Mitochondria isolated in nearly isotonic KCl buffer: Focus on cardiolipin and organelle morphology

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation.     

SMP30 deficiency in mice causes an accumulation of neutral lipids and phospholipids in the liver and shortens the life span

Senescence marker protein-30 (SMP30) is an androgen-independent factor that decreases with aging. SMP30-deficient (SMP30Y/-) mice are viable and fertile but lower in body weight and shorter in life span than the wild-type. In the electron microscope, hepatocytes from SMP30Y/- but not the wild-type mice at 12 months of age clearly contained many lipid droplets, abnormally enlarged mitochondria with indistinct cristae, and enlarged lysosomes filled with electron-dense bodies. In liver specimens from SMP30Y/- mice, the marked number of lipid droplets visible around the central vein increased notably in size and amount as the animals aged. Biochemical analysis of neutral lipids, total hepatic triglyceride, and cholesterol from SMP30Y/- mice showed approximately 3.6- and 3.3-fold higher levels, respectively, than those from age-matched wild-type mice. Moreover, values for total hepatic phospholipids from SMP30Y/- mice were approximately 3.7-fold higher than those for their wild-type counterparts. By thin-layer chromatography analysis, phosphatidylethanolamine, cardiolipin, phosphatidylcholine, phosphatidylserine, and sphingomyelin accumulations were detected separately in lipid extracts from SMP30Y/- mouse livers and provided results that strongly indicate the profound effect of an SMP30 deficiency on the metabolism of these neutral lipids and phospholipids. Conceivably, this abnormality of lipid metabolism is sufficient to curtail the life span of SMP30-deficient mice.     

Anoxia-reoxygenation-induced cytochrome c and cardiolipin release from rat brain mitochondria

Rat brain mitochondria were successively submitted to anoxia and reoxygenation. The main mitochondrial functions were assessed at different reoxygenation times. Although the respiratory control ratio decreased, the activity for each one of the enzymes participating in the respiratory chain was not affected. However, during reoxygenation, mitochondrial membrane lipoperoxidation quickly increased and was proportional to the decrease seen in membrane fluidity. Under the same conditions, cytochrome c and cardiolipin were released from mitochondria and their rate of release increased with reoxygenation time. The release of cytochrome c and cardiolipin was followed by the collapse of the membrane potential and it was not inhibited by cyclosporin A. Addition of the antioxidant alpha-tocopherol abolished all these reoxygenation-induced changes. These data indicate that, in this model, reoxygenation promotes the uncoupling of respiratory chain, and cytochrome c and cardiolipin releases. These events are not related to the membrane potential collapse but to an oxidative stress.     

The disulphide bonds of an Asian influenza virus neuraminidase

The arrangement of the disulphide bonds in the pronase-released neuraminidase heads of the Asian influenza virus A/Tokyo/3/67 have been examined by cyanogen bromide fragmentation, enzymic digestion and diagonal peptide mapping. There are 9 intrachain disulphide bridges and one interchain bridge which links pairs of monomers at the distal end of the stalk region of the neuraminidase tetramer. The disulphide bond arrangements of the remaining 3 half-cystine residues in the membrane-embedded stalk region of the neuraminidase were not examined.     

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 °C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.     

Cardiolipin remodeling and the function of tafazzin

Cardiolipin, the specific phospholipid of mitochondria, is involved in the biogenesis, the dynamics, and the supramolecular organization of mitochondrial membranes. Cardiolipin acquires a characteristic composition of fatty acids by post-synthetic remodeling, a process that is crucial for cardiolipin homeostasis and function. The remodeling of cardiolipin depends on the activity of tafazzin, a non-specific phospholipid–lysophospholipid transacylase. This review article discusses recent findings that suggest a novel function of tafazzin in mitochondrial membranes. By shuffling fatty acids between molecular species, tafazzin transforms the lipid composition and by doing so supports changes in the membrane conformation, specifically the generation of membrane curvature. Tafazzin activity is critical for the differentiation of cardiomyocytes, in which the characteristic cristae-rich morphology of cardiac mitochondria evolves. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Identification of a Mammalian-type Phosphatidylglycerophosphate Phosphatase in the Eubacterium Rhodopirellula baltica

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.     

Apoptosis-induced changes in mitochondrial lipids

Apoptosis is an active and tightly regulated form of cell death, which can also be considered a stress-induced process of cellular communication. Recent studies reveal that the lipid network within cells is involved in the regulation and propagation of death signalling. Despite the vast growth of our current knowledge on apoptosis, little is known of the specific role played by lipid molecules in the central event of apoptosis—the piercing of mitochondrial membranes. Here we review the information regarding changes in mitochondrial lipids that are associated with apoptosis and discuss whether they may be involved in the permeabilization of mitochondria to release their apoptogenic factors, or just lie downstream of this permeabilization leading to the amplification of caspase activation. We focus on the earliest changes that physiological apoptosis induces in mitochondrial membranes, which may derive from an upstream alteration of phospholipid metabolism that reverberates on the mitochondrial re-modelling of their characteristic lipid, cardiolipin. Hopefully, this review will lead to an increased understanding of the role of mitochondrial lipids in apoptosis and also help revealing new stress sensing mechanisms in cells. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Monte Carlo simulations of tBid association with the mitochondrial outer membrane

Bid, a BH3-only pro-apoptopic member of the BCL-2 protein family, regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 α-helices (H1–H8, respectively) and is soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid by caspase 8 in apoptosis yields activated “tBid” (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. The release of cytochrome c from mitochondria to the cytosol constitutes a critical control point in apoptosis that is regulated by interaction of tBid protein with mitochondrial membrane. tBid displays structural homology to channel-forming bacterial toxins, such as colicins or transmembrane domain of diphtheria toxin. By analogy, it has been hypothesized that tBid would unfold and insert into the lipid bilayer of the mitochondria outer membrane (MOM) upon membrane association. However, it has been shown recently that unlike colicins and the transmembrane domain of diphtheria toxin, tBid binds to the lipid bilayer maintaining α-helical conformation of its helices without adopting a transmembrane orientation by them. Here, the mechanism of the association of tBid with the model membrane mimicking the mitochondrial membrane is studied by Monte Carlo simulations, taking into account the underlying energetics. A novel two-stage hierarchical simulation protocol combining coarse-grained discretization of conformational space with subsequent refinements was applied which was able to generate the protein conformation and its location in the membrane using modest computational resources. The simulations show that starting from NMR-established conformation in the solution, the protein associates with the membrane without adopting the transmembrane orientation. The configuration (conformation and location) of tBid providing the lowest free energy for the system protein/membrane/solvent has been obtained. The simulations reveal that tBid upon association with the membrane undergoes significant conformational changes primarily due to rotations within the loops between helices H4 and H5, H6 and H7, H7 and H8. It is established that in the membrane-bound state of tBid-monomer helices H3 and H5 have the locations exposed to the solution, helices H6 and H8 are partly buried and helices H4 and H7 are buried into the membrane at shallow depth. The average orientation of tBid bound to the membrane in the most stable configuration reported here is in satisfactory agreement with the evaluations obtained by indirect experimental means. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of lipids strictly associated to PSII complexes: Focus on cardiolipin structural and functional role

In this work, lipid extracts from spinach membrane fragments enriched in Photosystem II (PSII) and from spinach PSII dimers were analyzed, by means of Thin Layer Chromatography (TLC) and Electro-Spray Ionization Mass Spectrometry. Cardiolipin found in association with PSII was isolated and purified by preparative TLC, then characterized by mass and mass-mass analyses. Cardiolipin structures with four unsaturated C18 acyl chains and variable saturation degrees were evidenced. Structural and functional effects of different phospholipids on PSII complexes were investigated by Fluorescence, Resonance Light Scattering and Oxygen Evolution Rate measurements. An increment of PSII thermal stability was observed in the presence of cardiolipin and phosphatidylglycerol.