Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.     

BRAIN MITOCHONDRIA : II. The Relationship of Brain Mitochondria to Glycolysis

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.     

Enolase takes part in a macromolecular complex associated to mitochondria in yeast

In eucaryotes, glycolytic enzymes are classically regarded as being localised in the cytosol. Recently, we have shown that part of the cellular pool of the glycolytic enzyme, enolase, is tightly associated with the mitochondrial surface in the yeast Saccharomyces cerevisiae (N. Entelis, I. Brandina, P. Kamenski, I.A. Krasheninnikov, R.P. Martin and I. Tarassov, A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae, Genes Dev. 20 (2006) 1609-1620). Here, using enzymatic assays, we show that all glycolytic enzymes are associated with mitochondria in yeast, to extents similar to those previously reported for Arabidopsis cells. Using separation of mitochondrial complexes by blue-native/SDS-PAGE and coimmunoprecipitation of mitochondrial proteins with anti-enolase antibodies, we found that enolase takes part in a large macromolecular complex associated to mitochondria. The identified components included additional glycolytic enzymes, mitochondrial membrane carriers, and enzymes of the TCA cycle. We suggest a possible role of the enolase complex in the channeling of pyruvate, the terminal product of glycolysis, towards the TCA cycle within mitochondria. Moreover, we show that the mitochondrial enolase-containing complex also contains the cytosolic tRNA(CUU)Lys, which is mitochondrially-imported, and its presumed import carrier, the precursor of the mitochondrial lysyl-tRNA synthetase. This suggests an unsuspected novel function for this complex in tRNA mitochondrial import.     

Broad Distribution of TPI-GAPDH Fusion Proteins among Eukaryotes: Evidence for Glycolytic Reactions in the Mitochondrion?

Glycolysis is a central metabolic pathway in eukaryotic and prokaryotic cells. In eukaryotes, the textbook view is that glycolysis occurs in the cytosol. However, fusion proteins comprised of two glycolytic enzymes, triosephosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were found in members of the stramenopiles (diatoms and oomycetes) and shown to possess amino-terminal mitochondrial targeting signals. Here we show that mitochondrial TPI-GAPDH fusion protein genes are widely spread across the known diversity of stramenopiles, including non-photosynthetic species (Bicosoeca sp. and Blastocystis hominis). We also show that TPI-GAPDH fusion genes exist in three cercozoan taxa (Paulinella chromatophora, Thaumatomastix sp. and Mataza hastifera) and an apusozoan protist, Thecamonas trahens. Interestingly, subcellular localization predictions for other glycolytic enzymes in stramenopiles and a cercozoan show that a significant fraction of the glycolytic enzymes in these species have mitochondrial-targeted isoforms. These results suggest that part of the glycolytic pathway occurs inside mitochondria in these organisms, broadening our knowledge of the diversity of mitochondrial metabolism of protists.     

Post-translational modifications of mitochondrial outer membrane proteins

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Post-translational modifications of mitochondrial outer membrane proteins

Abstract

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

The C-terminal region of TIM17 links the outer and inner mitochondrial membranes in Arabidopsis and is essential for protein import

The translocase of the inner membrane 17 (AtTIM17-2) protein from Arabidopsis has been shown to link the outer and inner mitochondrial membranes. This was demonstrated by several approaches: (i) In vitro organelle import assays indicated the imported AtTIM17-2 protein remained protease accessible in the outer membrane when inserted into the inner membrane. (ii) N-terminal and C-terminal tagging indicated that it was the C-terminal region that was located in the outer membrane. (iii) Antibodies raised to the C-terminal 100 amino acids recognize a 31-kDa protein from purified mitochondria, but cross-reactivity was abolished when mitochondria were protease-treated to remove outer membrane-exposed proteins. Antibodies to AtTIM17-2 inhibited import of proteins via the general import pathway into outer membrane-ruptured mitochondria, but did not inhibit protein import via the carrier import pathway. Together these results indicate that the C-terminal region of AtTIM17-2 is exposed on the outer surface of the outer membrane, and the C-terminal region is essential for protein import into mitochondria.     

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.     

MCC and PSC, the Putative Protein Import Channels of Mitochondria

All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in thecytoplasm and imported into the organelle. Water-filled channels are integral to the process oftranslocating proteins since channels can provide an aqueous pathway through the hydrophobicenvironment of the membrane. The MCC (multiple conductance channel) and PSC(peptide-sensitive channel) are two high-conductance channels previously identified inelectrophysiological studies of mitochondrial membranes. MCC and PSC are the putative pores of the importcomplexes of the inner and outer membranes, respectively. The genetic, biochemical, andbiophysical evidence regarding these assignments are summarized herein. These findingssupport the identification of MCC and PSC as the protein import channels of mitochondria.     

Biogenesis of beta-barrel membrane proteins of mitochondria

beta-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of beta-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of beta-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both beta-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane beta-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial beta-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.     

Glucose catabolism in brain. Intracellular localization of hexokinase

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.     

In Vitro Import of a Nuclearly Encoded tRNA into the Mitochondrion of Trypanosoma brucei

All of the mitochondrial tRNAs of Trypanosoma brucei have been shown to be encoded in the nucleus and must be imported into the mitochondrion. The import of nuclearly encoded tRNAs into the mitochondrion has been demonstrated in a variety of organisms and is essential for proper function in the mitochondrion. An in vitro import assay has been developed to study the pathway of tRNA import in T. brucei. The in vitro system utilizes crude isolated trypanosome mitochondria and synthetic RNAs transcribed from a cloned nucleus-encoded tRNA gene cluster. The substrate, composed of tRNA(Ser) and tRNA(Leu), is transcribed in tandem with a 59-nucleotide intergenic region. The tandem tRNA substrate is imported rapidly, while the mature-size tRNA(Leu) fails to be imported in this system. These results suggest that the preferred substrate for tRNA import into trypanosome mitochondria is a precursor molecule composed of tandemly linked tRNAs. Import of the tandem tRNA substrate requires (i) a protein component that is associated with the surface of the mitochondrion, (ii) ATP pools both outside and within the mitochondrion, and (iii) a membrane potential. Dissipation of the proton gradient across the inner mitochondrial membrane by treatment with an uncoupling agent inhibits import of the tandem tRNA substrate. Characterization of the import requirements indicates that mitochondrial RNA import proceeds by a pathway including a protein component associated with the outer mitochondrial membrane, ATP-dependent steps, and a mitochondrial membrane potential.     

Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain.

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity.     

Exploiting mitochondrial dysfunction for effective elimination of imatinib-resistant leukemic cells

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.     

Gazing at translocation in the mitochondrion

Most mitochondrial proteins are synthesized in the cytosol and imported into the mitochondrion via molecular machines called translocons on the outer and inner mitochondrial membranes. Alder et al. (2008b) examine protein translocation into intact mitochondria by adapting fluorescent techniques first used to study translocation in the endoplasmic reticulum.     

The role of the mitochondrial outer membrane in energy metabolism of tumor cells

The outer mitochondrial membrane contains a pore structure which is composed of a 30,000 Da protein, porin. The pore has an internal diameter of 2 nm and exhibits a molecular-sieving exclusion limit between 3000 and 6000 Da. These pores, therefore, provide the exit/entrance port for metabolites moving between mitochondria and the cytosol. Hexokinase binds to porin on the outer surface of mitochondria. The location of hexokinase has evoked a number of theories in which bound hexokinase is given a central role in regulating glycolysis, and, perhaps, the metabolic communication between oxidative and glycolytic metabolism. This is of particular importance in rapidly growing tumor cells in which the aerobic production of lactate and hexokinase activity are highly induced. In the present paper, we summarize the suggested roles of the outer membrane and bound hexokinase in regulation glycolysis of tumor cells. Experiments attempting to elucidate the role of hexokinase binding in the regulation of tumor cell metabolism are presented.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.