Side Of Inner Mitochondrial Membrane Research Articles

Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane

VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.

Structures of bovine cytochrome oxidase reveal proton active transports

Bovine cytochrome c oxidase (CcO) pumps four protons in each catalytic cycle through H-pathway including a hydrogen-bond network and a water channel in tandem. Protons, transferred through the water-channel from the negative side of mitochondrial inner membrane into the hydrogen-bond network, are pumped to the positive side of the membrane electrostatically by net positive charges on a heme (heme a) iron created upon electron transfer to the O2 reduction site. For blockage of backward proton leak from the hydrogen-bond network, which determines the proton-pumping direction, the water channel is closed after O2 binding to initiate proton-pump. Thus, four protons must be collected in the hydrogen-bond network before O2 binding. The X-ray structural analyses of the oxidized/reduced CcO at 1.5/1.6 Å resolution reveal a large cluster composed of ~21 water molecules and a Mg2+ site including Glu198 (Subunit II) bridging CuA and Mg2+. The cluster of the oxidized state have 20 water sites with full occupancy and two sites with partial occupies of water, while that of the reduced state have 19 water sites with full occupancy and 3 sites with partial occupancies. The carboxyl group of Glu198 changes its coordination structure to Mg2+ upon the reduction of the active centers. The cluster is tightly sealed sterically against proton exchanges with the cluster outside except for a short hydrogen-bond network connecting the cluster with H-pathway. Five proton-acceptable groups hydrogen-bonded with the cluster suggest sufficient storage capacity for four proton equivalents. The redox-coupled structural changes in the electron transfer pathway from CuA, the initial electron acceptor from cytochrome c, to heme a suggest redox-driven effective proton donations from the cluster to H-pathway, facilitated by Glu198. These results indicate that the cluster is a crucial element of the proton-pumping system of bovine CcO.

Nanosecond Time-Resolved Infrared Basis for a Bulge of the Transmembrane Helix Between Hemes A and A3 to Facilitate Highly Efficient Proton Pumping by Bovine Heart Cytochrome C Oxidase

Recent X-ray structural analyses show that net positive charges on a heme iron site (heme a) of bovine cytochrome c oxidase, created upon electron donation to the O2-reduction site, electrostatically drives proton-pump through a hydrogen-bond network to the positive side of mitochondrial inner membrane [1, 2]. The four electron equivalents for complete reduction of O2 at the fully reduced O2-reduction site are transferred one at a time, each, coupled with pumping of one proton equivalent, giving four intermediate species, F, O, E and R (from P). X-ray structures of P, F, O and R suggested that the water channel which connects the negative side space with the hydrogen bond network is kept closed by Ser382 bulge of the trans-membrane-helix during the transfer of four electron equivalents for complete reduction of the bound O2. The closure blocks effectively proton back-leakage from the hydrogen-bond network.The structural basis for complete protonation of the hydrogen-bond network before the O2-binding prerequisite for the efficient energy transduction was explored using a newly developed nanosecond time-resolved infrared apparatus for aqueous protein system. A transient CO-binding to CuB, after Fea3-CO photolysis, was discovered to open the water-channel by eliminating the Ser382 bulge. The infrared and X-ray structural results suggest that, sensing protonation state of the hydrogen-bond network, a relay system including CuB, O2, Fea3 and two a-helix turns extending to Ser382 facilitates effective proton collection and timely water-channel closure by conformational changes in the Ser382-containing segment, thereby ensuring efficient energy transduction.[1] Muramoto K., et al (2010) Proc. Natl. Acad. Sci. USA 107: 7740-7745.[2] Yoshikawa S., et al (2011) Ann. Rev. Biophys. 40: 205-223.

Nanosecond time-resolved infrared basis for a bulge of the transmembrane helix between hemes a and a3 to facilitate highly efficient proton pumping by bovine heart cytochrome c oxidase

Recent X-ray structural analyses show that net positive charges on a heme iron site (heme a) of bovine cytochrome c oxidase, created upon electron donation to the O2-reduction site, electrostatically drives proton-pump through a hydrogen-bond network to the positive side of mitochondrial inner membrane [1, 2]. The four electron equivalents for complete reduction of O2 at the fully reduced O2-reduction site are transferred one at a time, each, coupled with pumping of one proton equivalent, giving four intermediate species, F, O, E and R (from P). X-ray structures of P, F, O and R suggested that the water channel which connects the negative side space with the hydrogen bond network is kept closed by Ser382 bulge of the trans-membrane-helix during the transfer of four electron equivalents for complete reduction of the bound O2. The closure blocks effectively proton back-leakage from the hydrogen-bond network.The structural basis for complete protonation of the hydrogen-bond network before the O2-binding prerequisite for the efficient energy transduction was explored using a newly developed nanosecond time-resolved infrared apparatus for aqueous protein system. A transient CO-binding to CuB, after Fea3-CO photolysis, was discovered to open the water-channel by eliminating the Ser382 bulge. The infrared and X-ray structural results suggest that, sensing protonation state of the hydrogen-bond network, a relay system including CuB, O2, Fea3 and two a-helix turns extending to Ser382 facilitates effective proton collection and timely water-channel closure by conformational changes in the Ser382-containing segment, thereby ensuring efficient energy transduction.[1] Muramoto K., et al (2010) Proc. Natl. Acad. Sci. USA 107: 7740-7745.[2] Yoshikawa S., et al (2011) Ann. Rev. Biophys. 40: 205-223.

A new mitochondrial pH biosensor for quantitative assessment of pancreatic β-cell function

Mitochondrial pH is a key determinant of mitochondrial energy metabolism. We have developed a new fluorescence-based ratiometric pH biosensor using a chloride-insensitive and hydrogen-sensitive probe for direct, quantitative and bleaching-free measurement in a living cell. Fusing this biosensor with a mitochondrial localization signal (MTpHGV) allowed us to determine mitochondrial pH. This new system was applied to measure mitochondrial pH in pancreatic β-cells, in which mitochondrial function plays a pivotal role in insulin secretion. Rat INS1 cells and mouse MIN6 cells are transfected with MTpHGV stably to monitor mitochondrial pH. While carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rapidly decreased mitochondrial pH in cultured rat MTpHGV-INS-1 cells, MTpHGV-MIN6 cells showed a rapid increase. These data suggest that MTpHGV probe exist in matrix side in INS-1 cells, but on the outer side of mitochondrial inner membrane in MIN6 cells. Moreover, while MTpHGV-INS-1 cells showed a rapid increase of pH by glucose stimulation, mitochondrial pH decreased quickly by glucose stimulation in all MTpHGV-MIN6 cells examined and recovered smoothly. Perfusion study of glucose load in MTpHGV-MIN6 cells under aminooxyacetate (AOA) or 100μM diazoxide showed that this mitochondrial pH acidification was dependent on nicotinamide adenine dinucleotide (NADH) shuttle, but independent from KATP channel. This new system for measuring mitochondrial pH is sensitive across the range of physiologic conditions and may be a useful tool for evaluating mitochondrial function in living cells.

Living unicellular eukaryote Tetrahymena pyriformis as a model for study of mitochondrial energetics in mammalian cells under conditions of reduced oxidative metabolism.

AbstractSome “animal-like” protozoa are used instead of mammalian cells in diverse investigations. Tetrahymena pyriformis cells in stationary phase of growth and washed from oxidative substrates (T.pyriformis) function under conditions of reduced oxidative metabolism. To elucidate if T.pyriformis can be applied as a model for mitochondrial energetics study in mammalian cells during hibernation, the features of T.pyriformis mitochondria were compared with that of liver mitochondria isolated from hibernating animals; the published earlier data were used. Based on the respiration recording and observation of Mito Tracker Red fluorescence in living T.pyriformis we tentatively concluded that the mitochondrial electrical membrane potential, Δψ, is low, and that the most part of the proton motive force is stored as the difference of pH between the sides of inner mitochondrial membrane. A sharp decrease of the maximal uncoupler-stimulated respiration rates was observed in the liver mitochondria isolated from hibernating animals. These and other data are explained by hypothesis that remodeling the liver mitochondria to a condense configuration is prerequisite for reduction of the oxidative metabolism. The rearrangement possibly resembles the apoptosis with the initial Δψ decrease induced by the respiratory activity suppression; the process has been earlier studied by E. Gotlieb and coauthors. The data suggest that T.pyriformis are suitable to study of some aspects of mitochondrial role in cellular adaptation to metabolic depression; that the evolutionary ancient mechanisms are conserved in a modified form in the ciliate and mammalian cells.

Living unicellular eukaryote Tetrahymena pyriformis as a model for study of mitochondrial energetics in mammalian cells under conditions of reduced oxidative metabolism.

AbstractSome “animal-like” protozoa are used instead of mammalian cells in diverse investigations. Tetrahymena pyriformis cells in stationary phase of growth and washed from oxidative substrates (T.pyriformis) function under conditions of reduced oxidative metabolism. To elucidate if T.pyriformis can be applied as a model for mitochondrial energetics study in mammalian cells during hibernation, the features of T.pyriformis mitochondria were compared with that of liver mitochondria isolated from hibernating animals; the published earlier data were used. Based on the respiration recording and observation of Mito Tracker Red fluorescence in living T.pyriformis we tentatively concluded that the mitochondrial electrical membrane potential, Δψ, is low, and that the most part of the proton motive force is stored as the difference of pH between the sides of inner mitochondrial membrane. A sharp decrease of the maximal uncoupler-stimulated respiration rates was observed in the liver mitochondria isolated from hibernating animals. These and other data are explained by hypothesis that remodeling the liver mitochondria to a condense configuration is prerequisite for reduction of the oxidative metabolism. The rearrangement possibly resembles the apoptosis with the initial Δψ decrease induced by the respiratory activity suppression; the process has been earlier studied by E. Gotlieb and coauthors. The data suggest that T.pyriformis are suitable to study of some aspects of mitochondrial role in cellular adaptation to metabolic depression; that the evolutionary ancient mechanisms are conserved in a modified form in the ciliate and mammalian cells.

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 μM CAT and 75 μM Ca 2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca 2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca 2+ but the release of Ca 2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 μM CAT did not change the respiration, but increased the mitochondrial capacity for Ca 2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 μM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.

Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side-chain cleavage enzyme (P450scc) and of 11 beta-hydroxylase (P45011 beta), 3 beta-hydroxysteroid dehydrogenase/ delta 5-4 isomerase (3 beta HSD), and 21-hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata-reticularis. 17 alpha-Hydroxylase/C17-C20 lyase (P45017 alpha,lyase) is present in the zona fasciculata-reticularis cells of human, bovine, pig, and guinea-pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011 beta, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata-reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011 beta are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3 beta HSD, P450C21, and P45017 alpha, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes.