scholarly journals Ionic strength of the intermembrane space of intact mitochondria as estimated with fluorescein-BSA delivered by low pH fusion.

1991 ◽  
Vol 113 (6) ◽  
pp. 1331-1340 ◽  
Author(s):  
J D Cortese ◽  
A L Voglino ◽  
C R Hackenbrock
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Membrane Fusion ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Low Ph ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Fusion Procedure ◽  
Bulk Medium

The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the microM range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength-dependent change in fluorescence were found for bulk medium, soluble (9.8 +/- 0.8 mM) and intermembrane space-entrapped FITC-BSA (10.2 +/- 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within +/- 20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.

scholarly journals The Mitochondrial TOM Complex Is Required for tBid/Bax-induced Cytochrome c Release

2007 ◽  
Vol 282 (38) ◽  
pp. 27633-27639 ◽  
Author(s):  
Martin Ott ◽  
Erik Norberg ◽  
Katharina M. Walter ◽  
Patrick Schreiner ◽  
Christian Kemper ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Mitochondrial Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Outer Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Unknown Mechanism

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.

scholarly journals The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

1999 ◽  
Vol 147 (4) ◽  
pp. 809-822 ◽  
Author(s):  
Ruth M. Kluck ◽  
Mauro Degli Esposti ◽  
Guy Perkins ◽  
Christian Renken ◽  
Tomomi Kuwana ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Cytochrome C Release ◽  
Free System ◽  
Xenopus Egg ◽  
Apoptotic Proteins ◽  
Outer Membrane Permeability

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Effects of cytochrome c on the mitochondrial apoptosis-induced channel MAC

2004 ◽  
Vol 286 (5) ◽  
pp. C1109-C1117 ◽  
Author(s):  
Liang Guo ◽  
Dawn Pietkiewicz ◽  
Evgeny V. Pavlov ◽  
Sergey M. Grigoriev ◽  
John J. Kasianowicz ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Cell Types ◽  
Rnase A ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Mitochondrial Apoptosis ◽  
Noise Type ◽  
Voltage Dependent

Recent studies indicate that cytochrome c is released early in apoptosis without loss of integrity of the mitochondrial outer membrane in some cell types. The high-conductance mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane early in apoptosis of FL5.12 cells. Physiological (micromolar) levels of cytochrome c alter MAC activity, and these effects are referred to as types 1 and 2. Type 1 effects are consistent with a partitioning of cytochrome c into the pore of MAC and include a modest decrease in conductance that is dose and voltage dependent, reversible, and has an increase in noise. Type 2 effects may correspond to “plugging” of the pore or destabilization of the open state. Type 2 effects are a dose-dependent, voltage-independent, and irreversible decrease in conductance. MAC is a heterogeneous channel with variable conductance. Cytochrome c affects MAC in a pore size-dependent manner, with maximal effects of cytochrome c on MAC with conductance of 1.9–5.4 nS. The effects of cytochrome c, RNase A, and high salt on MAC indicate that size, rather than charge, is crucial. The effects of dextran molecules of various sizes indicate that the pore diameter of MAC is slightly larger than that of 17-kDa dextran, which should be sufficient to allow the passage of 12-kDa cytochrome c. These findings are consistent with the notion that MAC is the pore through which cytochrome c is released from mitochondria during apoptosis.

scholarly journals Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion

2011 ◽  
Vol 124 (7) ◽  
pp. 1126-1135 ◽  
Author(s):  
F. Anton ◽  
J. M. Fres ◽  
A. Schauss ◽  
B. Pinson ◽  
G. J. K. Praefcke ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane

The open reading frame 1-encoded (‘36K’) protein of Carnation Italian ringspot virus localizes to mitochondria

2001 ◽  
Vol 82 (1) ◽  
pp. 29-34 ◽  
Author(s):  
L. Rubino ◽  
F. Weber-Lotfi ◽  
A. Dietrich ◽  
C. Stussi-Garaud ◽  
M. Russo
Keyword(s):  
Outer Membrane ◽  
Fluorescent Protein ◽  
Amorphous Material ◽  
Subcellular Fractionation ◽  
Open Reading Frame ◽  
Ringspot Virus ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Reading Frame ◽  
Open Reading Frame 1

The localization of the 36 kDa (‘36K’) protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K–GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

scholarly journals Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli

2002 ◽  
Vol 159 (6) ◽  
pp. 923-929 ◽  
Author(s):  
Damien Arnoult ◽  
Philippe Parone ◽  
Jean-Claude Martinou ◽  
Bruno Antonsson ◽  
Jérôme Estaquier ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Actinomycin D ◽  
Membrane Permeabilization ◽  
Mitochondrial Outer Membrane ◽  
Simple Explanation ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Mitochondrial Inner Membrane ◽  
Apoptosis Inducing Factor

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

scholarly journals Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

1996 ◽  
Vol 16 (8) ◽  
pp. 4035-4042 ◽  
Author(s):  
D A Court ◽  
F E Nargang ◽  
H Steiner ◽  
R S Hodges ◽  
W Neupert ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Protein Translocation ◽  
Specific Binding ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Terminal Domain ◽  
Tom Complex

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.

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