Deterioration of Mitochondrial Membranes from Castor Bean Endosperm by Hydrogen Peroxide

Loss of membrane integrity by hydrogen peroxide (H₂O₂) was studiedin isolated mitochondria of castor bean. Incubation of mitochondriawith H₂O₂ resulted in the release of fumarase with a concomitantloss of phospho-lipids. Degradation of membrane was found tooccur independent of lipid peroxidation. H₂O₂-treated mitochondriawere able to degrade exogenous radiolabelled phospho-lipids. ³Corresponding author; fax 82-42-821-2391     

Transverse Distribution of Phospholipids in Organelle Membranes from Ricinus communis L. var. Hale Endosperm: MITOCHONDRIA AND GLYOXYSOMES

Phospholipase A₂ (Naja naja), the nonpenetrating dye trinitrobenzene sulfonate, and the penetrating dye dinitrofluorobenzene, were used to determine the transmembrane distributions of phospholipids of mitochondria and glyoxysomes isolated from endosperm tissue of castor bean (Ricinus communis L. var. Hale). These studies indicated that the phospholipid distributions were distinctly asymmetric in the accessible (reacted with the probes without total membrane disruption by detergents) pools of the glyoxysomal and inner mitochondrial membranes, but more nearly symmetric in the outer mitochondrial membrane. However, significant quantities of the phospholipids of the mitochondrial membranes were inaccessible to the probes used. An increased accessibility of the phospholipids of all membranes following Triton X-100 dispersion was found, and protein to phospholipid ratios in organelle membranes were found to correlate inversely with the accessibility of the phospholipids to the probes. The inaccessible phospholipids may be involved in lipid-protein interactions.     

Phosphatidylethanolamine synthesis by castor bean endosperm. Intracellular distribution and characteristics of CTP:ethanolaminephosphate cytidylyltransferase.

The intracellular distribution and catalytic properties of CTP: ethanolaminephosphate cytidylyltransferase from endosperm of castor bean (Ricinus communis L. var. Hale) have been studied. This enzyme was confined to membranes, with about 80% of the activity occurring in mitochondria and the rest in endoplasmic reticulum (ER) following sucrose density gradient centrifugation. The mitochondrial location of this enzyme was supported by further purifying mitochondria on Percoll density gradients. The mitochondrial cytidylyltransferase was detected largely in outer membrane fractions, and lost its activity after trypsin treatment, indicating that the active sites are exposed to the cytoplasm. Both mitochondrial and ER cytidylyltransferase required cations for activity; Mg2+ was preferred over Mn2+ and Ca2+. The pH optima both were 6.5. The apparent Km values for ethanolamine phosphate were 143 and 83 microM and those for CTP were 125 and 1010 microM, respectively, for the mitochondrial and ER activities. The mitochondrial cytidylyltransferase reached a maximal velocity of 3.0 nmol/min/mg protein, whereas ER cytidylyltransferase was 0.424 nmol/min/mg protein. These findings reveal that the majority of the cytidylyltransferase activity in castor bean endosperm is not closely associated with ethanolaminephosphotransferase (predominantly in ER) which catalyzes the subsequent reaction in the synthesis of phosphatidyl-ethanolamine by a nucleotide pathway. The possible roles of these enzymes in phosphatidylethanolamine synthesis in plants are discussed.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Intracellular Localization of Phospholipase D in Leaves and Seedling Tissues of Castor Bean

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Phospholipase A2 from sheep erythrocyte membrane CA2+ dependence and localization

The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A₂ specificity was found to be responsible for both phosphatidylethanolamine and phosphatidylcholine degradation.The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [¹⁴C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.     

Lipid Breakdown in Smooth Microsomal Membranes from Bean Cotyledons Alters Membrane Proteins and Induces Proteolysis

The effects of lipid degradation on proteins of smooth microsomalmembranes isolated from young bean cotyledons have been examinedby three techniques, viz. fluorescence energy transfer fromtryptophan to cis-parinaric acid; protein spin-labelling with3-maleimido PROXYL; and SDS-PAGE. Lipid degradation was inducedin isolated membranes by activating phospholipase D and phosphatidicacid phosphatase through the addition of Ca²⁺, by treatmentwith exogenous phospholipase C to simulate the concerted actionsof phospholipase D and phosphatidic acid phosphatase or by treatmentwith exogenous phospholipase A₂ to generate endogenous substratefor lipoxygenase. All of the treatments induced time-dependentchanges in lipid-protein interaction and in protein conformation,and the treatment with phospholipase A₂ also engendered proteolysis.The effects of the Ca²⁺ and phospholipase C treatments on lipid-proteininteraction and protein conformation can presumably be partlyattributed to an accumulation of diacylglycerol in the membrane,whereas the induction of proteolysis by phospholipase A₂ appearsto be due to activated oxygen derived from the lipoxygenasereaction and ensuing lipid peroxidation. Lipid degradation inducedby these treatments simulates that which occurs during naturalsenescence of the cotyledons and hence these observations suggestthat loss of protein function and proteolysis in senescing membranesis facilitated by lipolytic and peroxidative activity withinthe lipid bilayer. Key words: Activated oxygen, lipids, membranes, proteins, senescence     

Phospholipid metabolism in plant mitochondria: submitochondrial sites of synthesis

Intact mitochondria from the endosperm of castor bean were isolated on linear sucrose gradients. These mitochondria were ruptured and the membranes separated on discontinuous sucrose gradients into outer membrane, intact inner membrane, and ruptured inner membrane fractions. Each membrane fraction was examined for its capacity to synthesize phosphatidylglycerol, CDP-diglyceride, phosphatidylcholine via methylation, and phosphatidic acid. The syntheses of phosphatidylglycerol, CDP-diglyceride, and phosphatidylcholine were localized exclusively in the inner mitochondrial membrane fractions while phosphatidic acid synthesis occurred in both the inner and outer mitochondrial membranes.     

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg²⁺ free medium. Exogenous citrate suppressed H₂O₂ emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate’s effect. Exogenous MgCl₂ strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg²⁺ or other divalent cations.     

Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases,p38 and JNK

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H₂O₂ induced apoptosis were analyzed with Western blot. H₂O₂ induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H₂O₂ induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H₂O₂ caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H₂O₂ activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

干旱胁迫下水曲柳苗木细根线粒体的形态及活性变化

根系依赖根细胞内线粒体呼吸代谢产生的能量, 不断从土壤中获取养分。在胁迫条件下, 线粒体的结构和功能会发生一定的变化, 从而影响根系的功能。土壤干旱是最容易引起苗木细根衰老死亡的非生物胁迫因子之一。为了更好地认识干旱胁迫下细根线粒体的结构和功能变化, 对土壤干旱胁迫下水曲柳(Fraxinus mandshurica)不同颜色细根皮层薄壁细胞内线粒体的超微结构(线粒体数量、形态)、线粒体的呼吸功能、线粒体膜脂质氧化(膜透性变化、过氧化氢含量等)情况进行了研究。结果表明: (1)干旱胁迫下, 水曲柳白色及黄色根皮层薄壁细胞内线粒体形状、结构及分布数量与对照相似, 无显著差异。干旱胁迫下产生的褐色根皮层薄壁细胞线粒体数量减少, 分布密度也变小。线粒体内、外膜先后发生不同程度的解体, 最后消失。(2)干旱胁迫显著干扰了线粒体膜的正常呼吸耦联作用, 细根线粒体呼吸控制率(RCR)与磷氧比(无机磷酸/分子氧, P/O)均显著低于对照(p < 0.05)。随着细根颜色加深, 线粒体RCR和P/O值逐渐下降, 白色根﹥黄色根﹥褐色根。褐色根线粒体RCR值最低, 接近极值1。说明褐色根线粒体结构完整性最差, 能量转化效率最低。(3)干旱胁迫下, 不同颜色细根线粒体内的H₂O₂含量、线粒体膜透性、膜脂氧化产物丙二醛(MDA)含量均显著高于对照(p < 0.05)。且随着细根颜色加深, 各个值增加明显。分析可能是由于干旱胁迫导致线粒体内H₂O₂含量升高, 线粒体膜脂质过氧化(MDA含量升高), 膜结构受到破坏(膜透性增加) (电镜下可见部分线粒体内膜电子密度下降及外膜解体)。线粒体膜结构完整性的破坏, 直接影响了线粒体呼吸代谢反应, 使线粒体呼吸功能下降。     

Spherosomes of Castor Bean Endosperm: MEMBRANE COMPONENTS, FORMATION, AND DEGRADATION

The membrane components of the castor bean spherosomes were characterized. The storage triacylglycerols of isolated spherosomes were extracted with diethyl ether, and the membrane was isolated by sucrose gradient centrifugation. It had an apparent equilibrium density of 1.12 grams per cubic centimeter, and possessed an antimycin A-insensitive NADH cytochrome c reductase and an acid lipase. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in roughly equal amounts were the major phospholipids. The membrane proteins were resolved into several major and minor protein bands of molecular weights ranging from 10,000 to 70,000 by acrylamide gel electrophoresis, and the protein pattern in the gel was different from those of the endoplasmic reticulum, mitochondrial, and glyoxysomal membranes.     

Exogenous Hydrogen Peroxide Changes Antioxidant Enzyme Activity and Protects Ultrastructure in Leaves of Two Cucumber Ecotypes Under Osmotic Stress

Cucumber (Cucumis sativus L.) varieties cv. Jinchun no. 4 (a North China ecotype) and cv. Lvfeng no. 6 (a South China ecotype) were cultivated to explore the effects of osmotic stress on the ultrastructure of chloroplasts and mitochondria, as well as to assess the possible protective effect of exogenous hydrogen peroxide (H₂O₂). Under osmotic stress induced by 10% polyethylene glycol 6000, 84.3% of the chloroplasts in Jinchun no. 4 were abnormal, whereas 88.6% were abnormal in Lvfeng no. 6. Abnormal mitochondria occurred in these two strains at rates of 78.5 and 87.1%, respectively. The stress condition disintegrated the membranes of most chloroplasts and mitochondria in the leaf cells of both cucumber ecotypes, and it also increased the malondialdehyde (MDA) content. We subjected the two cultivars to a combined treatment with H₂O₂ and osmotic stress and made the following observations: (1) Abnormal chloroplasts occurred at rates of 25.7 and 28.6%, and abnormal mitochondria were observed at rates of 22.9 and 32.8%, respectively. (2) Most of the investigated membranes were well organized in leaves of Jinchun no. 4 and Lvfeng no. 6, and the levels of endogenous H₂O₂, superoxide anion, and MDA were lower. Osmotic stress and exogenous H₂O₂ both increased the activities of antioxidative enzymes such as manganese superoxide dismutase, glutathione peroxidase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and the antioxidants ascorbate and reduced glutathione. The combined effect of osmotic stress and exogenous H₂O₂ resulted in the highest antioxidant activities in both cucumber ecotypes. We propose that exogenous H₂O₂ increases antioxidant activity in cucumber leaves and thereby decreases lipid peroxidation to some extent, thus protecting the ultrastructure of most chloroplasts and mitochondria under osmotic stress.     

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H₂O₂ was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H₂O₂ for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H₂O₂ scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H₂O₂. H₂O₂ may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H₂O₂. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H₂O₂ formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H₂O₂ or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H₂O₂ through NADPH oxidase was inhibited, and H₂O₂ mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H₂O₂ was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H₂O₂ was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H₂O₂ in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H₂O₂. Results obtained suggest that H₂O₂ may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both pro-mitochondria and mitochondria obtained from O₂-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N₂) to elaidic (O₂)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O₂-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.