Phosphatidylglycerophosphate Synthase Research Articles

New insights into the role of mitochondrial metabolic dysregulation and immune infiltration in septic cardiomyopathy by integrated bioinformatics analysis and experimental validation

BackgroundSeptic cardiomyopathy (SCM), a common cardiovascular comorbidity of sepsis, has emerged among the leading causes of death in patients with sepsis. SCM’s pathogenesis is strongly affected by mitochondrial metabolic dysregulation and immune infiltration disorder. However, the specific mechanisms and their intricate interactions in SCM remain unclear. This study employed bioinformatics analysis and drug discovery approaches to identify the regulatory molecules, distinct functions, and underlying interactions of mitochondrial metabolism and immune microenvironment, along with potential interventional strategies in SCM.MethodsGSE79962, GSE171546, and GSE167363 datasets were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) and module genes were identified using Limma and Weighted Correlation Network Analysis (WGCNA), followed by functional enrichment analysis. Machine learning algorithms, including support vector machine–recursive feature elimination (SVM–RFE), least absolute shrinkage and selection operator (LASSO) regression, and random forest, were used to screen mitochondria-related hub genes for early diagnosis of SCM. Subsequently, a nomogram was developed based on six hub genes. The immunological landscape was evaluated by single-sample gene set enrichment analysis (ssGSEA). We also explored the expression pattern of hub genes and distribution of mitochondria/inflammation-related pathways in UMAP plots of single-cell dataset. Potential drugs were explored using the Drug Signatures Database (DSigDB). In vivo and in vitro experiments were performed to validate the pathogenetic mechanism of SCM and the therapeutic efficacy of candidate drugs.ResultsSix hub mitochondria-related DEGs [MitoDEGs; translocase of inner mitochondrial membrane domain-containing 1 (TIMMDC1), mitochondrial ribosomal protein S31 (MRPS31), F-box only protein 7 (FBXO7), phosphatidylglycerophosphate synthase 1 (PGS1), LYR motif containing 7 (LYRM7), and mitochondrial chaperone BCS1 (BCS1L)] were identified. The diagnostic nomogram model based on the six hub genes demonstrated high reliability and validity in both the training and validation sets. The immunological microenvironment differed between SCM and control groups. The Spearman correlation analysis revealed that hub MitoDEGs were significantly associated with the infiltration of immune cells. Upregulated hub genes showed remarkably high expression in the naive/memory B cell, CD14+ monocyte, and plasma cell subgroup, evidenced by the feature plot. The distribution of mitochondria/inflammation-related pathways varied across subgroups among control and SCM individuals. Metformin was predicted to be the most promising drug with the highest combined score. Its efficacy in restoring mitochondrial function and suppressing inflammatory responses has also been validated.ConclusionsThis study presents a comprehensive mitochondrial metabolism and immune infiltration landscape in SCM, providing a potential novel direction for the pathogenesis and medical intervention of SCM.

Pseudomonas aeruginosa adapts to octenidine via a combination of efflux and membrane remodelling

Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.

High-throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts.

Mutations in OPA1 cause autosomal dominant optic atrophy (DOA) as well as DOA+, a phenotype characterized by more severe neurological deficits. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae, respiration, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. We screened the entire known mitochondrial proteome (1,531 genes) to identify genes that control mitochondrial morphology using a first‐in‐kind imaging pipeline. We identified 145 known and novel candidate genes whose depletion promoted elongation or fragmentation of the mitochondrial network in control fibroblasts and 91 in DOA+ patient fibroblasts that prevented mitochondrial fragmentation, including phosphatidyl glycerophosphate synthase (PGS1). PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1‐deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology, or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation.

Analysis of miRNA-seq in the liver of common carp (Cyprinus carpio L.) in response to different environmental temperatures.

Water temperature affects the survival, growth, immunity, reproduction, and productivity of farmed fish. The temperature beyond suitable range will disrupt the normal physiological activity. Common carp (Cyprinus carpio L.) is a representative eurythermic fish; they are able to sense and respond to changes in water temperature by adjusting their physiology. To investigate the miRNAs in common carp at different temperatures, nine liver small-RNA libraries (5°C, 17°C, and 30°C, each group have three biological repetitions) were constructed and sequenced using high-throughput sequencing. A total of 110 known miRNAs were identified. Twenty-nine known miRNAs were differentially expressed compared with in control group. GO and KEGG analysis indicated that the miRNAs may play important roles in metabolism and environment information processing. Specifically, we considered the insulin-signaling and glycerophospholipid metabolism pathway, and the results show that in 30°C, miR-301a, miR-203b-5p, and miR-210-3p were upregulated; their target genes which are the mechanistic targets of the rapamycin kinase (mtor) gene and the protein kinase AMP-activated catalytic subunit alpha 1 (prkaa1) gene in the insulin-signaling pathway were downregulated. And miR-9-5p, miR-27d, miR-92b-3p, and miR-155 were upregulated; their target genes, 1-acylglycerol-3-phosphate O-acyltransferase 3 (agpat3), CDP-diacylglycerol-inositol 3-phosphatidyltransferase (cdipt), glycerol-3-phosphate acyltransferase mitochondrial (gpam), and phosphatidylglycerophosphate synthase 1 (pgs1), in glycerophospholipid metabolism pathway were downregulated. But in 5°C, the situation was opposite. These findings suggest that significant changes occur in energy metabolism and metabolic processes with components of the cell membrane in different temperatures, which significantly advance our understanding of the regulatory mechanisms underlying the physiological change of temperature stress-induced in liver, specifically with regard to miRNAs. These data provide a foundation for further studies of the role of miRNAs in environmental adaptation in fish.

Characterization of the Mechanisms of Daptomycin Resistance among Gram-Positive Bacterial Pathogens by Multidimensional Lipidomics.

Previous work suggests that altered lipid metabolism may be associated with daptomycin resistance in Gram-positive pathogens, but lipidomic changes underlying resistance are not fully understood. We performed untargeted lipidomics by using three-dimensional hydrophilic interaction liquid chromatography-ion mobility-mass spectrometry (HILIC-IM-MS) to characterize alterations in the lipidomes of daptomycin-susceptible and -resistant isogenic strain pairs of Enterococcus faecalis, Staphylococcus aureus, and Corynebacterium striatum. We first validated the HILIC-IM-MS method by replicating the expected alterations of phospholipid metabolism in the previously studied E.faecalis strain pairs, such as reduced phosphatidylglycerols (PGs), while also revealing additional changes in cardiolipins (CLs), lysyl-PGs, and glycolipids. Whole-genome sequencing of the S.aureus and C.striatum strains found that daptomycin resistance was associated with mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, as well as mutations in genes affecting fatty acid biosynthesis and cell wall metabolism. Lipidomics revealed significantly decreased levels of PGs, CLs, and amino acid-modified PGs, as well as accumulation of lipids upstream of PGs, such as glycolipids and phosphatidic acids, in the resistant strains. Notably, the glycolipids, diglucosyldiacylglycerols, were significantly elevated in a fatty acid-dependent manner in the daptomycin-resistant S.aureus strain. In daptomycin-resistant C.striatum, which has a unique cell envelope architecture, the glycolipids, glucuronosyldiacylglycerols, and phosphatidylinositols were significantly elevated. These results demonstrate that alteration of lipid metabolism via mutations in pgsA is a common mechanism of daptomycin resistance in two distinct species of Gram-positive bacteria and point to the potential contribution of altered glycolipid and fatty acid compositions to daptomycin resistance. IMPORTANCE This work comprehensively characterizes lipidomic changes underlying daptomycin resistance in three Gram-positive bacterial species, E.faecalis, S.aureus, and C.striatum, by using a novel three-dimensional lipidomics methodology based on advanced mass spectrometry. We demonstrated a number of advantages of our method in comparison with other methods commonly used in the field, such as high molecular specificity, sensitivity, and throughput. Whole-genome sequencing of the S.aureus and C.striatum strains identified mutations in pgsA, which encodes phosphatidylglycerophosphate synthase, in both resistant strains. Lipidomics revealed significantly decreased levels of lipids downstream of PgsA, as well as accumulation of lipids upstream of PgsA in the resistant strains. Furthermore, we found that changes in individual molecular species of each lipid class depend on the their specific fatty acid compositions. The characteristic changes in individual lipid species could be used as biomarkers for identifying underlying resistance mechanisms and for evaluating potential therapies.

Identification and characterization of a plastidial phosphatidylglycerophosphate phosphatase in Arabidopsis thaliana.

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation.

Functional Specificity of Cardiolipin Synthase Revealed by the Identification of a Cardiolipin Synthase CrCLS1 in Chlamydomonas reinhardtii

Phosphatidylglycerol (PG) and cardiolipin (CL) are two essential classes of phospholipid in plants and algae. Phosphatidylglycerophosphate synthase (PGPS) and cardiolipin synthase (CLS) involved in the biosynthesis of PG and CL belong to CDP-alcohol phosphotransferase and share overall amino acid sequence homology. However, it remains elusive whether PGPS and CLS are functionally distinct in vivo. Here, we report identification of a gene encoding CLS in Chlamydomonas reinhardtii, CrCLS1, and its functional compatibility. Whereas CrCLS1 did not complement the growth phenotype of a PGPS mutant of Synechocystis sp. PCC 6803, it rescued the temperature-sensitive growth phenotype, growth profile with different carbon sources, phospholipid composition and enzyme activity of Δcrd1, a CLS mutant of Saccharomyces cerevisiae. These results suggest that CrCLS1 encodes a functional CLS of C. reinhardtii as the first identified algal CLS, whose enzyme function is distinct from that of PGPSs from C. reinhardtii. Comparison of CDP-alcohol phosphotransferase motif between PGPS and CLS among different species revealed a possible additional motif that might define the substrate specificity of these closely related enzymes.

Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803.

Phosphatidylglycerol (PG) is an indispensable phospholipid class with photosynthetic function in plants and cyanobacteria. However, its biosynthesis in eukaryotic green microalgae is poorly studied. Here, we report the isolation and characterization of two homologs (CrPGP1 and CrPGP2) of phosphatidylglycerophosphate synthase (PGPS), the rate-limiting enzyme in PG biosynthesis, in Chlamydomonas reinhardtii. Heterologous complementation of Synechocystis sp. PCC 6803 pgsA mutant by CrPGP1 and CrPGP2 rescued the PG-dependent growth phenotype, but the PG level and its fatty acid composition were not fully rescued in the complemented strains. As well, oxygen evolution activity was not fully recovered, although electron transport activity of photosystem II was restored to the wild-type level. Gene expression study of CrPGP1 and CrPGP2 in nutrient-starved C. reinhardtii showed differential response to phosphorus and nitrogen deficiency. Taken together, these results highlight the distinct and overlapping function of PGPS in cyanobacteria and eukaryotic algae.

Enzymatic measurement of phosphatidylglycerol and cardiolipin in cultured cells and mitochondria.

Phosphatidylglycerol (PG) and cardiolipin (CL) are synthesized in mitochondria and regulate numerous biological functions. In this study, a novel fluorometric method was developed for measuring PG and CL using combinations of specific enzymes and Amplex Red. This assay quantified the sum of PG and CL (PG + CL) regardless of the species of fatty acyl chain. The calibration curve for PG + CL measurement was linear, and the detection limit was 1 μM (10 pmol in the reaction mixture). This new method was applied to the determinations of PG + CL content in HEK293 cells and CL content in purified mitochondria, because the mitochondrial content of PG is negligible compared with that of CL. We demonstrated that the PG+CL content was greater at low cell density than at high cell density. The overexpression of phosphatidylglycerophosphate synthase 1 (PGS1) increased the cellular contents of PG + CL and phosphatidylcholine (PC), and reduced that of phosphatidic acid. PGS1 overexpression also elevated the mitochondrial contents of CL and PC, but had no effect on the number of mitochondria per cell. In addition to the enzymatic measurements of other phospholipids, this simple, sensitive and high-throughput assay for measuring PG + CL can be used to understand cellular, physiological and pathological processes.

Phosphatidylglycerol biosynthesis is required for the development of embryos and normal membrane structures of chloroplasts and mitochondria in Arabidopsis

Phosphatidylglycerophosphate (PGP) synthase, encoded by PGP1 and PGP2 in Arabidopsis, catalyzes a committed step in the biosynthesis of phosphatidylglycerol (PG). In this study, we isolated a pgp1pgp2 double mutant of Arabidopsis to study the function of PG. In this mutant, embryo development was delayed and the majority of seeds did not germinate. Thylakoid membranes did not develop in plastids, mitochondrial membrane structures were abnormal in the mutant embryos, and radiolabeling of phospholipids showed that radioactivity was not significantly incorporated into PG. These results demonstrated that PG biosynthesis is essential for the development of embryos and normal membrane structures of chloroplasts and mitochondria.

Phosphatidylglycerophosphate synthase associates with a mitochondrial inner membrane complex and is essential for growth of Trypanosoma brucei

Maintenance of the lipid composition is important for proper function and homeostasis of the mitochondrion. In Trypanosoma brucei, the enzymes involved in the biosynthesis of the mitochondrial phospholipid, phosphatidylglycerol (PG), have not been studied experimentally. We now report the characterization of T. brucei phosphatidylglycerophosphate synthase (TbPgps), the rate-limiting enzyme in PG formation, which was identified based on its homology to other eukaryotic Pgps. Lipid quantification and metabolic labelling experiments show that TbPgps gene knock-down results in loss of PG and a reduction of another mitochondria-specific phospholipid, cardiolipin. Using immunohistochemistry and immunoblotting of digitonin-isolated mitochondria, we show that TbPgps localizes to the mitochondrion. Moreover, reduced TbPgps expression in T. brucei procyclic forms leads to alterations in mitochondrial morphology, reduction in the amounts of respiratory complexes III and IV and, ultimately, parasite death. Using native polyacrylamide gel electrophoresis we demonstrate for the first time in a eukaryotic organism that TbPgps is a component of a 720 kDa protein complex, co-migrating with T. brucei cardiolipin synthase and cytochrome c1, a protein of respiratory complex III.

Deficiency of Cardiolipin Synthase Causes Abnormal Mitochondrial Function and Morphology in Germ Cells of Caenorhabditis elegans

Cardiolipin (CL) is a major membrane phospholipid specifically localized in mitochondria. At the cellular level, CL has been shown to have a role in mitochondrial energy production, mitochondrial membrane dynamics, and the triggering of apoptosis. However, the in vivo role of CL in multicellular organisms is largely unknown. In this study, by analyzing deletion mutants of a CL synthase gene (crls-1) in Caenorhabditis elegans, we demonstrated that CL depletion selectively caused abnormal mitochondrial function and morphology in germ cells but not in somatic cell types such as muscle cells. crls-1 mutants reached adulthood but were sterile with reduced germ cell proliferation and impaired oogenesis. In the gonad of crls-1 mutants, mitochondrial membrane potential was significantly decreased, and the structure of the mitochondrial cristae was disrupted. Contrary to the abnormalities in the gonad, somatic tissues in crls-1 mutants appeared normal with respect to cell proliferation, mitochondrial function, and mitochondrial morphology. Increased susceptibility to CL depletion in germ cells was also observed in mutants of phosphatidylglycerophosphate synthase, an enzyme responsible for producing phosphatidylglycerol, a precursor phospholipid of CL. We propose that the contribution of CL to mitochondrial function and morphology is different among the cell types in C. elegans.

Genetic Evidence for Functional Interaction of the Escherichia coli Signal Recognition Particle Receptor with Acidic Lipids in Vivo

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.

Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control

p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC)-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Disruption of cardiolipin biosynthesis by phosphatidylglycero-phosphate synthase (PGS) or CDP-diacylglycerol synthase 2 (CDS-2) short hairpin RNA (shRNA) transfection eliminated the MMC-induced translocation of mitochondrial p53. The elimination of mitochondrial p53 translocation also reduced Bcl-xL and Bcl-2 mitochondrial distribution. In HEK 293T models with saturated p53 expression, the mitochondrial partition of p53, Bcl-xL, and Bcl-2 obviously decreased in their PGS shRNA- or CDS-2 shRNA-expressing stable clones. In p53-null H1299 models, both the mitochondrial partitions of Bcl-xL and Bcl-2 were strongly reduced in relation to the HEK 293T models. The Bcl-xL mitochondrial partition was elevated in H1299 models expressing pCEP4-p53wt suggesting the direct carrier role of p53 in transporting Bcl-xL to the mitochondria. We also found that the cytosolic pool of Bcl-xL and Bcl-2 remained unaffected in the low-dose MMC treatment but decreased in the high-dose MMC treatment. The cytosolic pool of Bcl-2 and Bcl-xL directly regulated their amounts in p53-dependent mitochondrial distribution. In the low-dose MMC treatment, the increased mitochondrial p53, Bcl-xL, and Bcl-2 could attenuate apoptosis. However, in the high-dose MMC treatment, only the p53 translocated to the mitochondria and resulted in apoptosis progression. On the basis of this study, we thought mitochondrial p53 might regulate apoptosis in a biphasic manner.

Induction of extracytoplasmic function sigma factors in Bacillus subtilis cells with membranes of reduced phosphatidylglycerol content

The Bacillus subtilis gene pgsA, which codes for the phosphatidylglycerophosphate synthase that catalyzes the committed step for the synthesis of phosphatidylglycerol (PG), is essential since Pspac-pgsA cells require IPTG for growth. Removal of the inducer caused a dramatic decrease of PG content in the membranes of cells and retarded growth. At 60 min and 120 min after removal, it was reduced to 14.1% and 8.9% of total lipid, respectively, from an initial content of 28.1%. We conjectured that the activity of some extracytoplasmic function (ECF) sigma factors, most of which are caught and regulated directly by cognate transmembrane anti-sigma factors, are affected by altered lipid composition of the membranes. Induction of the activities of ECF sigma factors (sigma(M) and sigma(V)) was observed after removal of IPTG, though that of sigma(V) was small. But other ECF sigma factors (sigma(W), sigma(X), sigma(Y), sigma(YlaC) and sigma(Z)) and the general stress sigmas sigma(B) and sigma(I) were not induced. Especially sigma(M) was activated strongly with the reduction of PG content and sustained a high level of activity, in contrast to the transient activation in PG normal cells after exposure to high salinity. This study demonstrates a new relationship between the alterations of lipid composition in the membranes and the activation of ECF sigma factors.

De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis.

Identification and functional characterization of hCLS1, a human cardiolipin synthase localized in mitochondria

In eukaryotic cells, CLS (cardiolipin synthase) is involved in the final step of cardiolipin synthesis by catalysing the transfer of a phosphatidyl residue from CDP-DAG (diacylglycerol) to PG (phosphatidylglycerol). Despite an important role of cardiolipin in regulating mitochondrial function, a gene encoding the mammalian CLS has not been identified so far. We report in the present study the identification and characterization of a human cDNA encoding the first mammalian CLS [hCLS1 (human CLS1)]. The predicted hCLS1 peptide sequence shares significant homology with the yeast and plant CLS proteins. The recombinant hCLS1 enzyme expressed in COS-7 cells catalysed efficiently the synthesis of cardiolipin in vitro using CDP-DAG and PG as substrates. Furthermore, overexpression of hCLS1 cDNA in COS-7 cells resulted in a significant increase in cardiolipin synthesis in intact COS-7 cells without any significant effects on the activity of the endogenous phosphatidylglycerophosphate synthase of the transfected COS-7 cells. Immunohistochemical analysis demonstrated that the recombinant hCLS1 protein was localized to the mitochondria when transiently expressed in COS-7 cells, which was further corroborated by results from subcellular fractionation analyses of the recombinant hCLS1 protein. Northern-blot analysis showed that the hCLS1 gene was predominantly expressed in tissues that require high levels of mitochondrial activities for energy metabolism, with the highest expression in skeletal and cardiac muscles. High levels of hCLS1 expression were also detected in liver, pancreas, kidney and small intestine, implying a functional role of hCLS1 in these tissues.

Ampicillin-induced bacteriolysis of Escherichia coli is not affected by reduction in levels of anionic phospholipids.

Anionic phospholipids have been shown to interact with both membrane-associated proteins and integral membrane proteins. The objective of this work was to determine whether bacteriolysis induced by treatment with ampicillin was influenced by the levels of anionic membrane phospholipids in Escherichia coli strain HDL11. The pgsA gene, encoding phosphatidylglycerophosphate synthase, in HDL11 is under the control of lacOP, and the levels of anionic membrane phospholipids are consequently dependent on IPTG. The results indicate that limiting the amounts of phosphatidylglycerol and cardiolipin did not affect the lysis process in both growing and nongrowing bacteria.

Phosphatidylethanolamine Domains and Localization of Phospholipid Synthases in Bacillus subtilis Membranes

Application of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange has recently revealed CL-rich domains in the septal regions and at the poles of the Bacillus subtilis membrane (F. Kawai, M. Shoda, R. Harashima, Y. Sadaie, H. Hara, and K. Matsumoto, J. Bacteriol. 186:1475-1483, 2004). This finding prompted us to examine the localization of another phospholipid, phosphatidylethanolamine (PE), with the cyclic peptide probe, Ro09-0198 (Ro), that binds specifically to PE. Treatment with biotinylated Ro followed by tetramethyl rhodamine-conjugated streptavidin revealed that PE is localized in the septal membranes of vegetative cells and in the membranes of the polar septum and the engulfment membranes of sporulating cells. When the mutant cells of the strains SDB01 (psd1::neo) and SDB02 (pssA10::spc), which both lack PE, were examined under the same conditions, no fluorescence was observed. The localization of the fluorescence thus evidently reflected the localization of PE-rich domains in the septal membranes. Similar PE-rich domains were observed in the septal regions of the cells of many Bacillus species. In Escherichia coli cells, however, no PE-rich domains were found. Green fluorescent protein fusions to the enzymes that catalyze the committed steps in PE synthesis, phosphatidylserine synthase, and in CL synthesis, CL synthase and phosphatidylglycerophosphate synthase, were localized mainly in the septal membranes in B. subtilis cells. The majority of the lipid synthases were also localized in the septal membranes; this includes 1-acyl-glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, diacylglycerol kinase, glucolipid synthase, and lysylphosphatidylglycerol synthase. These results suggest that phospholipids are produced mostly in the septal membranes and that CL and PE are kept from diffusing out to lateral ones.

The Phosphatidylglycerol/Cardiolipin Biosynthetic Pathway Is Required for the Activation of Inositol Phosphosphingolipid Phospholipase C, Isc1p, during Growth of Saccharomyces cerevisiae

Inositolsphingolipid phospholipase C (Isc1p) is the Saccharomyces cerevisiae member of the extended family of neutral sphingomyelinases that regulates the generation of bioactive ceramides. Recently, we reported that Isc1p is post-translationally activated in the post-diauxic phase of growth and that it localizes to mitochondria (Vaena de Avalos, S., Okamoto, Y., and Hannun, Y. A. (2004) J. Biol. Chem. 279, 11537-11545). In this study the in vivo mechanisms of activation and function of Isc1p were investigated. Deletion of ISC1 resulted in markedly lower growth in non-fermentable carbon sources. Interestingly, the growth defect of isc1Delta strains resembled that of pgs1Delta strains, lacking the committed step in the synthesis of phosphatidylglycerol (PG) and cardiolipin (CL), which were shown to activate Isc1p in vitro. Therefore, the role of Pgs1p in activation of Isc1p in vivo was investigated. The results showed that in the pgs1Delta strain, the growth-dependent activation of Isc1p was impaired as was the ISC1-dependent increase in the levels of phytoceramide during the post-diauxic phase, demonstrating that the activation of Isc1p in vivo is dependent on PGS1 and on the mitochondrial phospholipids PG/CL. Mechanistically, loss of Isc1p resulted in lower levels of mitochondrial cytochrome c oxidase subunits cox3p and cox4p, previously established targets of both PG and CL (Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W. (2001) J. Biol. Chem. 276, 25262-25272), thus suggesting that Isc1p mediates at least some functions downstream of PG/CL. This study provides the first evidence for the mechanism of in vivo activation and function of Isc1p. A model with endogenous PG/CL as the in vivo activator of Isc1p is proposed.