Mdm38 is a 14-3-3-like receptor and associates with the protein synthesis machinery at the inner mitochondrial membrane

Mitochondrial ribosomes synthesize core subunits of the inner membrane respiratory chain complexes. In mitochondria, translation is regulated by mRNA‐specific activator proteins and occurs on membrane‐associated ribosomes. Mdm38/Letm1 is a conserved membrane receptor for mitochondrial ribosomes and specifically involved in respiratory chain biogenesis. In addition, Mdm38 and its higher eukaryotic homolog Letm1, function as K⁺/H⁺ or Ca²⁺/H⁺ antiporters in the inner membrane. Here, we identify the conserved ribosome‐binding domain (RBD) of Mdm38 and determine the crystal structure at 2.1 Å resolution. Surprisingly, Mdm38^RBD displays a 14‐3‐3‐like fold despite any similarity to 14‐3‐3‐proteins at the primary sequence level and thus represents the first 14‐3‐3‐like protein in mitochondria. The 14‐3‐3‐like domain is critical for respiratory chain assembly through regulation of Cox1 and Cytb translation. We show that this function can be spatially separated from the ion transport activity of the membrane integrated portion of Mdm38. On the basis of the phenotypes observed for mdm38Δ as compared to Mdm38 lacking the RBD, we suggest a model that combining ion transport and translational regulation into one molecule allows for direct coupling of ion flux across the inner membrane, and serves as a signal for the translation of mitochondrial membrane proteins via its direct association with the protein synthesis machinery.     

Regulation of mitochondrial matrix volume

Mitochondrial volume homeostasis is a housekeeping cellular function essential for maintaining the structural integrity of the organelle. Changes in mitochondrial volume have been associated with a wide range of important biological functions and pathologies. Mitochondrial matrix volume is controlled by osmotic balance between cytosol and mitochondria. Any dysbalance in the fluxes of the main intracellular ion, potassium, will thus affect the osmotic balance between cytosol and the matrix and promote the water movement between these two compartments. It has been hypothesized that activity of potassium efflux pathways exceeds the potassium influx in functioning mitochondria and that potassium concentration in matrix could be actually lower than in cytoplasm. This hypothesis provides a clear-cut explanation for the mitochondrial swelling observed after mitochondrial depolarization, mitochondrial calcium overload, or opening of permeability transition pore. It should also be noted that the rate of water flux into or out of the mitochondrion is determined not only by the osmotic gradient that acts as the driving force for water transport but also by the water permeability of the inner membrane. Recent data suggest that the mitochondrial inner membrane has also specific water channels, aquaporins, which facilitate water movement between cytoplasm and matrix. This review discusses different phases of mitochondrial swelling and summarizes the potential effects of mitochondrial swelling on cell function. potassium homeostasis; depolarization; mitochondrial swelling     

Effect of Osmotic Stress on Ion Transport Processes and Phospholipid Composition of Wheat (Triticum aestivum L.) Mitochondria

The effect of osmotic stress on wheat (Triticum aestivum L.) mitochondrial activity and phospholipid composition was investigated. Preliminary growth measurements showed that osmotic stress (−0.25 or −0.5 megapascal external water potential) inhibited the rate of shoot dry matter accumulation while root dry matter accumulation was less sensitive. We have determined that differences in sensitivity to osmotic stress existed between tissues at the mitochondrial level. Mitochondria isolated from roots or shoots of stressed seedlings showed respiratory control and ADP/O ratios similar to control seedlings which indicates that stressed mitochondria were well coupled. However, under passive swelling conditions in a KCl reaction mixture, the rate and extent of valinomycin-induced swelling of shoot mitochondria were increased by osmotic stress while root mitochondria were largely unaffected. Active ion transport studies showed efflux transport by stressed-shoot mitochondria to be partially inhibited since mitochondrial contraction required the addition of N-ethylmaleimide or nigericin. Efflux ion transport by root mitochondria was not inhibited by osmotic stress which indicates that stress-induced changes in ion transport were largely limited to shoot mitochondria. Characterization of mitochondrial fatty acid and phospholipid composition showed an increase in the percentage of phosphatidylcholine in stressed shoot mitochondria compared to the control. Mitochondrial fatty acid composition was not markedly altered by stress. No significant changes in either the phospholipid or fatty acid composition of stressed root mitochondria were observed. Hence, these results suggest that a tissue-specific response to osmotic stress exists at the mitochondrial level.     

Loss of the m‐AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation

The m‐AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1‐deficient neurons, indicating that they are not caused by OMA1‐mediated degradation of the dynamin‐like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N‐acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2‐depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.     

Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability

The interference of glibenclamide, an antidiabetic sulfonylurea, with mitochondrial bioenergetics was assessed on mitochondrial ion fluxes (H+, K+, and Cl-) by passive osmotic swelling of rat liver mitochondria in K-acetate, KNO3, and KCl media, by O2 consumption, and by mitochondrial transmembrane potential (Deltapsi). Glibenclamide did not permeabilize the inner mitochondrial membrane to H+, but induced permeabilization to Cl- by opening the inner mitochondrial anion channel (IMAC). Cl- influx induced by glibenclamide facilitates K+ entry into mitochondria, thus promoting a net Cl-/K+ cotransport, Deltapsi dissipation, and stimulation of state 4 respiration rate. It was concluded that glibenclamide interferes with mitochondrial bioenergetics of rat liver by permeabilizing the inner mitochondrial membrane to Cl- and promoting a net Cl-/K+ cotransport inside mitochondria, without significant changes on membrane permeabilization to H+.     

MscS-like proteins control plastid size and shape in Arabidopsis thaliana

BACKGROUND: Mechanosensitive (MS) ion channels provide a mechanism for the perception of mechanical stimuli such as sound, touch, and osmotic pressure. The bacterial MS ion channel MscS opens in response to increased membrane tension and serves to protect against cellular lysis during osmotic downshock. MscS-like proteins are found widely in bacterial and archaeal species and have also been identified in fission yeast and plants. None of the eukaryotic members of the family have yet been characterized. RESULTS: Here, we characterize two MscS-like (MSL) proteins from Arabidopsis thaliana, MSL2 and MSL3. MSL3 can rescue the osmotic-shock sensitivity of a bacterial mutant lacking MS-ion-channel activity, suggesting that it functions as a mechanosensitive ion channel. Arabidopsis plants harboring insertional mutations in both MSL3 and MSL2 show abnormalities in the size and shape of plastids, which are plant-specific endosymbiotic organelles responsible for photosynthesis, gravity perception, and numerous metabolic reactions. MSL2-GFP and MSL3-GFP are localized to discrete foci on the plastid envelope and colocalize with the plastid division protein AtMinE. CONCLUSIONS: Our data support a model wherein MSL2 and MSL3 control plastid size, shape, and perhaps division during normal plant development by altering ion flux in response to changes in membrane tension. We propose that MscS family members have evolved new roles in plants since the endosymbiotic event that gave rise to plastids.     

Turgor pressure changes trigger characteristic changes in the electrical conductance of the tonoplast and the plasmalemma of the marine alga Valonia utricularis

The giant marine alga Valonia utricularis is capable of regulating its turgor pressure in response to changes in the osmotic pressure of the sea water. The turgor pressure response comprises two phases, a fast, exponential phase arising exclusively from water shifting between the vacuole and the external medium (time constant about 10 min) and a second very slow, almost exponential phase adjusting (but not always) the turgor pressure near to the original value by release or uptake of KCl (time constant about 5 h). The changes in the vacuolar membrane potential as well as in the individual conductances of the tonoplast and plasmalemma accompanying turgor pressure regulation were measured by using the vacuolar perfusion assembly (with integrated microelectrodes, pressure transducers and pressure‐regulating valves) as described by Wang et al. (J. Membrane Biology 157, 311–321, 1997). Measurements on pressure‐clamped cells gave strong evidence that the turgor pressure, but not effects related to water flow (i.e. electro‐osmosis or streaming potential) or changes in the internal osmotic pressure and in the osmotic gradients, triggers the cascade of osmotic and electrical events recorded after disturbance of the osmotic equilibrium. The findings definitely exclude the existence of osmosensors as postulated for other plant cells and bacteria. There was also evidence that turgor pressure signals were primarily sensed by ion transporters in the vacuolar membrane because conductance changes were first recorded in the many‐folded tonoplast and then significantly delayed in the plasmalemma independent of the direction of the osmotic challenge. Consistently, turgor pressure up‐regulation (but not down‐regulation) could be inhibited reversibly by external addition of the K⁺ transport inhibitor Ba²⁺ and/or by the Cl^– transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Extensive studies under iso‐, hyper‐ and hypo‐osmotic conditions revealed that K⁺ and Cl^– contribute predominantly to the plasmalemma conductance. Addition of 0.3 mm NaCN showed further that part of the K⁺ and Cl^– transporters depended on ATP. These transporters are apparently up‐regulated upon hyper‐osmotic, but not hypo‐osmotic challenge. These findings explain the strong increase of the K⁺ influx upon lowering turgor pressure and the less pronounced pressure‐dependence of the Cl^– influx of V. utricularis reported in the literature. The data derived from the blockage experiments under hypo‐osmotic conditions were also equally consistent with the experimental findings that the K⁺ efflux is solely passive and progressively increases with increasing turgor pressure due to an increase of the volumetric elastic modulus of the cell wall. However, despite unravelling some of the sequences and other components involved in turgor pressure regulation of V. utricularis the co‐ordination between the ion transporters in the tonoplast and plasmalemma remains unresolved because of the failure to block the tonoplast transporters by addition of Ba²⁺ and DIDS from the vacuolar side.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

A role for mitochondrial aquaporins in cellular life-and-death decisions?

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the K+ cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease.     

Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

Mechanosensitive channels protect plastids from hypoosmotic stress during normal plant growth

Cellular response to osmotic stress is critical for survival and involves volume control through the regulated transport of osmolytes. Organelles may respond similarly to abrupt changes in cytoplasmic osmolarity. The plastids of the Arabidopsis thaliana leaf epidermis provide a model system for the study of organellar response to osmotic stress within the context of the cell. An Arabidopsis mutant lacking two plastid-localized homologs of the bacteria mechanosensitive channel MscS (MscS-like [MSL] 2 and 3) exhibits large round epidermal plastids that lack dynamic extensions known as stromules. This phenotype is present under normal growth conditions and does not require exposure to extracellular osmotic stress. Here we show that increasing cytoplasmic osmolarity through a genetic lesion known to produce elevated levels of soluble sugars, exogenously providing osmolytes in the growth media, or withholding water rescues the msl2-1 msl3-1 leaf epidermal plastid phenotype, producing plastids that resemble the wild-type in shape and size. Furthermore, the epidermal plastids in msl2-1 msl3-1 leaves undergo rapid and reversible volume and shape changes in response to extracellular hypertonic or hypotonic challenges. We conclude that plastids are under hypoosmotic stress during normal plant growth and dynamic response to this stress requires MSL2 and MSL3.     

Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential

Background information. Human OPA1 (optic atrophy type 1) is a dynamin‐related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. Results. We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L‐OPA1 (long isoforms of OPA1) and three S‐OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L‐OPA1 to S‐OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy‐metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins‐associated rhomboid‐like protein) – the human orthologue of Pcp1/Rbd1 – and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix‐oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock‐down of YME1L (human yme1‐like protein), an IMS‐oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of ΔΨm (inner mitochondrial membrane potential) or OPA1 processing. Conclusions. Metalloprotease‐mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.     

Activation of the MAPKs ERK1/2 by cell swelling in turbot hepatocytes

Background information. Activation of MAPKs (mitogen‐activated protein kinases), in particular ERK1/2 (extracellular‐signal‐regulated kinase 1/2), has been reported to take place in a large variety of cell types after hypo‐osmotic cell swelling. Depending on cell type, ERK1/2 phosphorylation can then serve or not the RVD (regulatory volume decrease) process. The present study investigates ERK1/2 activation after aniso‐osmotic stimulations in turbot hepatocytes and the potential link between phosphorylation of these proteins and RVD. Results. In turbot hepatocytes, Western‐blot analysis shows that a hypo‐osmotic shock from 320 to 240 mOsm·kg^?1 induced a rapid increase in ERK1/2 phosphorylation, whereas a hyper‐osmotic shock from 320 to 400 mOsm·kg^?1 induced no significant change in the phosphorylation of these proteins. The hypo‐osmotic‐induced ERK1/2 phosphorylation was significantly prevented when hypo‐osmotic shock was performed in the presence of the specific MEK (MAPK/ERK kinase) inhibitor PD98059 (100 μM). In these conditions, the RVD process was not altered, suggesting that ERK1/2 did not participate in this process in turbot hepatocytes. Moreover, the hypo‐osmotic‐induced activation of ERK1/2 was significantly prevented by breakdown of extracellular ATP by apyrase (10 units·ml^?1), by inhibition of purinergic P₂ receptors by suramin (100 μM) or by calcium depletion using EGTA (1 mM) and thapsigargin (1 μM). Conclusions. In turbot hepatocytes, hypo‐osmotic swelling but not hyper‐osmotic shrinkage induced the activation of ERK1/2. However, these proteins do not seem to be involved in the RVD process. Their hypo‐osmotic‐induced activation is partially due to cascades of signalling events triggered by the binding of released ATP on purinergic P₂ receptors and requires the presence of calcium.     

Mechanism of action of agents which uncouple oxidative phosphorylation: Direct correlation between protoncarrying and respiratory-releasing properties using rat liver mitochondria

The proton-carrying properties of uncoupling agents were investigated by measuring passive mitochondrial swelling under conditions where electrogenic proton transport was rate limiting. The ability of uncoupling agents to transport protons into mitochondria, measured in this way, was compared with respiratory stimulation. The results show that with the single exception of arsenate, all agents tested which uncouple oxidative phosphorylation demonstrate a very close correlation between release of respiration and proton transport. These findings are in support of Mitchell's original proposal that uncoupling agents act by promoting electrogenic hydrogen ion transport across the mitochondrial inner membrane.     

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.     

ATP-sensitive potassium channel in mitochondria of the eukaryotic microorganism Acanthamoeba castellanii

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.     

Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity

Members of the MscS superfamily of mechanosensitive ion channels function as osmotic safety valves, releasing osmolytes under increased membrane tension. MscS homologs exhibit diverse topology and domain structure, and it has been proposed that the more complex members of the family might have novel regulatory mechanisms or molecular functions. Here, we present a study of MscS-Like (MSL)10 from Arabidopsis thaliana that supports these ideas. High-level expression of MSL10-GFP in Arabidopsis induced small stature, hydrogen peroxide accumulation, ectopic cell death, and reactive oxygen species- and cell death-associated gene expression. Phosphomimetic mutations in the MSL10 N-terminal domain prevented these phenotypes. The phosphorylation state of MSL10 also regulated its ability to induce cell death when transiently expressed in Nicotiana benthamiana leaves but did not affect subcellular localization, assembly, or channel behavior. Finally, the N-terminal domain of MSL10 was sufficient to induce cell death in tobacco, independent of phosphorylation state. We conclude that the plant-specific N-terminal domain of MSL10 is capable of inducing cell death, this activity is regulated by phosphorylation, and MSL10 has two separable activities—one as an ion channel and one as an inducer of cell death. These findings further our understanding of the evolution and significance of mechanosensitive ion channels.     

Failure of exogenous NADH and cytochrome c to support energy-dependent swelling of mitochondria

The possibility of direct oxidation of external NADH in rat liver mitochondria and of the inner membrane potential generation in this process is still not clear. In the present work, the energy-dependent swelling of mitochondria in the medium containing valinomycin and potassium acetate was measured as one of the main criteria of the proton-motive force generation by complex III, complex IV, and both complexes III and IV of the respiratory chain. Mitochondria swelling induced by external NADH oxidation was compared with that induced by succinate or ferrocyanide oxidation, or by electron transport from succinate to ferricyanide. Mitochondria swelling, nearly equal to that promoted by ferrocyanide oxidation, was observed under external NADH oxidation, but only after the outer mitochondrial membrane was ruptured as a result of the swelling-contraction cycle, caused by succinate oxidation and its subsequent inhibition. In this case, significantly accelerated intermembrane electron transport and well-detected inner membrane potential generation, in addition to mitochondria swelling, were also observed. Presented results suggest that exogenous NADH and cytochrome c do not support the inner membrane potential generation in intact rat liver mitochondria, because the external NADH-cytochrome c reductase system, oriented in the outer mitochondrial membrane toward the cytoplasm, is inaccessible for endogenous cytochrome c reduction; as well, the inner membrane cytochrome c oxidase is inaccessible for exogenous cytochrome c oxidation.