scholarly journals The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin.

1975 ◽  
Vol 65 (3) ◽  
pp. 615-630 ◽  
Author(s):  
C R Hackenbrock ◽  
K J Miller
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Membrane ◽  
High Density ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
Anionic Sites ◽  
Contact Sites ◽  
Intact Mitochondrion ◽  
Boundary Membrane

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.

scholarly journals Mitofilin complexes: conserved organizers of mitochondrial membrane architecture

2012 ◽  
Vol 393 (11) ◽  
pp. 1247-1261 ◽  
Author(s):  
Ralf M. Zerbes ◽  
Ida J. van der Klei ◽  
Marten Veenhuis ◽  
Nikolaus Pfanner ◽  
Martin van der Laan ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Membrane ◽  
Protein Complexes ◽  
Boundary Region ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Oligomeric Protein ◽  
Enzyme Complexes ◽  
Boundary Membrane

Abstract Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

scholarly journals A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria

2014 ◽  
Vol 25 (25) ◽  
pp. 3999-4009 ◽  
Author(s):  
Agnieszka Gornicka ◽  
Piotr Bragoszewski ◽  
Piotr Chroscicki ◽  
Lena-Sophie Wenz ◽  
Christian Schulz ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Membrane ◽  
Alternative Pathway ◽  
Intermembrane Space ◽  
Outer Mitochondrial Membrane ◽  
Membrane Translocation ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Core Components

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

scholarly journals Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain.

1988 ◽  
Vol 106 (5) ◽  
pp. 1499-1505 ◽  
Author(s):  
M Nguyen ◽  
A W Bell ◽  
G C Shore
Keyword(s):  
G Protein ◽  
Simple Extension ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
The Matrix ◽  
Targeting Signal ◽  
Vesicular Stomatitis ◽  
Matrix Precursor ◽  
Identical Fashion

Recently, we fused a matrix-targeting signal to a large fragment of vesicular stomatitis virus G protein, which contains near its COOH-terminus a well-characterized endoplasmic reticulum (ER) stop-transfer sequence; the hybrid G protein was sorted to the inner mitochondrial membrane (Nguyen, M., and G. C. Shore. 1987. J. Biol. Chem. 262:3929-3931). Here, we show that the 19 amino acid G stop-transfer domain functions in an identical fashion when inserted toward the COOH-terminus of an otherwise normal matrix precursor protein, pre-ornithine carbamyl transferase; after import, the mutant protein was found anchored in the inner membrane via the stop-transfer sequence, with its NH2 terminus facing the matrix and its short COOH-terminal tail located in the intermembrane space. However, when the G stop-transfer sequence was placed near the NH2 terminus, the protein was inserted into the outer membrane, in the reverse orientation (NH2 terminus facing out, with a large COOH-terminal fragment located in the intermembrane space). These observations for mitochondrial topogenesis can be explained by a simple extension of existing models for ER sorting.

scholarly journals Molecular mechanism of mitochondrial phosphatidate transfer by Ups1

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Jiuwei Lu ◽  
Chun Chan ◽  
Leiye Yu ◽  
Jun Fan ◽  
Fei Sun ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mitochondrial Membrane ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
Membrane Interaction ◽  
Detailed Mechanism ◽  
Physiological Regulator ◽  
Membrane Bound ◽  
Dynamics Simulations

AbstractCardiolipin, an essential mitochondrial physiological regulator, is synthesized from phosphatidic acid (PA) in the inner mitochondrial membrane (IMM). PA is synthesized in the endoplasmic reticulum and transferred to the IMM via the outer mitochondrial membrane (OMM) under mediation by the Ups1/Mdm35 protein family. Despite the availability of numerous crystal structures, the detailed mechanism underlying PA transfer between mitochondrial membranes remains unclear. Here, a model of Ups1/Mdm35-membrane interaction is established using combined crystallographic data, all-atom molecular dynamics simulations, extensive structural comparisons, and biophysical assays. The α2-loop, L2-loop, and α3 helix of Ups1 mediate membrane interactions. Moreover, non-complexed Ups1 on membranes is found to be a key transition state for PA transfer. The membrane-bound non-complexed Ups1/ membrane-bound Ups1 ratio, which can be regulated by environmental pH, is inversely correlated with the PA transfer activity of Ups1/Mdm35. These results demonstrate a new model of the fine conformational changes of Ups1/Mdm35 during PA transfer.

scholarly journals Membrane-tethering of cytochrome c accelerates regulated cell death in yeast

2020 ◽  
Vol 11 (9) ◽  
Author(s):  
Alexandra Toth ◽  
Andreas Aufschnaiter ◽  
Olga Fedotovskaya ◽  
Hannah Dawitz ◽  
Pia Ädelroth ◽  
...  
Keyword(s):  
Cell Death ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Membrane ◽  
Mitochondrial Respiratory Chain ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Fitness Advantage ◽  
Regulated Cell Death ◽  
Membrane Tethering ◽  
Membrane Anchoring

Abstract Intrinsic apoptosis as a modality of regulated cell death is intimately linked to permeabilization of the outer mitochondrial membrane and subsequent release of the protein cytochrome c into the cytosol, where it can participate in caspase activation via apoptosome formation. Interestingly, cytochrome c release is an ancient feature of regulated cell death even in unicellular eukaryotes that do not contain an apoptosome. Therefore, it was speculated that cytochrome c release might have an additional, more fundamental role for cell death signalling, because its absence from mitochondria disrupts oxidative phosphorylation. Here, we permanently anchored cytochrome c with a transmembrane segment to the inner mitochondrial membrane of the yeast Saccharomyces cerevisiae, thereby inhibiting its release from mitochondria during regulated cell death. This cytochrome c retains respiratory growth and correct assembly of mitochondrial respiratory chain supercomplexes. However, membrane anchoring leads to a sensitisation to acetic acid-induced cell death and increased oxidative stress, a compensatory elevation of cellular oxygen-consumption in aged cells and a decreased chronological lifespan. We therefore conclude that loss of cytochrome c from mitochondria during regulated cell death and the subsequent disruption of oxidative phosphorylation is not required for efficient execution of cell death in yeast, and that mobility of cytochrome c within the mitochondrial intermembrane space confers a fitness advantage that overcomes a potential role in regulated cell death signalling in the absence of an apoptosome.

Abstract 15070: Micu1 Regulates Mitochondrial Cristae Structure and Function Independent of the Mitochondrial Calcium Uniporter Channel

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Dhanendra Tomar ◽  
Manfred Thomas ◽  
Joanne Garbincius ◽  
Devin Kolmetzky ◽  
Oniel Salik ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Coiled Coil ◽  
Structure And Function ◽  
Membrane Dynamics ◽  
Size Exclusion ◽  
Null Mouse ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
And Function

Background: MICU1 is an EF-hand domain containing Ca 2+ -sensor regulating the mitochondrial Ca 2+ uniporter channel and mitochondrial Ca 2+ uptake. MICU1-null mouse and fly models display perinatal lethality with disorganized mitochondrial architecture. Interestingly, these phenotypes are distinct from other mtCU loss-of-function models ( MCU, MICU2, EMRE, MCUR1 ) and thus are likely not explained solely by changes in matrix Ca 2+ content. Using size-exclusion proteomics and co-immunofluorescence, we found that MICU1 localizes to mitochondrial complexes lacking MCU. These observations suggest that MICU1 may have additional cellular functions independent of the MCU. Methods: Biotin-based proximity labeling and proteomics, protein biochemistry, live-cell Ca 2+ imaging, electron microscopy, confocal and super-resolution imaging were utilized to identify and validate MICU1 novel functions. Results: The expression of a MICU1-BioID2 fusion protein in MCU +/+ and MCU -/- cells allowed the identification of the total vs. MCU-independent MICU1 interactome. LC-MS analysis of purified biotinylated proteins identified the mitochondrial contact site and cristae organizing system (MICOS) components Mitofilin (MIC60) and Coiled-coil-helix-coiled-coil helix domain containing 2 (CHCHD2) as MCU independent novel MICU1 interactors. We demonstrate that MICU1 is essential for proper organization of the MICOS complex and that MICU1 ablation results in altered cristae organization, mitochondrial ultrastructure, mitochondrial membrane dynamics, membrane potential, and cell death signaling. We hypothesize that MICU1 is a MICOS Ca 2+ - sensor since perturbing MICU1 is sufficient to modulate cytochrome c release independent of Ca 2+ uptake across the inner mitochondrial membrane. Conclusions: Here, we provide the first experimental evidence of an intermembrane space Ca 2+ - sensor regulating mitochondrial membrane dynamics, independent of changes in matrix Ca 2+ content. This study provides a novel paradigm to understand Ca 2+ -dependent regulation of mitochondrial structure and function and may help explain the mitochondrial remodeling reported to occur in numerous disease states.

scholarly journals Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (11) ◽  
pp. 5487-5496 ◽  
Author(s):  
M E Dumont ◽  
T S Cardillo ◽  
M K Hayes ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Mitochondrial Membrane ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Apocytochrome C ◽  
Cytochrome C Heme Lyase

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

scholarly journals Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

2007 ◽  
Vol 12 (4) ◽  
Author(s):  
Izabela Koszela-Piotrowska ◽  
Katarzyna Choma ◽  
Piotr Bednarczyk ◽  
Krzysztof Dołowy ◽  
Adam Szewczyk ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Chloride Channel ◽  
Chloride Channels ◽  
Mitochondrial Membrane ◽  
Disulfonic Acid ◽  
Mitochondrial Membranes ◽  
Rat Skeletal Muscle ◽  
Inner Mitochondrial Membrane ◽  
Stilbene Derivatives ◽  
Rat Heart Mitochondria

AbstractIon channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.

Stimulation of apoptosis by sulindac and piroxicam

1998 ◽  
Vol 95 (3) ◽  
pp. 385-388 ◽  
Author(s):  
William R. WADDELL
Keyword(s):  
Acetic Acid ◽  
Mitochondrial Membrane ◽  
Prostaglandin Synthesis ◽  
Intermembrane Space ◽  
Colon Polyps ◽  
Inner Mitochondrial Membrane ◽  
Normal Cells ◽  
Inhibition Of Prostaglandin Synthesis ◽  
Stimulation Of

1.Sulindac, cis-5-fluoro-2-methyl-1-(p-methylsulphinylbenzylidene)indene-3-acetic acid, inhibits growth of colon polyps and cancers. This effect has been attributed to inhibition of prostaglandin synthesis but more recent observations indicate that, in vitro, cells that do not have cyclo-oxygenase nor RNA for synthesis of such enzymes are affected by sulindac. Therefore the presumptive effect is probably not correct. 2.It has also been found that sulindac stimulates apoptosis. It is herein postulated that in tumour cells such effects may be due to interaction of the anionic form of the drug with protons in the intermembrane space of mitochondria to disrupt the potential across the inner mitochondrial membrane and thereby initiate apoptosis. Normal cells are not affected.

scholarly journals Cardiolipin, Non-Bilayer Structures and Mitochondrial Bioenergetics: Relevance to Cardiovascular Disease

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1721
Author(s):  
Edward S. Gasanoff ◽  
Lev S. Yaguzhinsky ◽  
Győző Garab
Keyword(s):  
Electron Transport Chain ◽  
Mitochondrial Membrane ◽  
Mitochondrial Bioenergetics ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
Atp Production ◽  
Functional Roles ◽  
Transport Chain ◽  
Energy Converting

The present review is an attempt to conceptualize a contemporary understanding about the roles that cardiolipin, a mitochondrial specific conical phospholipid, and non-bilayer structures, predominantly found in the inner mitochondrial membrane (IMM), play in mitochondrial bioenergetics. This review outlines the link between changes in mitochondrial cardiolipin concentration and changes in mitochondrial bioenergetics, including changes in the IMM curvature and surface area, cristae density and architecture, efficiency of electron transport chain (ETC), interaction of ETC proteins, oligomerization of respiratory complexes, and mitochondrial ATP production. A relationship between cardiolipin decline in IMM and mitochondrial dysfunction leading to various diseases, including cardiovascular diseases, is thoroughly presented. Particular attention is paid to the targeting of cardiolipin by Szeto–Schiller tetrapeptides, which leads to rejuvenation of important mitochondrial activities in dysfunctional and aging mitochondria. The role of cardiolipin in triggering non-bilayer structures and the functional roles of non-bilayer structures in energy-converting membranes are reviewed. The latest studies on non-bilayer structures induced by cobra venom peptides are examined in model and mitochondrial membranes, including studies on how non-bilayer structures modulate mitochondrial activities. A mechanism by which non-bilayer compartments are formed in the apex of cristae and by which non-bilayer compartments facilitate ATP synthase dimerization and ATP production is also presented.

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