Biosynthesis,remodeling and turnover of mitochondrial cardiolipin

Among mitochondrial lipids, cardiolipin occupies a unique place. It is the only phospholipid that is specific to mitochondria and although it is merely a minor component, accounting for 10-20% of the total phospholipid content, cardiolipin plays an important role in the molecular organization, and thus the function of the cristae. This review covers the formation of cardiolipin, a phospholipid dimer containing two phosphatidyl residues, and its assembly into mitochondrial membranes. While a large body of literature exists on this topic, the review focuses on papers that appeared in the past three years. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

细菌中I型毒素蛋白与细胞膜相互作用机制的研究进展

I型毒素-抗毒素（TA）系统在细菌基因组中广泛存在,在细菌的生长、生存中发挥多种生物学功能,包括抗菌、红细胞毒性、促进持留菌形成、抑制细菌生长或导致细菌休眠等。绝大部分I型毒素蛋白以细胞膜作为靶标,目前已知的一种作用机制是在细胞膜上形成孔洞结构,造成膜电位的下降或细胞膜的破坏,从而抑制ATP的合成或导致细菌死亡;另一种可能的作用机制是毒素蛋白作用在细胞膜上,改变细胞的形状,导致细胞进入休眠状态。I型毒素蛋白-细胞膜作用机制的复杂性和生物功能的多样性远超预期。因此,解析I型毒素蛋白在不同细胞膜中的组装机制及其所形成结构特征就变得非常重要,这也是揭示其结构-功能关系的关键。本文通过综述已报道的I型TA系统的结构特征与生物学功能,结合对其跨膜结构域的预测,探讨了其可能在细胞膜中形成的不同结构及其对功能的影响,分析了影响作用机制的关键因素。这些研究既给耐药细菌的治疗带来机遇,又为新型抗菌药物的研发带来思路。     

心磷脂重构与心血管疾病

心磷脂（cardiolipin, CL）是线粒体内膜的特征性磷脂,参与线粒体嵴的形成。心磷脂在线粒体内的合成伴随着特殊的分子重构过程,从而使其自身的4条酰基链形成特定的组成,以发挥其特殊的生理功能。研究发现,心磷脂重构对维持线粒体的形态及功能至关重要,其重构异常是大多数心血管疾病（cardiovascular disease, CVD）共有的病理现象,相应的分子机制研究得到了广泛关注。本文主要对心磷脂的理化特性及其生物合成途径,以及心磷脂重构在巴氏综合征（Barth syndrome, BTHS）、糖尿病心肌病（diabetic cardiomyopathy, DCM）以及心力衰竭（heart failure, HF）等心血管疾病的病理生理过程研究中的进展进行综述,以期为与心磷脂重构相关的心血管疾病的病理生理基础研究和药物干预的分子机制研究提供参考。     

Molecular symmetry in mitochondrial cardiolipins

Cardiolipin is a unique mitochondrial phospholipid with an atypical fatty acid profile, but the significance of its acyl specificity has not been understood. We explored the enormous combinatorial diversity among cardiolipin species, which results from the presence of four fatty acids in each molecule, by integrated use of high-performance liquid chromatography, mass spectrometry, diacylglycerol species analysis, fatty acid analysis, and selective cleavage of fatty acids by phospholipase A2. The most abundant cardiolipin species from various organisms and tissues (human heart, human lymphoblasts, rat liver, Drosophila, sea urchin sperm, yeast, mung bean hypocotyls) contained only one or two types of fatty acids, which generated a high degree of structural uniformity and molecular symmetry. However, an exception was found in patients with Barth syndrome, in whom an acyltransferase deficiency led to loss of acyl selectivity and formation of multiple molecular species. These results suggest that restriction of the number of fatty acid species, rather than the selection of a particular kind of fatty acid, is the common theme of eukaryotic cardiolipins. This limits the structural diversity of the cardiolipin species and creates molecular symmetry with implications for the stereochemistry of cardiolipin.     

Formation of molecular species of mitochondrial cardiolipin. 1. A novel transacylation mechanism to shuttle fatty acids between sn-1 and sn-2 positions of multiple phospholipid species

Mitochondrial cardiolipin undergoes extensive remodeling of its acyl groups to generate uniformly substituted species, such as tetralinoleoyl-cardiolipin, but the mechanism of this remodeling has not been elucidated, except for the fact that it requires tafazzin. Here we show that purified recombinant Drosophila tafazzin exchanges acyl groups between cardiolipin and phosphatidylcholine by a combination of forward and reverse transacylations. The acyl exchange is possible in the absence of phospholipase A₂ because it requires only trace amounts of lysophospholipids. We show that purified tafazzin reacts with various phospholipid classes and with various acyl groups both in sn-1 and sn-2 position. Expression studies in Sf9 insect cells suggest that the effect of tafazzin on cardiolipin species is dependent on the cellular environment and not on enzymatic substrate specificity. Our data demonstrate that tafazzin catalyzes general acyl exchange between phospholipids, which raises the question whether pattern formation in cardiolipin is the result of the equilibrium distribution of acyl groups between multiple phospholipid species.     

Model membrane morphology and crosslinking of oxidized lipids with proteins

Model membranes composed of thion-phosphatidylcholine, cardiolipin, and cytochrome c have been studied by 31P NMR, polyacrylamide gel electrophoresis, gel filtration, fluorescence, and freeze-fracturing. Covalent binding of oxidized phospholipids to cytochrome c was shown to result in the formation of high-molecular-weight oligomeric complexes via Schiff base formation between a protein molecule and aldehydes produced upon peroxidation of phospholipids. The initial steps of the protein oligomerization lead to the appearance of intramembranous particles (IMPs) of various size and distribution on freeze-fractured faces of these model membranes. In the final phase of the crosslinking between cytochrome c and oxidized products of cardiolipin there is a breakdown of membrane vesicles and formation of globular lipoprotein complexes which are seen as globular particles. It is believed that the covalent linking between the products of phospholipid peroxidation and membrane proteins causes the oligomerization of membrane proteins and structural alteration in the hydrophobic region of other models also and, perhaps, in biological membranes.     

The role of cardiolipin in the structural organization of mitochondrial membranes

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

Cellulosomes: microbial nanomachines that display plasticity in quaternary structure

The assembly of proteins that display complementary activities into supramolecular intra- and extracellular complexes is central to cellular function. One such nanomachine of considerable biological and industrial significance is the plant cell wall degrading apparatus of anaerobic bacteria termed the cellulosome. The Clostridium thermocellum cellulosome assembles through the interaction of a type I dockerin module in the catalytic entities with one of several type I cohesin modules in the non-catalytic scaffolding protein. Recent structural studies have provided the molecular details of how dockerin-cohesin interactions mediate both cellulosome assembly and the retention of the protein complex on the bacterial cell surface. The type I dockerin, which displays near-perfect sequence and structural symmetry, interacts with its cohesin partner through a dual binding mode in which either the N- or C-terminal helix dominate heterodimer formation. The biological significance of this dual binding mode is discussed with respect to the plasticity of the orientation of the catalytic subunits within this supramolecular assembly. The flexibility in the quaternary structure of the cellulosome may reflect the challenges presented by the degradation of a heterogenous recalcitrant insoluble substrate by an intricate macromolecular complex, in which the essential synergy between the catalytic subunits is a key feature of cellulosome function.     

The physicochemical properties of cardiolipin bilayers and cardiolipin-containing lipid membranes

In this review article, we summarize the current state of biophysical knowledge concerning the phase behavior and organization of cardiolipin (CL) and CL-containing phospholipid bilayer model membranes. We first briefly consider the occurrence and distribution of CL in biological membranes and its probable biological functions therein. We next consider the unique chemical structure of the CL molecule and how this structure may determine its distinctive physical properties. We then consider in some detail the thermotropic phase behavior and organization of CL and CL-containing lipid model membranes as revealed by a variety of biophysical techniques. We also attempt to relate the chemical properties of CL to its function in the biological membranes in which it occurs. Finally, we point out the requirement for additional biophysical studies of both lipid model and biological membranes in order to increase our currently limited understanding of the relationship between CL structure and physical properties and CL function in biological membranes.     

Cardiolipin content in mitochondria from cultured skin fibroblasts harboring mutations in the mitochondrial <Emphasis Type="Italic">ATP6</Emphasis> gene

The role of phospholipids in normal assembly and organization of the membrane proteins has been well documented. Cardiolipin, a unique tetra-acyl phospholipid localized in the inner mitochondrial membrane, is implicated in the stability of many inner-membrane protein complexes. Loss of cardiolipin content, alterations in its acyl chain composition and/or cardiolipin peroxidation have been associated with dysfunction in multiple tissues in a variety of pathological conditions. The aim of this study was to analyze the phospholipid composition of the mitochondrial membrane in the four most frequent mutations in the ATP6 gene: L156R, L217R, L156P and L217P but, more importantly, to investigate the possible changes in the cardiolipin profile. Mitochondrial membranes from fibroblasts with mutations at codon 217 of the ATP6 gene, showed a different cardiolipin content compared to controls. Conversely, results similar to controls were obtained for mutations at codon 156. These findings may be attributed to differences in the biosynthesis and remodeling of cardiolipin at the level of the inner mitochondrial transmembrane related to some mutations of the ATP6 gene.     

Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.     

Cardiolipin binding in bacterial respiratory complexes: Structural and functional implications

The structural and functional integrity of biological membranes is vital to life. The interplay of lipids and membrane proteins is crucial for numerous fundamental processes ranging from respiration, photosynthesis, signal transduction, solute transport to motility. Evidence is accumulating that specific lipids play important roles in membrane proteins, but how specific lipids interact with and enable membrane proteins to achieve their full functionality remains unclear. X-ray structures of membrane proteins have revealed tight and specific binding of lipids. For instance, cardiolipin, an anionic phospholipid, has been found to be associated to a number of eukaryotic and prokaryotic respiratory complexes. Moreover, polar and septal accumulation of cardiolipin in a number of prokaryotes may ensure proper spatial segregation and/or activity of proteins. In this review, we describe current knowledge of the functions associated with cardiolipin binding to respiratory complexes in prokaryotes as a frame to discuss how specific lipid binding may tune their reactivity towards quinone and participate to supercomplex formation of both aerobic and anaerobic respiratory chains. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Murine diet-induced obesity remodels cardiac and liver mitochondrial phospholipid acyl chains with differential effects on respiratory enzyme activity

Cardiac phospholipids, notably cardiolipin, undergo acyl chain remodeling and/or loss of content in aging and cardiovascular diseases, which is postulated to mechanistically impair mitochondrial function. Less is known about how diet-induced obesity influences cardiac phospholipid acyl chain composition and thus mitochondrial responses. Here we first tested if a high fat diet remodeled murine cardiac mitochondrial phospholipid acyl chain composition and consequently disrupted membrane packing, supercomplex formation and respiratory enzyme activity. Mass spectrometry analyses revealed that mice consuming a high fat diet displayed 0.8–3.3 fold changes in cardiac acyl chain remodeling of cardiolipin, phosphatidylcholine, and phosphatidylethanolamine. Biophysical analysis of monolayers constructed from mitochondrial phospholipids of obese mice showed impairment in the packing properties of the membrane compared to lean mice. However, the high fat diet, relative to the lean controls, had no influence on cardiac mitochondrial supercomplex formation, respiratory enzyme activity, and even respiration. To determine if the effects were tissue specific, we subsequently conducted select studies with liver tissue. Compared to the control diet, the high fat diet remodeled liver mitochondrial phospholipid acyl chain composition by 0.6–5.3-fold with notable increases in n-6 and n-3 polyunsaturation. The remodeling in the liver was accompanied by diminished complex I to III respiratory enzyme activity by 3.5-fold. Finally, qRT-PCR analyses demonstrated an upregulation of liver mRNA levels of tafazzin, which contributes to cardiolipin remodeling. Altogether, these results demonstrate that diet-induced obesity remodels acyl chains in the mitochondrial phospholipidome and exerts tissue specific impairments of respiratory enzyme activity.     

Known unknowns of cardiolipin signaling: The best is yet to come

Since its discovery 75 years ago, a wealth of knowledge has accumulated on the role of cardiolipin, the hallmark phospholipid of mitochondria, in bioenergetics and particularly on the structural organization of the inner mitochondrial membrane. A surge of interest in this anionic doubly-charged tetra-acylated lipid found in both prokaryotes and mitochondria has emerged based on its newly discovered signaling functions. Cardiolipin displays organ, tissue, cellular and transmembrane distribution asymmetries. A collapse of the membrane asymmetry represents a pro-mitophageal mechanism whereby externalized cardiolipin acts as an “eat-me” signal. Oxidation of cardiolipin's polyunsaturated acyl chains - catalyzed by cardiolipin complexes with cytochrome c. - is a pro-apoptotic signal. The messaging functions of myriads of cardiolipin species and their oxidation products are now being recognized as important intracellular and extracellular signals for innate and adaptive immune systems. This newly developing field of research exploring cardiolipin signaling is the main subject of this review. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

A novel function of the human CLS1 in phosphatidylglycerol synthesis and remodeling

Phosphatidylglycerol (PG) is a precursor for the biosynthesis of cardiolipin and a signaling molecule required for various cellular functions. PG is subjected to remodeling subsequent to its de novo biosynthesis in mitochondria to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. Yet, a gene encoding a mitochondrial LPG acyltransferase has not been identified. In this report, we identified a novel function of the human cardiolipin synthase (hCLS1) in regulating PG remodeling. In addition to the reported cardiolipin synthase activity, the recombinant hCLS1 protein expressed in COS-7 cells and Sf-9 insect cells exhibited a strong acyl-CoA-dependent LPG acyltransferase activity, which was further confirmed by purified hCLS1 protein overexpressed in Sf-9 cells. The recombinant hCLS1 displayed an acyl selectivity profile in the order of in the order of C18:1 > C18:2 > C18:0 > C16:0, which is similar to that of hCLS1 toward PGs in cardiolipin synthesis, suggesting that the PG remodeling by hCLS1 is an intrinsic property of the enzyme. In contrast, no significant acyltransferase activity was detected from the recombinant hCLS1 enzyme toward lysocardiolipin which shares a similar structure with LPG. In support of a key function of hCLS1 in PG remodeling, overexpression of hCLS1 in COS-7 cells significantly increased PG biosynthesis concurrent with elevated levels of cardiolipin without any significant effects on the biosynthesis of other phospholipids. These results demonstrate for the first time that hCLS1 catalyzes two consecutive steps in cardiolipin biosynthesis by acylating LPG to PG and then converting PG to cardiolipin.     

Cardiolipin-cholesterol interactions in the liquid-crystalline phase: a steady-state and time-resolved fluorescence anisotropy study with cis- and trans-parinaric acids as probes

The potency of cholesterol to affect the acyl chain order and dynamics of cardiolipin membranes in the liquid-crystalline state was monitored by steady-state and time-resolved fluorescence anisotropy as well as excited-state lifetime measurements with cis- and trans-parinaric acids as probes. Up to a cholesterol mole fraction (mean chl) of congruent to 0.20, no measurable effect on any of the fluorescence parameters of either probe in cardiolipin bilayers was evidenced. This was in striking contrast to the situation in dioleoylphosphatidylcholine (DOPC), for which a cholesterol mole fraction of 0.20 corresponded to the half-maximal effect on the fluorescence parameters, reflecting the classical ordering effect of cholesterol observed in lecithin systems in the liquid-crystalline phase. Whereas in DOPC bilayers this order effect plateaued at mean chl = 0.50, in cardiolipins the increase in acyl chain order was observable up to a mole fraction as high as 0.80. This indicated that cardiolipins were able to incorporate about 4 mol of cholesterol/mol of cardiolipin (i.e., 1 mol of cholesterol per fatty acyl chain). Besides, 31P NMR spectra of multilamellar liposomes obtained from pure cardiolipins and cardiolipin--cholesterol mixtures evidenced a line shape characteristic of lamellar structures. These results clearly indicate that the presence of high levels of cardiolipins in inner mitochondrial membrane does not impede cholesterol uptake by these membranes. However, the absence of an effect of cholesterol in the physiological range of the cholesterol mole fraction (congruent to 0.20) would signify weaker sterol-cardiolipin interactions than with lecithins and in turn would explain the relative dearth of cholesterol in these membranes.     

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and (19)F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The (19)F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.     

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.     

Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes

Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I.