Treatment Of Intact Mitochondria Research Articles

Cyclophilin D Modulates Mitochondrial FOF1 ATP Synthase by Interacting with the Lateral Stalk of the Complex

Blue-native gel electrophoresis purification and immunoprecipitation of FOF1 ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate crosslinked CyPD with the lateral stalk of ATP synthase, while no interactions with F1 sector subunits, the ATP synthase natural inhibitor protein IF1 and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis, and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes, and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.

Cyclophilin D Modulates Mitochondrial F0F1-ATP Synthase by Interacting with the Lateral Stalk of the Complex

Blue native gel electrophoresis purification and immunoprecipitation of F(0)F(1)-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F(1) sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.

IκB-α, the NF-κB Inhibitory Subunit, Interacts with ANT, the Mitochondrial ATP/ADP Translocator

The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators. An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space. IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.

Purification of All Forms of HeLa Cell Mitochondrial DNA and Assessment of Damage to It Caused by Hydrogen Peroxide Treatment of Mitochondria or Cells

A purification scheme for mitochondrial DNA (mtDNA) was designed which maximized the yield of all forms of the DNA while minimizing damage to the DNA during its isolation. Treatment of intact mitochondria with DNase I removed nuclear DNA and the avoidance of phenol and the isolation by CsCl density gradients in the absence of ethidium bromide and subsequent detection by Southern Hydridization dot-blots minimized DNA damage. Four different mtDNA forms free of apparent nuclear DNA were obtained: closed circular (I), open circular (II), linear (III), and a large multimer complex (C) which were characterized by agarose gel electrophoresis and electron microscopy. Using this procedure, mtDNA was obtained from both whole cells or intact mitochondria treated with H2O2. Significant fragmentation was observed after treatment at 37 degrees C, but not at 0 degrees C, and more damage was observed when treating whole cells than isolated mitochondria. Very low levels of 8-hydroxydeoxyguanosine were observed in all cases. However, at doses of H2O2 which were just lethal, neither increased DNA damage nor inactivation of cytochrome c oxidase was observed.

Cholate extracts of mitochondrial outer membranes increase inhibition by malonyl-CoA of carnitine palmitoyltransferase-I by a mechanism involving phospholipids.

It has been reported that sodium cholate can separate the catalytic component of carnitine palmitoyltransferase-I (CPT-I) from a putative malonyl-CoA-binding regulatory protein capable of conferring sensitivity to malonyl-CoA on CPT-II. We found that cholate preferentially extracted a contaminating malonyl-CoA-sensitive CPT from mitochondrial inner membranes. When cholate extracts of outer membranes were incubated either with cholate extracts of inner membranes or with osmotically swollen mitochondria, inhibition of CPT by malonyl-CoA was increased. Treatment of intact mitochondria with subtilisin abolished the increased inhibition by malonyl-CoA, suggesting that the outer-membrane CPT-I was responsible for the increased inhibition. Incubation of cholate extracts with proteinase K did not prevent the increased inhibition. Fractionation of the cholate extract indicated the presence of phospholipids. Addition of cardiolipin or phosphatidylglycerol to osmotically swollen mitochondria increased sensitivity of CPT to malonyl-CoA, but several other phospholipids did not. When cardiolipin was added to intact mitochondria from either starved or fed rats, there were large increases in inhibition by malonyl-CoA; sensitivity in mitochondria from starved rats increased to that normally observed with mitochondria from fed rats. These results suggest that phospholipids are responsible for the increased inhibition of CPT by malonyl-CoA with added cholate extracts and that changes in membrane composition may be involved in the physiological regulation of CPT-I.

Location of the dicyclohexylcarbodiimide-reactive glutamate residue in the bovine heart mitochondrial porin

The mitochondrial porin or VDAC (Voltage-Dependent Anion Channel), the pore-forming structure responsible for the high permeability of the outer mitochondrial membrane, was found to be one of only three mitochondrial proteins bound by [14C]dicyclohexylcarbodiimide (DCCD) at low dosages (1.5 nmol/mg of mitochondrial porin) (De Pinto, V., Tommasino, M., Benz, R., and Palmieri, F. (1985) Biochim. Biophys. Acta 813, 230-242). Treatment of intact mitochondria with DCCD results in the inhibition of their ability to binding hexokinase (Nakashima, R. A., Mangan, P. S., Colombini, M., and Pedersen, P. L. (1986) Biochemistry 25, 1015-1021). In the present study, mitochondrial porin was purified from [14C]DCCD-labeled mitochondria. The purified labeled porin was treated with the cleavage reagent CNBr and with the endoproteases trypsin and V8 from Staphylococcus aureus and blotted to polyvinylidene difluoride membrane. The transferred peptides were detected with Coomassie Blue dye, excised, and sequenced. The sequences of several labeled and unlabeled peptides were obtained and then overlapped. The region containing the [14C]DCCD radioactivity was limited to 50 amino acid residues and completely sequenced. Covalently incorporated [14C]DCCD was exclusively released at the position corresponding to glutamate 72. The DCCD-reactive residue is located in the 4th of 16 predicted transmembrane amphipathic beta-strands. When the sequence surrounding the DCCD site was compared to those surrounding the DCCD-reactive residue of other membrane proteins, no homology was apparent.

Localization of the NADH Kinase in the Inner Membrane of Yeast Mitochondria

The bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against the NADH kinase caused inactivation of the enzyme. On the contrary, no influence was observed upon the same treatment of intact mitochondria. p-Chloromercuribenzoate and eosin-5-maleimide inactivated the enzyme without affecting the matrix ATPase. The NADH kinase was enzymatically iodinated in mitoplasts, but not in the intact mitochondria. These results support the conclusion that NADH kinase is localized and functions at the intermembrane space side of the mitochondrial inner membrane. It is evident that the NADH kinase is encoded by nuclear gene(s) because it is synthesized in the presence of chloramphenicol or acriflavine, and a significant amount of the enzyme was detected in mitochondrial DNA-deficient mutants.

Inheritance of large mitochondrial RNA's in alfalfa

Several large RNA molecules that migrated to electrophoretic positions ranging from 1.7-10 kb were observed in preparation of alfalfa (Medicago sativa) mitochondria. F1 progenies inherited the RNA's from both maternal and paternal parents (Fig. 1). Treatment of intact mitochondria with RNase A failed to remove the RNA's, indicating that they were contained within an RNase impermeable compartment. Further purification of mitochondria in linear sucrose gradients failed to separate the RNA's from mitochondria. Transmission electron microscopic examination of sucrose gradient purified mitochondria revealed that mitochondria were free of contamination by virus-like particles, indicating that the RNA's were contained within the mitochondrion. Biparental inheritance of large mitochondrial RNA's in alfalfa provides evidence that mitochondria are inherited biparentally in this species.

Submitochondrial localization and asymmetric disposition of two peripheral cyclic nucleotide phosphodiesterases.

There are two distinct cyclic AMP phosphodiesterases associated with the liver mitochondrion: one with the outer membrane and one with the inner membrane. No activity is associated with the lysosomal fraction. Both of the enzymes are peripheral proteins and can be released from the membranes by high-ionic-strength treatment. Treatment of intact mitochondria with trypsin and insoluble trypsin localizes these enzymes to the cytosol-facing surface of their respective membranes. The enzymes differ in regard to sedimentation coefficient, thermostability and susceptibility to inactivation by trypsin. Both enzymes degrade cyclic AMP and cyclic GMP. Whereas the outer-membrane enzyme displays Michaelis kinetics and appears to be a low-affinity enzyme, the inner-membrane enzyme displays kinetics indicative of apparent negative co-operativity.

Mitochondrial biogenesis during fungal spore germination: Products of mitochondrial protein synthesis in vivo

Germinated spores of the fungus Botryodiplodia theobromae synthesized about eight to ten mitochondrial proteins in the presence of cycloheximide as shown by acrylamidegel electrophoresis of [ 3H]leucine-labeled, detergent-solubilized mitochondrial extracts. The apparent molecular weights of these proteins (whose synthesis was blocked by chloramphenicol) ranged from 85,100 to 8400. The 8400 molecular weight protein was soluble in chloroform-methanol, while other mitochondrial proteins (including also those proteins synthesized only in the absence of cycloheximide) were insoluble in this solvent system. Treatment of intact mitochondria with trichloroacetic acid before detergent extraction caused conversion of the higher molecular weight radioactivity to the 8400 molecular weight form. Labeling kinetics of 300-min (germinated) spores indicated that the 8400 molecular weight protein was the first to contain [ 3H]leucine, and with increasing time of incubation the label appeared in the higher molecular weight proteins. The mitochondrial ribosomes of the spores were not active during the first 60 min of germination, although products of the cytoplasmic ribosomes were incorporated into or associated with mitochondria during this interval; during the second 60-min interval of germination, function of mitochondrial ribosomes was observed by synthesis of the 8400 molecular weight, chloroform-methanol-soluble protein. In three subsequent 60-min intervals of germination, radioactivity from [ 3H]leucine was incorporated into the higher molecular weight proteins. The significance of this mitochondrial protein synthesis in relation to other metabolic activities of the germinating spores is discussed.

Translocation of Inducible Tyrosine Aminotransferase to the Mitochondrial Fraction: FACILITATION BY ACUTE UREMIA AND OTHER CONDITIONS

Abstract Acute uremia induced both soluble and mitochondrial tyrosine aminotransferase activity, EC 2.6.1.5, in rat liver. Such induction was demonstrated to be independent of glucocorticoid action. Mitochondrial aspartate aminotransferase, EC 2.6.1.1, although identical with mitochondrial tyrosine aminotransferase, was not elevated during acute uremia. Purified antibody specific against cytosol tyrosine aminotransferase cross-reacted significantly with the mitochondrial enzyme from uremic rats, suggesting that the elevation of activity was due to translocation of the induced cytosol enzyme form to the mitochondria. Considerable translocation was also caused by a combination of fasting together with some factor known to increase urea synthesis in the liver, such as high nitrogen loads or intraperitoneal injection of hydrocortisone. Intravenous injection of rats with Triton WR-1339 resulted in the separation of lysosomes from mitochondria during sucrose density gradient centrifugation. All of the tyrosine aminotransferase activity was associated with the mitochondria and not with the lysosomes. Treatment of intact mitochondria with the antisoluble tyrosine aminotransferase antibody resulted in inhibition of about 50% of the total inhibitable mitochondrial activity, suggesting that some of the translocated cytosol enzyme may not be exposed and thus may be unavailable for antibody cross-reaction. In vitro crossover experiments using induced cytosol tyrosine aminotransferase with normal mitochondria resulted in an elevation of the mitochondrial activity, whereas incubation of noninduced cytosol enzyme with mitochondria from induced rats did not. Consequently, the translocation phenomenon is a function of altered cytosol tyrosine aminotransferase rather than altered mitochondria.