Changes in freeze-fractured mitochondrial membranes correlated to their energetic state: Dynamic interactions of the boundary membranes

Structural changes of mitochondria in correlation to their energetic state have been observed as matrix expansion and condensation. In this communication we describe a morphological correlation in freeze-fractured mitochondrial membranes which is also dependent on the metabolic state of the organelle: the frequency by which the fracture plane following the inner or outer boundary membrane deviates by jumping from one membrane to the other is higher in phosphorylating mitochondria when compared to freshly isolated or energized mitochondria. These deflections of the fracture plane occur mostly in minimal, short steps showing close apposition of the two boundary membranes. We therefore conclude that the observed change in morphological appearance is produced by a change in interactions between the inner and the outer membranes correlated to the different functional states of the inner membrane.     

Enrichment and biochemical characterization of boundary membrane contact sites from rat-liver mitochondria

A subfraction of mitochondrial membranes was prepared from osmotically lysed rat liver mitochondria by density gradient centrifugation which contained the inner boundary membrane and the contact sites between this membrane and the outer membrane. The fraction was composed of inner and outer limiting membrane components as shown by the presence of specific marker enzymes, monoamine oxidase and glycerolphosphate oxidase. Surface proteolysis analysis, studies of cytochrome c permeability, and electron microscopy revealed the localization of the inner membrane component within a right-side-out outer membrane vesicle. Moreover, the outer membrane component in this fraction exhibited a higher capacity to bind hexokinase and had a higher specific activity of glutathione transferase than the pure outer membrane. In freeze-fracture analyses the fraction showed fracture plane deflections which may be specific for hydrophobic interactions between the two membranes.     

Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub- mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.     

The effect of lactoperoxidase-catalyzed iodination on the integrity of mitochondrial membranes.

The effect of lactoperoxidase-catalyzed iodination on rat liver mitochondria was investigated. A change from the condensed to the swollen conformation is observed by electron microscopy after extensive iodination of the mitochondria. The outer membrane breaks after incorporation of 0.2 nmol or more iodine atoms per mg of mitochondrial protein releasing adenylate kinase, a soluble enzyme located in the intermembrane space. Further iodination of the mitochondria ruptures the inner membrane, releasing proteins such as glutamic dehydrogenase from the matrix space. Lipid peroxides and I₂ are not intermediates in the disruptive effect of extensive lactoperoxidase-catalyzed iodination on the membranes. During iodination at pH 6.5 almost no release of protein or glutamic dehydrogenase activity is detectable and the loss of adenylate kinase activity from the particulate is diminished. The effect of extensive iodination on mitochondrial membranes limits the amount of iodide which can be incorporated with the lactoperoxidase membrane-labeling procedure when this technique is used as a surface probe of mitochondrial membranes.     

Separate fusion of outer and inner mitochondrial membranes

Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of the outer membrane, was abolished by dissipation of the inner membrane potential with K+ (valinomycin) or H+ ionophores (cccp). In addition, outer and inner membrane fusion proceeded separately in the absence of any drug. The separate fusion of outer and inner membranes and the different requirements of these fusion reactions point to the existence of fusion machineries that can function separately.     

大鼠心肌线粒体内、外膜磷脂动态结构的研究

我们以DPH为荧光探针.用毫微秒荧光分光光度计测定了大鼠心肌线粒体及线粒体内、外膜的动态微细结构;用HPLC分析了磷脂组成.实验结果提示.完整线粒体膜流动性主要反映了线粒体外膜的运动状态.线粒体内膜微粘度及磷脂分子摇动角大于外膜,扩散速率小于外膜.除去了蛋白质的线粒体内、外膜磷脂脂质体膜流动性无明显差异.提示线粒体内膜的高微粘度与膜中所含有的多量蛋白有关.     

Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria.

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.     

A morphometric analysis of inner membranes related to biochemical characteristics of mitochondria from heart muscle and liver in mice

Mitochondria of liver and heart muscle of mice—processed in the tissue or after isolation—were analysed by quantitative electron microscopy, and their morphometric characteristics were compared with pertinent biochemical parameters. The surface density (per unit mitochondrial volume) of the inner membranes (cristae) of heart-muscle mitochondria was found to be about twice that of liver mitochondria. No significant difference was noted in the surface densities of the outer and inner boundary membranes of either heart muscle or liver mitochondria. Isolated mitochondria, metabolically transformed to the orthodox conformation, showed morphometric characteristics very similar to mitochondria from tissue samples. The surface densities of the inner membranes showed a striking correlation with the content of insoluble mitochondrial protein and the levels of succinate dehydrogenase and cytochrome aa3 reported earlier.     

Dual Electron Microscopic Localization of Mitochondrial Creatine Kinase in Brain Mitochondria

Mitochondrial creatine kinase in brain mitochondria appears to be located at two different intramitochondrial sites. By using immunogold-labeling techniques, a peripheral immunoreactivity was localized between the two boundary membranes, while an additional, central immunoreactivity was found at the crista surface. The peripheral enzyme was accessible to the antibodies after treatment of the brain mitochondria with 100-300 μg digitonin/mg mitochondrial protein, which left 75% of the activity bound to the membranes. Electron microscopic analyses revealed that 43% of the labeled, peripheral creatine kinase was bound at those places where outer membrane vesicles remained attached to the inner envelope membrane, suggesting that the enzyme is in involved in contact formation between outer and inner mitochondrial membranes. Postembedding staining of mitochondria on thin sections of brain tissue or in the isolated state led to the observation of a second location of creatine kinase inside the mitochondria, along the cristae, which was not accessible to the antibodies in isolated, digitonin-treated mitochondria.     

Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Radical initiated alpha-tocopherol depletion and lipid peroxidation in mitochondrial membranes

The relationships between antioxidant status, lipid peroxidation and membrane protein integrity have been studied in an isolated mitochondrial membrane system. Tocopherol was shown to be present in both the outer and inner membrane of normal rat liver mitochondria; 77.3 and 22.3% of the total alpha-tocopherol was present in the outer and inner membranes, respectively. The endogenous alpha-tocopherol was depleted in a time-dependent manner by low levels of ferrous iron and by irradiation in the presence or absence of ferrous iron. This antioxidant depletion was followed by the appearance of lipid hydroperoxides. Fragmentation of monoamine oxidase, an integral outer membrane protein, was observed at irradiation doses that caused by antioxidant depletion and peroxide generation.     

Possible role of non-bilayer lipids in the structure of mitochondria. A freeze-fracture electron microscopy study

The possible role of non-bilayer phospholipids on the structure of isolated rat liver mitochondria has been morphologically studied. Freshly isolated freeze-fractured mitochondria show smooth fracture faces with particles, representing the limiting membranes. The frequency and size of the particles is representative for the various membrane faces. Distinctly large particles and pits represent the attachment sites of cristae to the inner membrane. Liposome-like structures in the matrix are found upon incubation with Ca2+ and Mn2+. At 5 mM Mn2+ and more, curved hexagonal (HII) phase tubes are observed. Subsequent addition of 1 mM EDTA results in disappearance of the HII tubes, and liposomal structures can again be seen. These findings are interpreted in terms of an Mn2+-induced lamellar to HII phase transition. Patchwork-like structures characterize the membranes of mitochondria, quenched from 37 degrees C, as well as those incubated with Ca2+, Mn2+, Mg2+ and apo- or cytochrome c. This phenomenon is interpreted as being the result of the fracture plane, jumping from the outer to the inner limiting membrane and vice versa at sites of contact. A semi-fusion model, in which non-bilayer lipids are involved, is proposed for these contact sites.     

Freeze-fracture electron microscopic analysis of ultrarapidly frozen envelope membranes on intact chloroplasts and after purification

Summary Freeze-fracture electron microscopy of ultrarapidly frozen intact pea chloroplasts has been used to characterize the supramolecular architecture of their outer and inner envelope membranes, to follow changes in these membranes caused by experimental treatments, and to identify the composition of purified envelope membrane subfractions. Examination of intact chloroplasts revealed that the two membranes exhibit dramatically different densities of intramembrane particles, with the inner membrane particle density approximately fourfold that of the outer. Analysis of purified envelope membrane subfractions indicates that the low bouyant density fraction (1.08 g/cm³) corresponds to the outer envelope membrane, whereas the relatively higher bouyant density fraction (1.13 g/cm³) is predominantly inner membrane. From qualitative and quantitative morphological data we conclude that the outer membrane subfraction is pure whereas the inner membrane subfraction is significantly contaminated by outer membrane. These results confirm conclusions reached from biochemical analysis of these membranes.During the course of the studies on intact chloroplasts, sites were observed where the outer and inner envelope membranes appear to adhere to each other (contact sites). Some of the contact sites observed on intact chloroplasts survived the envelope purification procedures as evidenced by their presence on a small number of vesicles in inner membrane preparations. The practical significance of these putative contact sites is discussed.     

Effect of aging on the composition of mitochondrial membranes from potato slices

The phospholipid content of mitochondrial membranes from slices of potato tuber (Solanum tuberosum) remains stable during aging. The phospholipid compositions of whole mitochondria and inner membranes do not vary during aging whereas the concentrations of phosphatidylinositol and phosphatidyl-glycerol in outer membranes are slightly amplified. The saturation of outer membrane fatty acids is slightly increased during aging. Gel electrophoresis of mitochondrial membrane proteins show slight variations of one polypeptide in outer membranes and of three polypeptides in inner membranes. These results suggest parallel variations of lipids and proteins in membranes during aging, in marked contrast with the large modifications observed in mitochondrial activities.     

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.     

Mitochondrial membranes modify mutant huntingtin aggregation

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.     

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.     

The morphology and configurational states of isolated heavy beef heart mitochondria by the freeze fracture technique

Configurational changes of glutaraldehyde fixed heavy beef heart mitochondria are confirmed using the freeze fracture technique. Large amplitude swelling occurred after unfixed mitochondria were suspended in 30% glycerol. Fine structure of the outer and inner mitochondrial membranes is described using unfixed heavy beef heart mitochondria by the freeze fracture technique. The matrix side of the inner membrane appears to be covered with 90 Å particles while the opposite side (cytochromec side) is also particulate covered by a high density of lower profile particles with a smooth underlying mosaic layer beneath. The outer surface of the outer membrane is smooth with particles embedded within the membrane. Possible structure of the membrane is discussed.