transplantation. The characteristics of these diseases can be elucidated in vivo, though animal-free in vitro experiments, with primary adult or neonatal cardiomyocytes, the rat ventricular H9c2 cell line or the mouse atrial HL-1 cells, providing intriguing experimental alternatives. Currently, it is not clear how H9c2 and HL-1 cells mimic the responses of primary cardiomyocytes to hypoxia and oxidative stress. In the present study, we show that H9c2 cells are more similar to primary cardiomyocytes than HL-1 cells with regard to energy metabolism patterns, such as cellular ATP levels, bioenergetics, metabolism, function and morphology of mitochondria. In contrast to HL-1, H9c2 cells possess beta-tubulin II, a mitochondrial isoform of tubulin that plays an important role in mitochondrial function and regulation. We demonstrate that H9c2 cells are significantly more sensitive to hypoxia-reoxygenation injury in terms of loss of cell viability and mitochondrial respiration, whereas HL-1 cells were more resistant to hypoxia as evidenced by their relative stability. In comparison to HL-1 cells, H9c2 cells exhibit a higher phosphorylation (activation) state of AMP-activated protein kinase, but lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha levels, suggesting that each cell type is characterized by distinct regulation of mitochondrial biogenesis. Our results provide evidence that H9c2 cardiomyoblasts are more energetically similar to primary cardiomyocytes than are atrial HL-1 cells. H9c2 cells can be successfully used as an in vitro model to simulate cardiac ischemia-reperfusion injury.