Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.     

Conserved sequence blocks 5′ to start codons of plant mitochondrial genes

Three sequence blocks of 10–12 bp are conserved in sequence and order 5 to putative start codons of several higher-plant mitochondrial genes. At least 25 examples were found, primarily associated with coxII, atp6, and orf25, in monocotyledons and dicotyledons. The proximal block can be 9 bp from start codons, and the three blocks generally occur within 100 bp 5 of start codons. In three examples 5 termini of the blocks represent recombination breakpoints, resulting in conservation of the blocks in resultant configurations. The two proximal blocks can form a secondary structure motif. The occurrence of the blocks near start codons, and conserved sequence and order, is consistent with a possible role in translation initiation or regulation.     

Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.     

Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase?

The interactions between the pyrophosphate (PPi) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PPi per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PPi/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PPi-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PPi. The addition of PPi at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PPi to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PPi binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.     

Incorporation of β-sitosterol into mitochondrial membrane enhances mitochondrial function by promoting inner mitochondrial membrane fluidity

Recent findings suggest that mitochondrial membrane fluidity could influence mitochondrial energy metabolism. β-sitosterol (BS) is a common plant sterol that is prevalent in plant oils, nuts, cereals and plant food products. Its chemical structure is very similar to that of cholesterol. As a cholesterol analog, BS is highly lipid soluble and largely resides in the membranes of cells or organelles where it may have an influence on the membrane fluidity. The present study reports that, with the cholesterol chelator 2-hydroxypropyl-β-cyclodextrin (HPβCD) as its carrier, BS is able to increase the fluidity of the inner mitochondrial membrane (IMM) without affecting the fluidity of the outer mitochondrial membrane (OMM), and consequently to increase the mitochondrial membrane potential (?Ψm) and mitochondrial ATP content. It has been previously proposed that a therapeutical boost in adenosine triphosphate (ATP) levels in mitochondria may be beneficial for neurodegenerative diseases such as Alzheimer’s disease (AD). Given that dietary administration of plant sterols could increase brain BS concentrations, these results may provide a better understanding of the beneficial effects of plant sterol-enriched nutrients on neurodegenerative diseases such as AD.     

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe²⁺) ≪ ferric chloride (Fe³⁺) < copper sulfate (Cu²⁺) < zinc sulfate (Zn²⁺) < manganese chloride (Mn²⁺) < calcium chloride (Ca²⁺) < magnesium sulfate (Mg²⁺). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe²⁺ compared with any other metal ions, including Fe³⁺.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

A Mössbauer spectroscopic study of the form of iron in iron overload

There are major differences in the temperature dependence of the Mössbauer spectra of ferritin and haemosiderin extracted from the organs of humans suffering from transfusional iron overload. Iron overload can also occur in animal systems as a result of artificial treatments or dietary factors. None of the animal systems which were investigated in the present study showed evidence in their Mössbauer spectra for the presence of the haemosiderin found in transfusional iron overload in humans. This suggests that the haemosiderin which occurs in the case of human transfusional iron overload may be specific to that situation.     

Capacity of siderophore — producing alkalophilic bacteria to accumulate iron,gallium and aluminum

Summary The ability of alkalophilic bacteria to remove iron, gallium and aluminium from culture media is reported. Of six bacterial strains grown in the presence of iron, gallium or aluminium (10 M), five were able to accumulate iron or gallium, but only two depleted the aluminium stock. A comparison of gallium removal under low (< 1 gmM) or high (10 gmM) iron conditions showed that two isolates accumulated gallium only under low-Fe conditions. One isolate, a coryneform bacterium, was able to grow in the presence of 1, 10 and 100 mg gallium/l, but growth and siderophore production were affected at high gallium concentration. Similar concentrations of gallium were accumulated from cultures initially containing 1 or 100 mg gallium/l. Offsprint requests to: D. J. Gascoyne     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.     

Towards the development of a new generation of whole-cell bioreporters to sense iron bioavailability in oceanic systems—learning from the case of Synechococcus sp. PCC7002 iron bioreporter

Whole-cell bioreporters are genetically modified micro-organisms designed to sense bioavailable forms of nutrients or toxic compounds in aquatic systems. As they represent the most promising cost-efficient tools available for such purpose, engineering and use of bioreporters is rapidly growing in association with wide applicability. Bioreporters are urgently needed to determine phytoplankton iron (Fe) limitation, which has been reported in up to 30% of the ocean, with consequences affecting Earth's global carbon cycle and climate. This study presents a critical evaluation and optimization of the only Cyanobacteria bioreporter available to sense Fe limitation in marine systems (Synechococcus sp. PCC7002). The nonmonotonic biphasic dose–response curve between the bioreporters’ signal and Fe bioavailability impairs an appropriate data interpretation, highlighting the need for new carefully designed bioreporters. Here, limitations under low Fe concentrations were related to cellular energy stress, nonlinear expression of the targeted promoter and siderophore expression. Furthermore, we provide critical standard criteria for the development of new Fe bioreporters. Finally, based on gene expression data under a range of marine Fe concentrations, we propose novel sensor genes for the development of new Cyanobacteria Fe bioreporters for distinct marine regions.     

Biosorption of iron(III)–cyanide complex anions to Rhizopus arrhizus: application of adsorption isotherms

The biosorption of iron(III)–cyanide complex anions to Rhizopus arrhizus was investigated. The iron(III)–cyanide complex ion binding capacity of the biosorbent was a function of initial pH, initial iron(III)–cyanide complex ion and biosorbent concentration. These results indicated that a significant reduction of iron(III)–cyanide complex ions was achieved at pH 13, a highly alkaline condition. The maximum loading capacity of biosorbent was 612·2 mg g⁻¹ at 1996·2 mg litre⁻¹ initial iron(III)–cyanide complex ion concentration at this pH. The Freundlich, Langmuir and Redlich–Peterson adsorption models were fitted to the equilibrium data at pH 3·0, 7·0 and 13·0. The equilibrium data could be best fitted to by all the adsorption models over the entire concentration range (50–2000 mg litre⁻¹) at pH 13.     

Cyclic AMP-dependent phosphorylation of the precursor to beta subunit of mitochondrial F1-ATPase: a physiological mistake?

By using the purified rat liver protein for reference in electrophoresis and peptide mapping experiments, I have identified the beta subunit of mitochondrial F1-ATPase and its cytoplasmic precursor in two-dimensional gel patterns of proteins from S49 mouse lymphoma cells. The beta subunit precursor is a substrate for cAMP-dependent phosphorylation during its synthesis. Normally, both nonphosphorylated and phosphorylated forms of beta subunit precursor are processed rapidly to the smaller, more acidic forms of mature beta subunit. When processing is inhibited with valinomycin, both nonphosphorylated and phosphorylated forms of beta subunit precursor are stabilized. Nonphosphorylated beta subunit is one of the most stable of cellular proteins, but the phosphorylated form is eliminated within minutes of processing. This suggests that phosphorylated beta subunit is recognized as aberrant and excluded from assembly into the ATPase complex. These results argue that cAMP-dependent phosphorylation of the beta subunit precursor is a physiological mistake that is remedied after mitochondrial import and processing.     

Does high bicarbonate supply to roots change availability of iron in the leaf apoplast?

The role of the leaf apoplast in iron (Fe) uptake into the leaf symplast is insufficiently understood, particularly in relation to the supposed inactivation of Fe in leaves caused by elevated bicarbonate in calcareous soils. It has been supposed that high bicarbonate supply to roots increases the pH of the leaf apoplast which decreases the physiological availability of Fe in leaf tissues. The study reported here has been carried out with sunflower plants grown in nutrient solution and with grapevine plants grown on calcareous soil under field conditions. The data obtained clearly show that the pH of the leaf apoplastic fluid was not affected by high bicarbonate supply in the root medium (nutrient solution and field experiments). The concentrations of total, symplastic and apoplastic Fe were decreased in chlorotic leaves of both sunflower (nutrient solution experiment) and grapevine plants in which leaf expansion was slightly inhibited (field experiment). However, in grapevine showing severe inhibition of leaf growth, total Fe concentration in chlorotic leaves was the same or even higher than in green ones, indicative to the so-called `chlorosis paradox'. The findings do not support the hypothesis of Fe inactivation in the leaf apoplast as the cause of Fe deficiency chlorosis since no increase was found in the relative amount of apoplastic Fe (% of total leaf Fe) either in the leaves of sunflower or grapevine plants. It is concluded that high bicarbonate concentration in the soil solution does not decrease Fe availability in the leaf apoplast.     

Do indicators of maternal iron status reflect placental iron status at delivery?

Indicators of maternal iron (Fe) status were studied in relation to placental Fe (Pl-Fe) status. Placental (Pl) and maternal (M) venous blood samples were obtained from primiparous women ($n=38$), with normal delivery at Paroissien Hospital, Argentina. Maternal hemoglobin (M Hb), soluble transferrin receptor (M sTfR) (ELISA) and serum ferritin (M S-Ft) were studied in relation to Pl-Fe, ferritin (Pl-Ft) and transferrin receptor (Pl-TfR). Pl-TfR was measured by dot blot assay, Pl-Ft and M S-Ft by immunoassay (IRMA) and Pl-Fe by atomic absorption spectrometry. Fe status indicators were, respectively, (mean±SD): M Hb 113±16 g/L; M S-Ft 36±42 μg/L; M sTfR 6.3±3.1 mg/L; Pl-Fe 170±56 μg/g placenta; Pl-Ft 33±18 μg/g placenta; Pl-TfR 18±18 (range 0–58) μg/g placenta. Pl-Fe, Pl-Ft and Pl-TfR did not correlate to M Hb, M S-Ft and M sTfR. Women with Pl- Fe, Pl-Ft and Pl-TfR above or below the corresponding median values did not show any statistical significant difference in M Hb, M sTfR or M S-Ft values. Pl-Ft concentration was lower in women with Hb<110 g/L than in women with normal values: 26±13 vs. 38±20 μg/g, respectively ($p=0.021$). When Pl-TfR, Pl-Ft and Pl-Fe were compared in women with M S-Ft above or below the cut-off point of 10 or 20 μg/L, no significant difference was found for Pl-TfR neither for Pl-Ft nor Pl-Fe. These results suggest that maternal indicators of Fe status, particularly M sTfR and M S-Ft, do not reflect Fe status of the placenta at delivery.     

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2

Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O₂ consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A₂ (PLA₂) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA₂ inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA₂.     

The accessory subunit of mitochondrial DNA polymerase γ determines the DNA content of mitochondrial nucleoids in human cultured cells

The accessory subunit of mitochondrial DNA polymerase γ, POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLGα. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.