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At least 217 records · Page 12Linked to original sources
PubMed1999

Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22.54 complex.

Members of the mitochondrial carrier family such as the ADP/ATP carrier (AAC) are composed of three structurally related modules. Here we show that each of the modules contains a mitochondrial import signal recognized by Tim10 and Tim12 in the intermembrane space. The first and the second module are translocated across the outer membrane independently of the membrane potential, DeltaDeltapsipsi, but they are not inserted into the inner membrane. The third module interacts tightly with the TOM complex and thereby prevents complete translocation of the precursor across the outer membrane. At this stage, binding of a TIM9.10 complex confers a topology to the translocation intermediate which reflects the modular structure of the AAC. The precursor is then transferred to the TIM9.10.12 complex, still interacting with the TOM complex. Release of the precursor from the TOM complex and insertion into the inner membrane by the TIM22.54 complex requires a DeltaDeltapsipsi-responsive signal in the third module.

Binding Sites↗
PubMed2003

Rotating disk potentiometry for inner solution optimization of low-detection-limit ion-selective electrodes.

The extent of optimization of the lower detection limit of ion-selective electrodes (ISEs) can be assessed with an elegant new method. At the detection limit (i.e., in the absence of primary ions in the sample), one can observe a reproducible change in the membrane potential upon alteration of the aqueous diffusion layer thickness. This stir effect is predicted to depend on the composition of the inner solution, which is known to influence the lower detection limit of the potentiometric sensor dramatically. For an optimized electrode, the stir effect is calculated to be exactly one-half the value of the case when substantial coextraction occurs at the inner membrane side. In contrast, there is no stir effect when substantial ion exchange occurs at the inner membrane side. Consequently, this experimental method can be used to determine how well the inner filling solution has been optimized. A rotating disk electrode was used in this study because it provides adequate control of the aqueous diffusion layer thickness. Various ion-selective membranes with a variety of inner solutions that gave different calculated concentrations of the complex at the inner membrane side were studied to evaluate this principle. They contained the well-examined silver ionophore O,O' '-bis[2-(methylthio)ethyl]-tert-butylcalix[4]arene, the potassium ionophore valinomycin, or the iodide carrier [9]mercuracarborand-3. Stir effects were determined in different background solutions and compared to theoretical expectations. Correlations were good, and the results encourage the use of such stir-effect measurements to optimize ISE compositions for real-world applications. The technique was also found to be useful in estimating the level of primary ion impurities in the sample. For an iodide-selective electrode measured in phosphoric acid, for example, apparent iodide impurity levels were calculated as 5 x 10(-10) M.

Ion-Selective Electrodes↗
PubMed2003

Ischemia-reperfusion injury in the aged heart: role of mitochondria.

The aged heart sustains greater injury during ischemia and reperfusion compared to the adult heart. Aging decreases oxidative phosphorylation and the activity of complexes III and IV only in interfibrillar mitochondria (IFM) that reside among the myofibrils, whereas subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. The peptide subunit composition of complexes III and IV is intact in aging. The aging defect in complex IV is in the inner membrane lipid environment. The defect in complex III is within the ubiquinol binding site of the cytochrome b subunit. Following ischemia, in the aged heart both SSM and IFM sustain additional decreases in complex III and complex IV activity. In contrast to the aging defect, with ischemia the subunits of complex IV appear to be damaged. Ischemia inactivates the iron-sulfur peptide subunit in complex III. Mitochondria are the major source of the reactive oxygen species that are generated during myocardial ischemia. Complex III is the major site of mitochondrial oxyradical production during ischemia in the adult heart. The role of complex III in the oxidative damage sustained by the aged heart during ischemia, as well as the potential contribution of aging defects in electron transport to ischemic damage in the aged heart, deserves further study. We propose that following ischemic damage to the electron transport chain, the production and release of reactive oxygen species increases from mitochondria in the aged heart, leading to additional damage during reperfusion.

Age Factors↗
PubMed2001

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria.

The type II secretion machinery allows most Gram-negative bacteria to deliver virulence factors into their surroundings. We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. The GspE-GspF-GspL-GspM complex was demonstrated by two methods: (i) co-immunoprecipitation of GspE-GspF-GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two-hybrid system between GspF and GspE, GspF and GspL, GspL and GspM. GspL was found to have an essential role in complex formation. We propose a model in which the GspE-GspF-GspL-GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles. By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus.

Bacterial Outer Membrane Proteins↗
PubMed2004

tRNA-triggered ATP hydrolysis and generation of membrane potential by the leishmania mitochondrial tRNA import complex.

Translocation of tRNAs across mitochondrial membranes is a receptor-mediated active transport process requiring ATP. A large tRNA import complex from the inner membrane of Leishmania mitochondria catalyzes translocation into phospholipid vesicles. In this reconstituted system, the import substrate tRNA(Tyr)(GUA) specifically stimulated hydrolysis of ATP within the vesicles, with the subsequent generation of a membrane potential by pumping out of protons, as shown by the protonophore-sensitive uptake of the potential-sensitive dye rhodamine 123. Generation of membrane potential was dependent on ATP hydrolysis, and inhibited by oligomycin, recalling the proton-translocation mechanism of the respiratory F(1)-F(0)-ATPase. For translocation of tRNA, ATP could be replaced by low pH of the medium, but proton-dependent import was resistant to oligomycin. Moreover, ATP hydrolysis, generation of membrane potential and tRNA uptake were inhibited by carboxyatractyloside, a specific inhibitor of mitochondrial ATP-ADP translocase, implying an ATP requirement within the vesicles. These observations imply a gating mechanism in which tRNA, on binding to its receptor, triggers the energetic activation of the complex, leading to the opening of import channels.

Adenosine Triphosphatases↗
PubMed2003

The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans.

Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multicellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.

Amino Acid Sequence↗
PubMed1995

The C terminus of SecA is involved in both lipid binding and SecB binding.

Using C-terminal deletion mutations in secA, we localized the previously proposed (Breukink, E., Keller, R.C. A., and de Kruijff, B. (1993), FEBS Lett. 331, 19-24) second lipid binding site on SecA. Since removal of these residues completely abolished the property of SecA to cause aggregation of negatively charged phosphatidyl-glycerol vesicles, we conclude that the C-terminal 70 amino acid residues of SecA are involved in lipid-binding. The C-terminal 70 amino acid residues of SecA are important for efficient in vitro translocation of the SecB-dependent precursor of PhoE across inverted inner membrane vesicles. Moreover, in vivo studies showed that this region is essential for growth. SecB and a SecB-precursor complex were shown to inhibit the SecA-mediated lipid vesicle aggregation, suggesting that the overall acidic SecB protein binds at or near the second lipid binding site on SecA. This together with the observation that the SecA mutant protein lacking the C-terminal 70 residues had a strongly reduced ability to mediate binding of SecB-precursor complexes to inverted inner membrane vesicles demonstrates that the C terminus of SecA is also involved in SecB binding.

Adenosine Triphosphatases↗
PubMed2005

A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex.

Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.

ATP-Dependent Proteases↗
PubMed2002

Su e of the yeast F1Fo-ATP synthase forms homodimers.

The yeast F(1)F(o)-ATP synthase forms a dimeric complex in the mitochondrial inner membrane. Dimerization of two F(1)F(o) monomeric complexes involves the physical association of two membrane-embedded F(o) sectors and in a manner, which is dependent on the F(o) subunit, Su e. Sequence analysis of Su e protein family members indicated the presence of a conserved coiled-coil motif. As this motif is often the basis for protein homodimerization events, it was hypothesized that Su e forms homodimers in the inner membrane and that formation of Su e dimers between two neighboring F(o) complexes would facilitate dimerization of the F(1)F(o)-ATP synthase complex (Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Schägger, H. (1998) EMBO J. 17, 7170-7178). Using a histidine-tagged derivative of yeast Su e, Su e-His(12), combined with cross-linking and affinity purification approaches, we have directly demonstrated the ability of the yeast Su e protein to form homodimers. Functionality of the Su e-His(12) derivative was confirmed by its ability to assemble into the ATP synthase complex and to support its dimerization in the Deltasu e null mutant yeast cells. The close association of two neighboring Su e proteins was also demonstrated using cross-linking with Cu(2+), which binds and cross-links a unique Cys residue in neighboring Su e proteins. Finally, we propose a model for the molecular basis of the homodimerization of the Su e proteins.

Dimerization↗
PubMed1985

Adriamycin as a probe for the transversal distribution of cardiolipin in the inner mitochondrial membrane.

The ability of adriamycin to complex cardiolipin was used to determine the distribution of cardiolipin across the inner membrane of rat liver and heart mitochondria. In both mitochondrial types, about 57 +/- 5% of the total cardiolipin was found to be located in the cytoplasmic face of the inner membrane. Mitochondria and mitoplasts were used to study the cytoplasmic face of the inner membrane, purified submitochondrial vesicles with inverted membrane orientation for the matrix face. The cardiolipin amount titrated by adriamycin in the latter was found to be complementary to the amount titrated in the cytoplasmic face. The adriamycin association constant determined for the first saturation level of mitochondria was in good agreement with the value published by Goormaghtigh et al. (Goormaghtigh, E., Chatelain, P., Caspers, J., and Ruysschaert, J. M. (1980) Biochim. Biophys. Acta 597, 1-14) for cardiolipin in artificial membranes. Two binding plateaus were observed when increasing amounts of adriamycin were added to mitochondria. The plateau at higher concentrations is conveniently explained by the penetration of adriamycin into mitochondria and the titration of cardiolipin in the matrix face. Scatchard plot analysis of the binding curves leading to the two plateaus produced almost identical association constants. The total amount of cardiolipin in mitochondria calculated from curves of this type corresponded to the total amount of cardiolipin determined by phosphate analysis of extracts, analyzed by thin layer chromatography.

Animals↗
PubMed1981

Orientation of complex III in the yeast mitochondrial membrane: labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate.

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [125I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochrome b revealed that the two core proteins and cytochrome b were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochrome c1 and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromes b and c1 was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15-30% of ;the total dithionite-reducible b and 100% of the cytochrome c1: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochrome b but insignificant amounts of cytochrome c1. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.

Diazonium Compounds↗
PubMed1999

Mechanisms of protein translocation into mitochondria.

Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins. At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified. These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix. The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components. Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP. The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase. The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex. A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins.

Animals↗
PubMed1994

Modulation of rat heart mitochondrial function and the production of reactive oxygen by vitamin E deficiency.

Vitamin E, an antioxidant present in all cellular membranes, is associated with protein complexes in the inner mitochondrial membranes and may affect oxidative changes which occur in these organelles when heart tissue is subjected to hypoxia. The effect of 60 min hypoxia, after a 30 min normoxic equilibration period, on the function and the production of reactive oxygen species (ROS) by cardiac mitochondria from rats fed vitamin E sufficient or deficient diets for 9 weeks was examined. Mitochondria from the hearts of rats fed vitamin E deficient diets had 40-fold less vitamin E and were more susceptible to lipid peroxidation, as compared to heart mitochondria from rats fed vitamin E sufficient diet. Perfusion with normoxic, but not hypoxic, media significantly decreased cardiac vitamin E in deficient, but not sufficient rats. Hypoxia decreased the production of ROS by mitochondria from vitamin E sufficient hearts, compared to normoxia. A similar level of ROS production was seen after hypoxia in mitochondria from vitamin E deficient hearts. However, vitamin E deficiency alone decreased the production of ROS by mitochondria from normoxic hearts, relative to vitamin E sufficient animals. Under all conditions where the production of ROS was decreased, 1 microM calcium increased production to the maximum levels seen in vitamin E sufficient, normoxic heart mitochondria. Mitochondrial function was depressed in mitochondria from hypoxic hearts as compared to mitochondria from normoxic hearts of vitamin E sufficient rats. A similar depression of mitochondrial function was not seen in mitochondria from hypoxic hearts of vitamin E deficient rats. Compensatory changes in response to long-term vitamin E deficiency may be responsible for the differences in response to hypoxia of mitochondria from vitamin E sufficient and deficient rats.

Animals↗
PubMed2000

The Agrobacterium T-DNA transport pore proteins VirB8, VirB9, and VirB10 interact with one another.

The VirB proteins of Agrobacterium tumefaciens form a transport pore to transfer DNA from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, VirB7, VirB8, VirB9 and VirB10. Using the yeast two-hybrid assay, we observed that VirB8, VirB9, and VirB10 interact with one another. In vitro studies using protein fusions demonstrated that VirB10 interacts with VirB9 and itself. These results suggest that the outer membrane VirB7-VirB9 complex interacts with the inner membrane proteins VirB8 and VirB10 for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of VirB8 and VirB9. All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of VirB9 are involved in its homotypic interaction, suggesting that VirB9 forms a higher oligomer. We observed that the alteration of serine at position 87 of VirB8 to leucine abolished its DNA transfer function. Studies on the interaction of the mutant protein with the other VirB proteins showed that the VirB8S87L mutant is defective in interaction with VirB9. The mutant, however, interacted efficiently with VirB8 and VirB10, suggesting that the VirB8-VirB9 interaction is essential for DNA transfer.

Bacterial Proteins↗
PubMed2006

The mitochondrial energy transduction system and the aging process.

Aged mammalian tissues show a decreased capacity to produce ATP by oxidative phosphorylation due to dysfunctional mitochondria. The mitochondrial content of rat brain and liver is not reduced in aging and the impairment of mitochondrial function is due to decreased rates of electron transfer by the selectively diminished activities of complexes I and IV. Inner membrane H(+) impermeability and F(1)-ATP synthase activity are only slightly affected by aging. Dysfunctional mitochondria in aged rodents are characterized, besides decreased electron transfer and O(2) uptake, by an increased content of oxidation products of phospholipids, proteins and DNA, a decreased membrane potential, and increased size and fragility. Free radical-mediated oxidations are determining factors of mitochondrial dysfunction and turnover, cell apoptosis, tissue function, and lifespan. Inner membrane enzyme activities, such as those of complexes I and IV and mitochondrial nitric oxide synthase, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests. The same enzymatic activities correlated positively with mice survival and negatively with the mitochondrial content of lipid and protein oxidation products. Conditions that increase survival, as vitamin E dietary supplementation, caloric restriction, high spontaneous neurological activity, and moderate physical exercise, ameliorate mitochondrial dysfunction in aged brain and liver. The pleiotropic signaling of mitochondrial H(2)O(2) and nitric oxide diffusion to the cytosol seems modified in aged animals and to contribute to the decreased mitochondrial biogenesis in old animals.

Aging↗
PubMed1988

Mitochondrial autoantigens in primary biliary cirrhosis. Association of disease-specific determinants with a subunit of complex I (NADH-ubiquinone reductase) of the inner mitochondrial membrane.

Anti-mitochondrial autoantibodies (AMA) from patients with primary biliary cirrhosis (PBC) were analysed for fine specificity by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA showed that complex I (NADH-ubiquinone reductase) from beef heart mitochondria completely inhibited the binding of AMA to mitochondrial inner membranes (SMP), indicating that the major mitochondrial antigens are located in complex I. Immunoblot analysis of beef heart SMP, complex I and the iron sulphur (IP) subfraction of complex I revealed several antigens, one of which (75 kDa) reacted with all PBC sera but not with the additional autoimmune sera tested. Resolution of SMP or complex I by two-dimensional electrophoresis yielded in both preparations a polypeptide of 75 kDa with an isoelectric point of 6.4, which reacted with PBC serum and with rabbit antisera raised against the 75,000 subunit of complex I. In immunoblot experiments, the antigenicity of the 75,000 polypeptide in SMP, complex I, and the IP subfraction is increased by prior reduction of the sample with mercaptoethanol. This suggests a similarity to the PBC-specific 'M-2' antigen, which is also sensitive to sulphur reagents. The data indicate that the 75 kDa polypeptide of complex I is a major mitochondrial antigen binding AMA in PBC sera, and allows us to identify the location and probable function of the PBC antigen.

Animals↗
PubMed2003

Mitochondrial biogenesis and the role of the protein import pathway.

PURPOSE: The importance of the mitochondrial protein import pathway, discussed relative to other steps involved in the overall biogenesis of the organelle, are reviewed. RESULTS: Mitochondrial biogenesis is a product of complex interactions between the nuclear and mitochondrial genomes. Signaling pathways, such as those activated by exercise, initiate the activation of transcription factors that increase the production of mRNA from nuclear and mitochondrial DNA. Nuclear gene products are translated in the cytosol as precursor proteins with inherent targeting signals. These precursor proteins interact with molecular chaperones that direct them to the import machinery of the outer membrane (Tom complex). The precursor is unfolded and transferred through the outer membrane, across the intermembrane space to the mitochondrial inner membrane translocases (Tim complex). Intramitochondrial components (mtHSP70) pull the precursor into the matrix, cleave off the targeting sequence (mitochondrial processing peptidase), and refold the protein (HSP60, cpn10) into its mature conformation. Physiological stressors such as contractile activity and thyroid hormone accelerate protein import into the mitochondria, coincident with an increase in the expression of some components of the import machinery. This is important for the overall expansion of the mitochondrial reticulum. Conversely, impairments in the import process can be a cause of mitochondrial dysfunction and disease. CONCLUSIONS: Efforts to further characterize the components of the import machinery, to define the role of specific machinery components on the import rate, and to examine protein import function in a variety of mitochondrial diseases are warranted.

Carrier Proteins↗
PubMed1989

Biochemical characterization of avirulent Agrobacterium tumefaciens chvA mutants: synthesis and excretion of beta-(1-2)glucan.

The chvA gene product of Agrobacterium tumefaciens is required for virulence and attachment of bacteria to plant cells. Three chvA mutants were studied. In vivo, they were defective in the synthesis, accumulation, and secretion of beta-(1-2)glucan; however, the 235-kilodalton (kDa) protein known to be involved in the synthesis of beta-(1-2)glucan (A. Zorreguieta and R. Ugalde, J. Bacteriol. 167:947-951, 1986) was present and active in vitro. was present and active in vitro. Two molecular forms of cyclic beta-(1-2)glucan, designated types I and II, were resolved by gel chromatography. Type I beta-(1-2)glucan was substituted with nonglycosidic residues, and type II beta-(1-2)glucan was nonsubstituted. Wild-type cells accumulated type I beta-(1-2)glucan, and chvA mutant cells accumulated mainly type II beta-(1-2)glucan and a small amount of type I beta-(1-2)glucan. Inner membranes of wild-type and chvA mutants formed in vitro type II nonsubstituted beta-(1-2)glucan. A 75-kDa inner membrane protein is proposed to be the chvA gene product. chvA mutant inner membranes had increased levels of 235-kDa protein; partial trypsin digestion patterns suggested that the 235-kDa protein (the gene product of the chvB region) and the gene product of the chvA region form a complex in the inner membrane that is involved in the synthesis, secretion, and modification of beta-(1-2)glucan. All of the defects assigned to the chvA mutation were restored after complementation with plasmid pCD522 containing the entire chvA region.

Carrier Proteins↗