Phosphatidylglycerolphosphate Synthase Research Articles

THYLAKOID FORMATION 1 interacts with FLOWERING LOCUS T and modulates temperature-responsive flowering inArabidopsis.

The intracellular localization of the florigen FLOWERING LOCUS T (FT) is important for its long-distance transport toward the shoot apical meristem. However, the mechanisms regulating the FT localization remain poorly understood. Here, we discovered that in Arabidopsis thaliana, the chloroplast-localized protein THYLAKOID FORMATION 1 (THF1) physically interacts with FT, sequestering FT in the outer chloroplast envelope. Loss of THF1 function led to temperature-insensitive flowering, resulting in early flowering, especially under low ambient temperatures. THF1 mainly acts in the leaf vasculature and shoot apex to prevent flowering. Mutation of CONSTANS or FT completely suppressed the early flowering of thf1-1 mutants. FT and THF1 interact via their anion binding pocket and coiled-coil domain (CCD), respectively. Deletion of the CCD in THF1 by gene editing caused temperature-insensitive early flowering similar to that observed in the thf1-1 mutant. FT levels in the outer chloroplast envelope decreased in the thf1-1 mutant, suggesting that THF1 is important for sequestering FT. Furthermore, THF1 protein levels decreased in seedlings grown at high ambient temperature, suggesting an explanation for its role in plant responses to ambient temperature. A thf1-1 phosphatidylglycerolphosphate synthase 1 (pgp1) double mutant exhibited additive acceleration of flowering at 23 and 16°C, compared to the single mutants, indicating that THF1 and phosphatidylglycerol (PG) act as independent but synergistic regulators of temperature-responsive flowering. Collectively, our results provide an understanding of the genetic pathway involving THF1 and its role in temperature-responsive flowering and reveal a previously unappreciated additive interplay between THF1 and PG in temperature-responsive flowering.

Origin and diversification of the cardiolipin biosynthetic pathway in the Eukarya domain.

Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are important anionic phospholipids widely distributed throughout all domains of life. They have key roles in several cellular processes by shaping membranes and modulating the activity of the proteins inserted into those membranes. They are synthesized by two main pathways, the so-called eukaryotic pathway, exclusively found in mitochondria, and the prokaryotic pathway, present in most bacteria and archaea. In the prokaryotic pathway, the first and the third reactions are catalyzed by phosphatidylglycerol phosphate synthase (Pgps) belonging to the transferase family and cardiolipin synthase (Cls) belonging to the hydrolase family, while in the eukaryotic pathway, those same reactions are catalyzed by unrelated homonymous enzymes: Pgps of the hydrolase family and Cls of the transferase family. Because of the enzymatic arrangement found in both pathways, it seems that the eukaryotic pathway evolved by convergence to the prokaryotic pathway. However, since mitochondria evolved from a bacterial endosymbiont, it would suggest that the eukaryotic pathway arose from the prokaryotic pathway. In this review, it is proposed that the eukaryote pathway evolved directly from a prokaryotic pathway by the neofunctionalization of the bacterial enzymes. Moreover, after the eukaryotic radiation, this pathway was reshaped by horizontal gene transfers or subsequent endosymbiotic processes.

Myostatin deficiency is associated with lipidomic abnormalities in skeletal muscles

Myostatin (Mstn) deficiency leads to skeletal muscle overgrowth and Mstn inhibition is considered as a promising treatment for muscle-wasting disorders. Mstn gene deletion in mice also causes metabolic changes with decreased mitochondria content, disturbance in mitochondrial respiratory function and increased muscle fatigability. However the impact of MSTN deficiency on these metabolic changes is not fully elucidated. Here, we hypothesized that lack of MSTN will alter skeletal muscle membrane lipid composition in relation with pronounced alterations in muscle function and metabolism. Indeed, phospholipids and in particular cardiolipin mostly present in the inner mitochondrial membrane, play a crucial role in mitochondria function and oxidative phosphorylation process. We observed that Mstn KO muscle had reduced fat membrane transporter levels (FAT/CD36, FABP3, FATP1 and FATP4) associated with decreased lipid oxidative pathway (citrate synthase and β-HAD activities) and impaired lipogenesis (decreased triglyceride and free fatty acid content), indicating a role of mstn in muscle lipid metabolism. We further analyzed phospholipid classes and fatty acid composition by chromatographic methods in muscle and mitochondrial membranes. Mstn KO mice showed increased levels of saturated and polyunsaturated fatty acids at the expense of monounsaturated fatty acids. We also demonstrated, in this phenotype, a reduction in cardiolipin proportion in mitochondrial membrane versus the proportion of others phospholipids, in relation with a decrease in the expression of phosphatidylglycerolphosphate synthase and cardiolipin synthase, enzymes involved in cardiolipin synthesis. These data illustrate the importance of lipids as a link by which MSTN deficiency can impact mitochondrial bioenergetics in skeletal muscle.

RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell’s rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.

Regulation of hepatic cardiolipin metabolism by TNFα: Implication in cancer cachexia

Cardiolipin (CL) content accumulation leads to an increase in energy wasting in liver mitochondria in a rat model of cancer cachexia in which tumor necrosis factor alpha (TNFα) is highly expressed. In this study we investigated the mechanisms involved in liver mitochondria CL accumulation in cancer cachexia and examined if TNFα was involved in this process leading to mitochondrial bioenergetics alterations. We studied gene, protein expression and activity of the main enzymes involved in CL metabolism in liver mitochondria from a rat model of cancer cachexia and in HepaRG hepatocyte-like cells exposed to 20ng/ml of TNFα for 12h. Phosphatidylglycerolphosphate synthase (PGPS) gene expression was increased 2.3-fold (p<0.02) and cardiolipin synthase (CLS) activity decreased 44% (p<0.03) in cachectic rat livers compared to controls. CL remodeling enzymes monolysocardiolipin acyltransferase (MLCL AT-1) activity and tafazzin (TAZ) gene expression were increased 30% (p<0.01) and 50% (p<0.02), respectively, in cachectic rat livers compared to controls. Incubation of hepatocytes with TNFα increased CL content 15% (p<0.05), mitochondrial oxygen consumption 33% (p<0.05), PGPS gene expression 44% (p<0.05) and MLCL AT-1 activity 20% (p<0.05) compared to controls. These above findings strongly suggest that in cancer cachexia, TNFα induces a higher energy wasting in liver mitochondria by increasing CL content via upregulation of PGPS expression.

The essential roles of cytidine diphosphate‐diacylglycerol synthase in bloodstream form Trypanosoma brucei

Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host in several fundamental ways. This has lead to the validation of a plethora of novel drug targets, giving hope of novel chemical intervention against this neglected disease. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate for several pathways in both prokaryotes and eukaryotes, being produced by CDP-DAG synthase (CDS). However, nothing is known about the single T. brucei CDS gene (Tb927.7.220/EC 2.7.7.41) or its activity. In this study we show TbCDS is functional by complementation of a non-viable yeast CDS null strain and that it is essential in the bloodstream form of the parasite via a conditional knockout. The TbCDS conditional knockout showed morphological changes including a cell-cycle arrest due in part to kinetoplast segregation defects. Biochemical phenotyping of TbCDS conditional knockout showed drastically altered lipid metabolism where reducing levels of phosphatidylinositol detrimentally impacted on glycoylphosphatidylinositol biosynthesis. These studies also suggest that phosphatidylglycerol synthesized via the phosphatidylglycerol-phosphate synthase is not synthesized from CDP-DAG, as was previously thought. TbCDS was shown to localized the ER and Golgi, probably to provide CDP-DAG for the phosphatidylinositol synthases.

EXPRESSION OF CARDIOLIPIN BIOSYNTHESIS AND REMODELING ENZYMES IN ADULT HEART FAILURE

BackgroundCardiolipin (CL) is a unique phospholipid that is an essential component of the inner‐mitochondrial membrane critical for normal energy metabolism. Biosynthesis of CL occurs via an enzymatic pathway or through remodeling of existing CLs. The content of total CL and (18:2) 4CL (tetralinoleic form, normally predominant in the heart) are lower in ventricular tissue from adults with heart failure secondary to idiopathic dilated cardiomyopathy (IDC).PurposeThe aim of this study was to determine the expression levels of CL biosynthetic and remodeling enzymes associated with CL content changes in IDC.MethodsmRNA was isolated from the left ventricle (LV) of adult IDC patients at transplant (n= 27; mean age = 51±14) and non‐failing control LV from donor hearts (n= 15, mean age = 43±8). RT‐PCR was used to measure expression of CL biosynthesis and remodeling enzymes.ResultsExpression of biosynthetic enzymes, CDP diacylglycerol synthase 2 and phosphatidylglycerolphosphate synthase, were both 33% lower in IDC LV (p&lt;0.05). Remodeling enzymes, monolyso‐CL acyltransferase and tafazzin, were also down‐regulated (66% and 43% respectively, p&lt;0.01) in IDC LV compared to controls.ConclusionsThese results demonstrate that biosynthetic and remodeling CL abnormalities are present in failing IDC hearts and may contribute to mitochondrial CL abnormalities in heart failure. Supported by NIH/NCATS Colorado CTSI Grant Number UL1 TR000154.

Expression of Cardiolipin Biosynthesis and Remodeling Enzymes in Adult Heart Failure

Purpose Cardiolipin (CL) is a unique phospholipid that is an essential component of the inner-mitochondrial membrane (IMM) and is critical to normal energy metabolism. In the heart, CL exists predominantly in the tetralinoleic form, (18:2) 4CL, which has the greatest affinity for the IMM and protection against cellular apoptosis. Biosynthesis of CL occurs by means of a five-step enzymatic pathway. Tetralinoleic CL is formed by the remodeling of existing CL. Total CL content is lower in ventricular tissue from adult humans with idiopathic dilated cardiomyopathy (IDC). Purpose: The aim of this study was to determine which mitochondrial CL biosynthetic and linoleic remodeling enzyme levels are dysregulated in adult IDC. Methods and Materials mRNA was isolated from failing adult left ventricle (LV) of patients with IDC obtained at the time of transplant (n=27; mean age = 51±14) and non-failing control LV from donor hearts not implanted for technical reasons (n=15, mean age = 43±8). RT-PCR was employed to measure gene expression of CL biosynthesis and (18:2) 4CL remodeling enzymes. Results In the biosynthesis pathway we found 37% lower CDP diacylglycerol synthase 2 and 35% lower phosphatidylglycerolphosphate synthase (both P Conclusions These results demonstrate that biosynthetic and remodeling CL abnormalities are present in the IDC adult failing heart and may contribute to mitochondrial CL abnormalities in heart failure. Future investigations will compare enzymatic dysregulation between these adult groups with pediatric IDC samples for potential age-related patterns in dysregulation. Ultimately, we hope to elucidate mechanisms of CL dysregulation and targets for pharmacological therapy.

Absence of anionic phospholipids inKluyveromyces lactiscells is fatal without F1-catalysed ATP hydrolysis

We have shown in previous research that the loss of phosphatidylglycerol and cardiolipin caused by disruption of the PGS1 gene is lethal for the petite-negative yeast Kluyveromyces lactis . This present study demonstrates the role and mechanism of atp2.1 in the suppression of pgs1 lethality in K. lactis cells. Phenotypic characterization has shown that a strain lacking the phosphatidylglycerolphosphate synthase (atp2.1pgs1Δ) possessed a markedly impaired respiratory chain, very low endogenous respiration, and uncoupled mitochondria. As a result the mutant strain was unable to generate a sufficient mitochondrial membrane potential via respiration. The atp2.1 suppressor mutation enabled an increase in the affinity of F(1)-ATPase for ATP in the hydrolytic reaction, resulting in the maintenance of sufficient membrane potential for the biogenesis of mitochondria and survival of cells lacking anionic phospholipid biosynthesis.

Mutation in the β subunit of F1 ATPase allows Kluyveromyces lactis to survive the disruption of the KlPGS1 gene

The petite-negative yeast Kluyveromyces lactis does not tolerate the loss of phosphatidylglycerol (PG). We demonstrate that the lethality of PG loss is suppressed in strains carrying a mutation in the beta subunit of F(1) ATPase (mgi1-1). Phenotypic characterization shows that the strain lacking the phosphatidylglycerolphosphate synthase gene (KlPGS1) is able to grow only on glucose, but significantly more slowly and to substantially lower densities than the parental mgi1-1 strain. In addition, oxygen consumption in the DeltaKlpgs1 strain is <1% of the parental strain. Western blot analysis of mitochondrial membrane proteins shows that the amounts of some proteins are substantially decreased or even not detectable in this mutant. However, overexpression of the KlPGS1 gene under the inducible GAL1 promoter does not restore the ability of DeltaKlpgs1 cells to grow on galactose, indicating the presence of some other mutations and/or deletions in genes involved in oxidative phosphorylation. We also demonstrate that DeltaKlpgs1 cells do not spontaneously lose mtDNA, but are able to survive its loss after ethidium bromide mutagenesis. Deletion of the cardiolipin synthase gene (KlCLS1) in mgi1-1 has only a minimal effect on mitochondrial physiology, and additional experiments show that this deletion is also viable in wild-type K. lactis.

The dynamics of cardiolipin synthesis post-mitochondrial fusion

Alteration in mitochondrial fusion may regulate mitochondrial metabolism. Since the phospholipid cardiolipin (CL) is required for function of the mitochondrial respiratory chain, we examined the dynamics of CL synthesis in growing Hela cells immediately after and 12 h post-fusion. Cells were transiently transfected with Mfn-2, to promote fusion, or Mfn-2 expressing an inactive GTPase for 24 h and de novo CL biosynthesis was examined immediately after or 12 h post-fusion. Western blot analysis confirmed elevated Mfn-2 expression and electron microscopic analysis revealed that Hela cell mitochondrial structure was normal immediately after and 12 h post-fusion. Cells expressing Mfn-2 exhibited reduced CL de novo biosynthesis from [1,3- 3H]glycerol immediately after fusion and this was due to a decrease in phosphatidylglycerol phosphate synthase (PGPS) activity and its mRNA expression. In contrast, 12 h post-mitochondrial fusion cells expressing Mfn-2 exhibited increased CL de novo biosynthesis from [1,3- 3H]glycerol and this was due to an increase in PGPS activity and its mRNA expression. Cells expressing Mfn-2 with an inactive GTPase activity did not exhibit alterations in CL de novo biosynthesis immediately after or 12 h post-fusion. The Mfn-2 mediated alterations in CL de novo biosynthesis were not accompanied by alterations in CL or monolysoCL mass. [1- 14C]Oleate incorporation into CL was elevated at 12 h post-fusion indicating increased CL resynthesis. The reason for the increased CL resynthesis was an increased mRNA expression of tafazzin, a mitochondrial CL resynthesis enzyme. Ceramide-induced expression of PGPS in Hela cells or in CHO cells did not alter expression of Mfn-2 indicating that Mfn-2 expression is independent of altered CL synthesis mediated by elevated PGPS. In addition, Mfn-2 expression was not altered in Hela cells expressing phospholipid scramblase-3 or a disrupted scramblase indicating that proper CL localization within mitochondria is not essential for Mfn-2 expression. The results suggest that immediately post-mitochondrial fusion CL de novo biosynthesis is “slowed down” and then 12 h post-fusion it is “upregulated”. The implications of this are discussed.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

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.

Lipidomic analysis reveals that phosphatidylglycerol and phosphatidylethanolamine are newly generated phospholipids in an early-divergent protozoan, Giardia lamblia

The pathogenic protozoan Giardia lamblia is known to not synthesize membrane lipids de novo. Therefore, it is possible that lipids in the small intestine, where trophozoites colonize, play key roles in regulating the growth and differentiation of this important pathogen. The focus of the current study is to conduct a complete lipidomic analysis and to test the hypothesis that Giardia has some ability to generate new phospholipids (PLs). Using mass spectrometry, now we show that phosphatidylglycerols (PGs) are major PLs followed by phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in non-encysting and encysting trophozoites, as well in cysts. The fatty acids attached to these PLs consist mostly of palmitate, palmitoleate, oleate, and linoleate. Results also indicate that PGs and PEs, unlike PCs, are not present in bovine bile and serum, the major sources of lipids of the culture medium, and that they could therefore be produced by fatty acid and headgroup remodeling reactions, circumventing the synthesis of entirely new PLs via de novo pathways. Genomic and transcriptional analyses show the presence of giardial phosphatidylglycerolphosphate synthase (gpgps) and phosphatidylserine decarboxylase (gpsd) genes, which are expressed throughout the life cycle. Bioinformatic and phylogenetic analyses further indicated that both genes are of prokaryotic origin and that they have undergone duplication in the course of evolution. Our studies suggest that the abundance of PG in Giardia is unique among eukaryotes and that its synthesis thus could serve as a potential target for developing new therapies against this waterborne parasite.

Molecular and phenotypic analysis of mutations causing anionic phospholipid deficiency in closely related yeast species

The pel1 mutation in Saccharomyces cerevisiae and the Cgpgs1Delta mutation in Candida glabrata result in deficiency of mitochondrial phosphatidylglycerolphosphate synthase and lack of two anionic phospholipids, phosphatidylglycerol and cardiolipin. DNA sequence analysis of the PCR-amplified pel1 mutant allele revealed that the pel1 mutation resulted from a single amino-acid substitution (Glu(463)Lys) in the C-terminal part of encoded enzyme. The CgPGS1 gene cloned in a centromeric pFL38 vector functionally complemented the pel1 mutation in S. cerevisiae. Likewise, the ScPGS1 gene cloned in pCgACU5 plasmid fully complemented the Cgpgs1Delta mutation in C. glabrata. This mutation increased the cell surface hydrophobicity and decreased biofilm formation. These results support a close evolutionary relatedness of S. cerevisiae and C. glabrata and point to the relationship between expression of virulence factors and anionic phospholipid deficiency in pathogenic C. glabrata.

Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease.

Regulation of phosphatidylglycerolphosphate synthase in aerobic yeast Kluyveromyces lactis

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase (PGPS) is essential for the viability and multiplication of Kluyveromyces lactis. Regulation of PGPS expression by factors affecting mitochondrial development (C source, growth phase) and general phospholipid biosynthesis was followed. PGS1 mRNA levels were not altered as cells progressed from the exponential to the stationary phase of growth in glucose. PGS1 mRNA abundance was nearly identical in cells growing in a medium with glucose or glycerol as the sole C source during the different growth phases. Regulation of PGS1 expression by exogenous myo-inositol and choline was not mediated at the transcriptional level, the PGPS activity dropped to 70 % after myo-inositol addition.

Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism

The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidyl-glycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1Delta strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism.

RhoGAP mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells

RhoGAP mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells

Functional characterization of the CgPGS1 gene reveals a link between mitochondrial phospholipid homeostasis and drug resistance in Candida glabrata

Cardiolipin and its precursor phosphatidylglycerol are two anionic phospholipids that are essential for the biogenesis of functional mitochondria. To assess their role in mitochondrial and cellular functions in the pathogenic yeast Candida glabrata, a functional characterization of the CgPGS1 gene encoding the phosphatidylglycerolphosphate synthase has been carried out. Transposon insertion mutation in CgPGS1 resulted in the loss of phosphatidylglycerolphosphate synthase activity and in deficiency of both phosphatidylglycerol and cardiolipin. The Cgpgs1 Delta mutant cells displayed reduced amounts of cytochrome b and cytochrome a, and had impaired growth on minimal media containing non-fermentable carbon and energy sources. They did not grow at elevated temperatures and failed to form colonies after induction of mitochondrial DNA deletions. The mutant cells also displayed a decreased susceptibility to fluconazole, ketoconazole, clotrimazole, voriconazole and posaconazole. In the Cgpgs1 Delta mutant, a quantitative real time PCR revealed enhanced mRNA levels for multidrug resistance associated genes such as CgPDR1 encoding transcriptional activator and CgCDR1, CgPDH1 and CgSNQ2 coding for drug efflux transporters. These results indicate that CgPGS1 and anionic phospholipids are required for optimal mitochondrial functions and maintenance of yeast susceptibility to azole antifungals.

StARD13(Dlc-2) RhoGap Mediates Ceramide Activation of Phosphatidylglycerolphosphate Synthase and Drug Response in Chinese Hamster Ovary Cells

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C(2)-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1-2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C(2)-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.