Search

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial [Ca.sup.2+] overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential ([DELTA][[PSI].sub.m]) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS. [Ca.sup.2+], and mPTP opening. The magnitude of APC-induced decrease in [DELTA][[PSI].sub.m] was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in [DELTA][[PSI].sub.m]. In cardiomyocytes, [DELTA][[PSI].sub.m], ROS, mPTP opening, and cytosolic and mitochondrial [Ca.sup.2+] were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and [Ca.sup.2+] uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not [Ca.sup.2+] uptake. However, in stressed cardiomyocytes, a similar decrease in [DELTA][[PSI].sub.m] attenuated both cytosolic and mitochondrial [Ca.sup.2+] accumulation. In conclusion, a partial decrease in [DELTA][[PSI].sub.m] underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in [DELTA][[PSI].sub.m] primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial [Ca.sup.2+] uptake. cardioprotection; oxidative stress; mitochondria; reactive oxygen species doi: 10.1152/ajpcell.00006.2010.

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step--specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. doi/10.1083/jcb.200905134

Two dynamin-related protein (DRP) families are essential for fusion of the outer and inner mitochondrial membranes, Fzol (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opal (mammals), respectively. Fzo1/Mfns possess two medial transmembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/ Opal are present in cells as long (I) isoforms that are anchored via the N terminus to the inner membrane, and short (s) isoforms were predicted to be soluble in the intermembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in membrane fusion. Our analysis indicates that in the absence of a membrane, I-and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

Transcriptional upregulation of the proapoptotic BCL2 family protein NIX limits red blood cell formation and can cause heart failure by inducing cell death, but the requisite molecular events are poorly defined. Here, we show complementary mechanisms for NIX-mediated cell death involving direct and ER/sarcoplasmic reticulum--mediated (ER/SR-mediated) mitochondria disruption. Endogenous cardiac NIX and recombinant NIX localize both to the mitochondria and to the ER/SR. In genetic mouse models, cardiomyocyte ER/SR calcium stores are proportional to the level of expressed NIX. Whereas Nix ablation was protective in a mouse model of apoptotic cardiomyopathy, genetic correction of the decreased SR calcium content of Nix-null mice restored sensitivity to cell death and reestablished cardiomyopathy. Nix mutants specific to ER/SR or mitochondria activated caspases and were equally lethal, but only ER/SR-Nix caused loss of the mitochondrial membrane potential. These results establish a new function for NIX as an integrator of transcriptional and calcium-mediated signals for programmed cell death., Introduction Apoptosis is essential for normal organism development and tissue homeostasis but creates or contributes to disease when it deviates from normal physiological constraints. Members of the BCL2 family of [...]

Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity, as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 effector molecules, BAX and BAK. Sustainable cellular stress induces proteins (e.g., BID, BIM, and cytosolic p53) capable of directly activating BAX and/or BAK, but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g., BCL-2, BCL-xL, and MCL-1). In the event of accumulated or marked cellular stress, a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator protein-mediated MOMP. We examined the effect of ABT-737, a BCL-2 antagonist, and PUMA, a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire, with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis, which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data show that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires 'covert' levels of direct activators of BAX and BAK at the OMM. BCL-2 family | mitochondria | MOMP | PUMA

Kidney proximal tubules develop a severe but highly reversible energetic deficit due to nonesterified fatty acid (NEFA)-induced dissipation of mitochondrial membrane potential ([DELTA][PSI]m) during reoxygenation after severe hypoxia. To assess the mechanism for this behavior, we have compared the efficacies of different NEFA for inducing mitochondrial deenergization in permeabilized tubules measured using safranin O uptake and studied the modification of NEFA-induced deenergization by inhibitors of the ADP/ATP carrier and glutamate using both normoxic tubules treated with exogenous NEFA and tubules deenergized during hypoxia-reoxygenation (H/R). Among the long-chain NEFA that accumulate during H/R of isolated tubules and ischemia-reperfusion of the kidney in vivo, oleate, linoleate, and arachidonate had strong effects to dissipate [DELTA][[PSI].sub.m] that were slightly greater than palmitate, while stearate was inactive at concentrations reached in the cells. This behavior correlates well with the protonophoric effects of each NEFA. Inhibition of the ADP/ATP carrier with either carboxyatractyloside or bongkrekic acid or addition of glutamate to compete for the aspartate/ glutamate carrier improved [DELTA][[PSI].sub.m] in the presence of exogenous oleate and after H/R. Effects on the two carders were additive and restored safranin O uptake to as much as 80% of normal under both conditions. The data strongly support NEFA cycling across the inner mitochondrial membrane using anion carriers as the main mechanism for NEFA-induced deenergization in this system and provide the first evidence for a contribution of this process to pathophysiological events that impact importantly on energetics of intact cells. acute renal failure; kidney; membrane potential; mitochondria doi:10.1152/ajprenal.00476.2006.

Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252-E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X2-(S/T)-[X.sub.3]-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mitoNEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates. mitochondria | oxidative phosphorylation | pioglitazone

Mitochondria are central for various cellular processes that include ATP production, intracellular [Ca.sup.2+] signaling, and generation of reactive oxygen species. Neurons critically depend on mitochondrial function to establish membrane excitability and to execute the complex processes of neurotransmission and plasticity. While much information about mitochondrial properties is available from studies on isolated mitochondria and dissociated cell cultures, less is known about mitochondrial function in intact neurons in brain tissue. However, a detailed description of the interactions between mitochondrial function, energy metabolism, and neuronal activity is crucial for the understanding of the complex physiological behavior of neurons, as well as the pathophysiology of various neurological diseases. The combination of new fluorescence imaging techniques, electrophysiology, and brain slice preparations provides a powerful tool to study mitochondrial function during neuronal activity, with high spatiotemporal resolution. This review summarizes recent findings on mitochondrial [Ca.sup.2+] transport, mitochondrial membrane potential ([DELTA][[PSI].sub.m]), and energy metabolism during neuronal activity. We will first discuss interactions of these parameters for experimental stimulation conditions that can be related to the physiological range. We will then describe how mitochondrial and metabolic dysfunction develops during pathological neuronal activity, focusing on temporal lobe epilepsy and its experimental models. The aim is to illustrate that 1) the structure of the mitochondrial compartment is highly dynamic in neurons, 2) there is a fine-tuned coupling between neuronal activity and mitochondrial function, and 3) mitochondria are of central importance for the complex behavior of neurons. brain slice preparation; electrophysiology; NADH; mitochondrial calcium transport; temporal lobe epilepsy

The human isoform 2 of pantothenate kinase (PanK2) is localized to the mitochondria, and mutations in this protein are associated with a progressive neurodegenerative disorder. PanK2 inhibition by acetyl-CoA is so stringent (IC50 < 1 [micro]M) that it is unclear how the enzyme functions in the presence of intracellular CoA concentrations. Palmitoylcarnitine was discovered to be a potent activator of PanK2 that functions to competitively antagonize acetyl-CoA inhibition. Acetyl-CoA was a competitive inhibitor of purified PanK2 with respect to ATP. The interaction between PanK2 and acetylCoA was stable enough that a significant proportion of the purified protein was isolated as the PanK2*acetyl-CoA complex. The longchain acylcarnitine activation of PanK2 explains how PanK2 functions in vivo, by providing a positive regulatory mechanism to counteract the negative regulation of PanK2 activity by acetyl-CoA. Our results suggest that PanK2 is located in the mitochondria to sense the levels of palmitoylcarnitine and up-regulate CoA biosynthesis in response to an increased mitochondrial demand for the cofactor to support [beta]-oxidation. carnitine | coenzyme A | [beta]-oxidation | pantothenate kinase-associated neurodegeneration

A study was conducted to clarify the biological role for the interaction between Bcl-[X.sub.L] and voltage-dependent anion channel (VDAC). The results suggest that Bcl-[X.sub.L] could bind to one or two VDAC molecules forming heterodimers and heterotrimers.

Mitochondrial ion channels are the hub of all cellular functions, crucial to the mechanism of energy supply and demand matching. They play a central role as determinants of cell life and death in a variety of pathologies. A review of research on mitochondrial ion channels and efforts to identify their molecular correlates is provided.

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

We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial [Ca.sup.2+]-sensitive [K.sup.+] (mt[K.sub.Ca]) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mt[K.sub.Ca] channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial [K.sup.+] influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential ([DELTA][[psi].sub.m]). To test this hypothesis, we measured effects of NS-1619, a putative mt[K.sub.Ca] channel opener, and valinomycin, a [K.sup.+] ionophore, on mitochondrial respiration, [DELTA][[psi].sub.m], and ROS generation in guinea pig heart mitochondria. NS-1619 (30 [micro]M) increased state 2 and 4 respiration by 5.2 [+ or -] 0.9 and 7.3 [+ or -] 0.9 nmol [O.sub.2]*[min.sup.-1]*mg [protein.sup.-1], respectively, with the NADH-linked substrate pyruvate and by 7.5 [+ or -] 1.4 and 11.6 [+ or -] 2.9 nmol [O.sub.2]* [min.sup.-1]*mg [protein.sup.-1], respectively, with the FAD[H.sub.2]-linked substrate succinate (+ rotenone); these effects were abolished by the mt[K.sub.Ca] channel blocker paxilline. [DELTA][[psi].sub.m] was not decreased by 10-30 [micro]M NS-1619 with either substrate, but [H.sub.2][O.sub.2] release was increased by 44.8% (65.9 [+ or -] 2.7% by 30 [micro]M NS-1619 vs. 21.1 [+ or -] 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase [H.sub.2][O.sub.2] release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained [DELTA][[psi].sub.m], despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix [K.sup.+] influx during states 2 and 4 increases mitochondrial respiration while maintaining [DELTA][[psi].sub.m]; this allows singlet electron uptake by [O.sub.2] and ROS generation. mitochondrial bioenergetics; heart mitochondria

A cornucopia of human pathologies, including several for which no efficient therapy is currently available, exhibit impaired apoptosis. The description of the multiple signaling pathways leading to apoptosis and the [...]

I. Introduction II. Calcium Binding Mitochondrial Carrier Gene and Proteins III. Aspartate-Ghitamate Carriers A. Mammalian AGCs B. Nonmammalian AGCs C. [Ca.sup.2+] regulation of the MAS D. Other mechanisms of [Ca.sup.2+] [...]

I. Introduction II. An Overview of Cell Death Pathways A. Apoptosis B. Autophagic cell death C. Necrosis D. Mitotic catastrophe III. Detection of Mitochondrial Membrane Permeabilization A. Signs of outer [...]

Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results in localized oxidative stress but does not impair the ability of the plant to withstand a wide range of abiotic stresses. However, absence of UCP1 results in a photosynthetic phenotype. Specifically there is a restriction in photorespiration with a decrease in the rate of oxidation of photorespiratory glycine in the mitochondrion. This change leads to an associated reduced photosynthetic carbon assimilation rate. Collectively, these results suggest that the main physiological role of UCP1 in Arabidopsis leaves is related to maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism. mitochondria | Arabidopsis | electron transport | reactive oxygen species | photorespiration

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Here we show that phosphorylation and cleavage of the vertebrate-specific P[beta] domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position [Ser.sup.77]-[Ala.sup.78] that is required to initiate PARL-induced mitochondrial fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology. protein evolution | protein phosphorylation | rhomboids | mitochondrial dynamics | intramebrane proteolysis

Capillary electrophoresis (CE) with postcolumn laser-induced fluorescence detection (LIF) was used to analyze single skeletal muscle fibers from young and old rats. Due to selective labeling of mitochondria with 10-N-nonyl acridine orange, the zeptomole ([10.sup.-21] mole) sensitivity, and the high separation power, three properties of individual mitochondrial particles were revealed: the number, the distributions of cardiolipin, and their electrophoretic mobilities. Type I fibers had more mitochondrial particles and cardiolipin per particle than did type IIb fibers from rats of similar age. Individual fibers of the same fiber type from young rats contained more mitochondrial particles and cardiolipin per particle than did fibers from old rats. There were fiber typespecific and age-specific differences in the electrophoretic mobility of individual mitochondrial particles. The CE-LIF results of individual mitochondrial particles are the first of their kind in that they reveal fiber type-specific and age-specific differences that are not obviously noticed in bulk measurements of heterogeneous tissues.

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions ([Zn.sup.2+]) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free [Zn.sup.2+] with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (Zn[Cl.sub.2]) to PAEC. We observed that [Zn.sup.2+]-exposed PAECs exhibited a dose-dependent increase in superoxide ([O.sup.-.sub.2]*) generation that was localized to the mitochondria. Furthermore, we found [Zn.sup.2+]-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the [Zn.sup.2+]-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a [Zn.sup.2+]-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and [Zn.sup.2+]-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of [Zn.sup.2+] homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of [Zn.sup.2+] levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress. reactive nitrogen species; apoptosis; mitochondrial dysfunction doi:10.1152/ajpcell.00509.2005

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215-232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.

Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating 'assembly on demand' of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process.

Fatty acid translocase (FAT/ CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 [micro]M) palmitate oxidation was decreased by 95% (P < 0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P < 0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation. fatty acid translocase; long-chain fatty acid

Mitochondrial carriers link biochemical pathways in the cytosol and mitochondrial matrix by transporting substrates across the inner mitochondrial membrane. Substrate recognition is specific for each carrier, but sequence similarities suggest the carriers have similar structures and mechanisms of substrate translocation. By considering conservation of amino acids, distance and chemical constraints, and by modeling family members on the known structure of the ADP/ATP translocase, we have identified a common substrate binding site. It explains substrate selectivity and proton coupling and provides a mechanistic link to carrier opening by substrate-induced perturbation of the salt bridges that seal the pathway to and from the mitochondrial matrix. It enables the substrate specificity of uncharacterized mitochondrial carriers to be predicted. comparative modeling | membrane protein | sequence alignment

Saccharomyces cerevisiae Mdm38 and YIh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and YIh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm384 mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas YIh47 plays a minor role in this process. Both Mdm38 and YIh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.

Kidney proximal tubules exhibit decreased ATP and reduced, but not absent, mitochondrial membrane potential ([DELTA][psi]m) during reoxygenation after severe hypoxia. This energetic deficit, which plays a pivotal role in overall cellular recovery, cannot be explained by loss of mitochondrial membrane integrity, decreased electron transport, or compromised [F.sub.1][F.sub.0]-ATPase and adenine nucleotide translocase activities. Addition of oleate to permeabilized tubules produced concentration-dependent decreases of [DELTA][psi]m measured by safranin O uptake (threshold for oleate = 0.25 [micro]M, 1.6 nmol/mg protein; maximal effect = 4 [micro]M, 26 nmol/mg) that were reversed by delipidated BSA (dBSA). Cell nonesterified fatty acid (NEFA) levels increased from < 1 to 17.4 nmol/mg protein during 60-min hypoxia and remained elevated at 7.6 nmol/mg after 60 min reoxygenation, at which time ATP had recovered to only 10% of control values. Safranin O uptake in reoxygenated tubules, which was decreased 85% after 60-min hypoxia, was normalized by dBSA, which improved ATP synthesis as well. dBSA also almost completely normalized [DELTA][psi]m when the duration of hypoxia was increased to 120 min. In intact tubules, the protective substrate combination of [alpha]-ketoglutarate + malate ([alpha]-KG/MAL) increased ATP three- to fourfold, limited NEFA accumulation during hypoxia by 50%, and lowered NEFA during reoxygenation. Notably, dBSA also improved ATP recovery when added to intact tubules during reoxygenation and was additive to the effect of [alpha]-KG/MAL. We conclude that NEFA overload is the primary cause of energetic failure of reoxygenated proximal tubules and lowering NEFA substantially contributes to the benefit from supplementation with [alpha]-KG/MAL. acute renal failure; kidney; mitochondrial membrane potential

The retrograde signaling in the budding yeast Saccharomyces cerevisiae which involves multiple factors that sense and transmit mitochondrial signals to effect changes in nuclear gene expression is explored. Theses changes lead to a reconfiguration of metabolism to accommodate cells to defects in mitochondria.

ATP-sensitive [K.sup.+] channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance [Ca.sup.2+]-sensitive [K.sup.+] channel (B[K.sub.ca]) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the B[K.sub.ca] channel on inducing mitochondrial preconditioning during I/R and the role of [O.sub.2]-derived free radicals in modulating protection by putative mitochondrial (m)B[K.sub.ca] channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide ([O.sup.-.sub.2]*), and m[[Ca.sup.2+]] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. B[K.sub.ca] channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl) -5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of [O.sup.-.sub.2]*, or an antagonist of the B[K.sub.ca] channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less [O.sup.-.sub.2] * generation, decreased m[[Ca.sup.2+]], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mB[K.sub.ca] channels; 2) NS effects are blocked by B[K.sub.ca] antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning. cell signaling; ischemic biology; oxidant stress; energy metabolism; heart; mitochondria; reactive oxygen species; redox balance

We examined the effects of different physiological concentrations of glucose on cytoplasmic [Ca.sup.2+] handling and mitochondrial membrane potential ([DELTA][[psi].sub.m]) and insulin secretion in single mouse islet cells. The threshold for both glucose-induced changes in [Ca.sup.2+] and [DELTA][[psi].sub.m] ranged from 6 to 8 mM. Glucose step-jumps resulted in sinusoidal oscillations of cytoplasmic [Ca.sup.2+], whereas [DELTA][[psi].sub.m] reached sustained plateaus with oscillations interposed on the top of these plateaus. The amplitude of the [Ca.sup.2+] rise (height of the peak) did not vary with glucose concentration, suggesting a 'digital' rather than 'analog' character of this aspect of the oscillatory [Ca.sup.2+] response. The average glucose-dependent elevation of cytoplasmic [Ca.sup.2+] concentration during glucose stimulation reached saturation at 8 mM stimulatory glucose, whereas [DELTA][[psi].sub.m] showed a linear glucose dose-response relationship over the range of stimulatory glucose concentrations (4-16 mM). Glucose-dependent increases in insulin secretion correlated well with [DELTA][[psi].sub.m], but not with average [Ca.sup.2+] concentration. These data show that an ATP-dependent [K.sup.+] channel-independent pathway is operative at the single cell level and suggest mitochondrial metabolism may be a determining factor in explaining graded, glucose concentration-dependent increases in insulin secretion.

Stimulation of [beta]-adrenergic receptors ([beta]-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits [beta]-AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with [[beta].sub.1] integrins. Herein we tested the hypothesis that MMP-2 impairs [[beta].sub.1] integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited [beta]-AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated [beta]-AR-stimulated decreases in mitochondrial membrane potential. Overexpression of [[beta].sub.1] integrins using adenoviruses expressing the human [[beta].sub.1A]-integrin decreased [beta]-AR-stimulated cytochrome c release and apoptosis. Overexpression of [[beta].sub.1] integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the [[beta].sub.1] integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun N[H.sub.2]-terminal kinase; cytochrome c

A multisubunit translocase of the outer mitochondrial membrane (TOM complex) mediates both the import of mitochondrial precursor proteins into the internal compartments of the organelle and the insertion of proteins residing in the mitochondrial outer membrane. The proposed [beta]-barrel structure of Tom40, the pore-forming component of the translocase, raises the question of how the apparent uninterrupted [beta]-barrel topology can be compatible with a role of Tom40 in releasing membrane proteins into the lipid core of the bilayer. In this review, I discuss insertion mechanisms of proteins into the outer membrane and present alternative models based on the opening of a multisubunit [beta]-barrel TOM structure or on the interaction of outer membrane precursors with the outer face of the Tom40 [beta]-barrel structure.

Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)--Iike fold and a short N[H.sub.2]-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p-Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p N[H.sub.2]-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.

Nitric oxide (NO) modulates cellular metabolism by competitively inhibiting the reduction of [O.sub.2] at respiratory complex IV. The aim of this study was to determine whether this effect could enhance cell survival in the hypoxic solid tumor core by inducing a state of metabolic arrest in cancer cells. Mitochondria from human alveolar type II-like adenocarcinoma (A549) cells showed a fourfold increase in NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescence and sixfold increase in [Ca.sup.2+]-insensitive NO synthase (NOS) activity during equilibration from [Po.sub.2]s of 100 [right arrow] 23 mmHg, which was abolished by [N.sup.[omega]]-nitro-L-arginine methyl ester-HCl (L-NAME) and the inducible NOS (iNOS) inhibitor, [N.sup.6]-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL). Similarly, cytosolic and compartmented DAF-FM fluorescence increased in intact cells during a transition between ambient [Po.sub.2] and 23 mmHg and was abolished by transfection with iNOS antisense oligonucleotides (AS-ODN). In parallel, mitochondrial membrane potential ([DELTA][[PSI].sub.m]), measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), decreased to a lower steady state in hypoxia without change in glycolytic rate, adenylate energy charge, or cell viability. However, L-NAME or iNOS AS-ODN treatment maintained [DELTA][[PSI].sub.m] at normoxic levels irrespective of hypoxia and caused a marked activation of glycolysis, destabilization energy charge, and cell death. Comparison with other cancer-derived (H441) or native tissue-derived (human bronchial epithelial; alveolar type II) lung epithelial cells revealed that the hypoxic suppression of [DELTA][[PSI].sub.m] was common to cells that expressed iNOS. The controlled dissipation of [DELTA][[PSI].sub.m], absence of an overt glycolytic activation, and conservation of viability suggest that A549 cells enter a state of metabolic suppression in hypoxia, which inherently depends on the activation of iNOS as [Po.sub.2] falls. cancer; oxygen conformation; mitochondrial nitric oxide synthase; mitochondrial metabolism

Apoptosis and other forms of cell death are often controlled at one or more crucial steps involving the mitochondria. Mitochondrial outer membrane permeabilization (MOMP), cytochrome c-induced activation of APAF-1 and caspase activation is some of the procedures, which leads to the death of the cell.

Although recent studies focused on the contribution of mitochondrial [Ca.sup.2+] to the mechanisms of ischemia-reperfusion injury, the regulation of mitochondrial [Ca.sup.2+] under pathophysiological conditions remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential ([DELTA][[PSI].sub.m]) and mitochondrial [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.m]) at the physiological range of cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.c]; 300 nM) and investigated the regulation of [[[Ca.sup.2+]].sub.m] during both normal and dissipated [DELTA][[PSI].sub.m]. When [DELTA][[PSI].sub.m] was partially depolarized by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 0.01-0.1 [micro]M), there were dose-dependent decreases in [[[Ca.sup.2+]].sub.m]. When complete [[DELTA][PSI].sub.m] dissipation was achieved by FCCP (0.3-1 [micro]M), [[[Ca.sup.2+]].sub.m] remained at one-half of the control level despite no [Ca.sup.2+] influx via the [Ca.sup.2+] uniporter. The [DELTA][[PSI].sub.m] dissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, which indicates that [DELTA][[PSI].sub.m] dissipation opened the mitochondrial permeability transition pore (mPTP). After FCCP addition, inhibition of the mPTP by CsA caused further [[[Ca.sup.2+]].sub.m] reduction; however, inhibition of mitochondrial [Na.sup.+]/[Ca.sup.2+] exchange (mitoNCX) by a [Na.sup.+]-free solution abolished this [[[Ca.sup.2+]].sub.m] reduction. Cytosolic [Na.sup.+] concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal [DELTA][[PSI].sub.m] and 7.6 mM at [DELTA][[PSI].sub.m] dissipation. We conclude that 1) the mitochondrial [Ca.sup.2+] uniporter accumulates [Ca.sup.2+] in a manner that is dependent on [DELTA][[PSI].sub.m] at the physiological range of [[[Ca.sup.2+]].sub.c]; 2) [DELTA][[PSI].sub.m] dissipation opens the mPTP and results in [Ca.sup.2+] influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes [Ca.sup.2+] from the matrix even after [DELTA][[PSI].sub.m] dissipation. permeability transition pore; [Na.sup.+]/[Ca.sup.2+] exchange; depolarization; ischemia-reperfusion injury

The steady-state kinetics of nicotinamide adenine dinucleotide (NADH) entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools.

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial [beta]-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial [beta]-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the [beta]-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of [beta]-barrel proteins in the outer membrane.

The binding of mitochondrial presequences to the mitochondrial membrane occurs via hydrophobic rather than ionic interactions. The presequence forms an amphiphilic helical structure with hydrophobic leucines that interact with a hydrophobic patch in the receptor groove.

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of approximately 2.1 nm and a height of approximately 7 nm. Tom40 is the key structural element of the TOM core complex.

Research was conducted to examine Ca2+-induced changes of membrane molecular organization which may possibly be caused by the interaction between the cation and Ca2+-cardiolipin (CL). Inverted bovine heart submitochondrial particles were used as samples. Results provide strong evidence supporting Ca2+ binding to CL as one of the initial steps in the molecular mechanism of Ca2+-induced nonspecific inner mitochondrial membrane permeabilization.

It has been possible to identify a new principle of organization of a multi-subunit preprotein translocase. It was found that the multidomain protein Tom22, required for the higher-level organization of the translocase of the outer membrane machinery, acts not only acts as a receptor and trans binding site for preproteins, but also organizes the interaction between the channel and receptor subcomplexes at two levels. It was also established that Tom22 is needed to negatively regulate the open probability of the Tom40 channel.

Research was conducted to examine the dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition process. The steady-state fluorescence anisotropy of two probes, namely, 1,6-diphenyl-1,3,5-hexatriene and hematoporphyrin were investigated. Results indicate that the mitochondrial permeability transition is accompanied by a remarkable change in the membrane fluidity as probed by the anisotropy changes of labeled mitochondria.

A prominent beta-barrel channel protein spanning the mitochondrial outer membrane, human voltage-dependent anion-selective channel (VDAC), has been shown to bypass the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein. Synthetic liposomes harboring the cystosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP.

Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-[x.sub.L] synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-[x.sub.L], created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-[x.sub.L] did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore - the apoptotic pore - large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole.