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

In this issue, Qi et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201609019) provide structural insights into the mechanisms of mitochondrial outer membrane fusion by investigating the structure of mitofusin 1 (MFN1). This work proposes a new model to explain the important and elusive process of MFN-mediated mitochondrial fusion.

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

The Journal of Cell Biology ◽

10.1083/jcb.113.6.1331 ◽

1991 ◽

Vol 113 (6) ◽

pp. 1331-1340 ◽

Cited By ~ 31

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.

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A catalytic domain variant of mitofusin requiring a wildtype paralog for function uncouples mitochondrial outer-membrane tethering and fusion

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006347 ◽

2019 ◽

Vol 294 (20) ◽

pp. 8001-8014 ◽

Cited By ~ 9

Author(s):

Emily A. Engelhart ◽

Suzanne Hoppins

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Self Assembly ◽

Catalytic Domain ◽

Mitochondrial Fusion ◽

Mitochondrial Outer Membrane ◽

Fusion Activity ◽

Mutant Variant ◽

Membrane Tethering

Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health. In Mfn1 and Mfn2 paralogs, a conserved phenylalanine (Phe-202 (Mfn1) and Phe-223 (Mfn2)) located in the GTPase domain on a conserved β strand is part of an aromatic network in the core of this domain. To gain insight into the poorly understood mechanism of Mfn-mediated membrane fusion, here we characterize a Mitofusin mutant variant etiologically linked to Charcot–Marie–Tooth syndrome. From analysis of mitochondrial structure in cells and mitochondrial fusion in vitro, we found that conversion of Phe-202 to leucine in either Mfn1 or Mfn2 diminishes the fusion activity of heterotypic complexes with both Mfn1 and Mfn2 and abolishes fusion activity of homotypic complexes. Using coimmunoprecipitation and native gel analysis, we further dissect the steps of mitochondrial fusion and demonstrate that the mutant variant has normal tethering activity but impaired higher-order nucleotide-dependent assembly. The defective coupling of tethering to membrane fusion observed here suggests that nucleotide-dependent self-assembly of Mitofusin is required after tethering to promote membrane fusion.

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Sequential requirements for the GTPase domain of the mitofusin Fzo1 and the ubiquitin ligase SCFMdm30 in mitochondrial outer membrane fusion

Journal of Cell Science ◽

10.1242/jcs.079293 ◽

2011 ◽

Vol 124 (9) ◽

pp. 1403-1410 ◽

Cited By ~ 54

Author(s):

M. M. Cohen ◽

E. A. Amiott ◽

A. R. Day ◽

G. P. Leboucher ◽

E. N. Pryce ◽

...

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Ubiquitin Ligase ◽

Mitochondrial Outer Membrane ◽

Gtpase Domain

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Cryo-Electron Microscopy and Correlation Averaging of Frozen-Hydrated, Polymorphic 2D Crystals of the Mitochondrial Outer Membrane Channel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179257 ◽

1990 ◽

Vol 48 (1) ◽

pp. 100-101

Author(s):

Xiao-Wei Guo

Keyword(s):

Outer Membrane ◽

Hexagonal Lattice ◽

Mitochondrial Outer Membrane ◽

Rectangular Lattice ◽

Electron Microscopic ◽

Cryo Electron Microscopy ◽

Voltage Dependent ◽

Outer Membrane Channel ◽

2D Crystals ◽

Vdac Channel

Voltage-dependent, anion-selective channels (VDAC) are formed in the mitochondrial outer membrane (mitOM) by a 30-kDa polypeptide. These channels form ordered 2D arrays when mitOMs from Neurospora crassa are treated with soluble phospholipase A2. We obtain low-dose electron microscopic images of unstained specimens of VDAC crystals preserved in vitreous ice, using a Philips EM420 equipped with a Gatan cryo-transfer stage. We then use correlation analysis to compute average projections of the channel crystals. The procedure involves Fourier-filtration of a region within a crystal field to obtain a preliminary average that is subsequently cross-correlated with the entire crystal. Subregions are windowed from the crystal image at coordinates of peaks in the cross-correlation function (CCF, see Figures 1 and 2) and summed to form averages (Figure 3).The VDAC channel forms several different types of crystalline arrays in mitOMs. The polymorph first observed during phospholipase treatment is a parallelogram array (a=13 run, b=11.5 run, θ==109°) containing 6 water-filled pores per unit cell. Figure 1 shows the CCF of a sub-field of such an “oblique” array used to compute the correlation average of Figure 3A. With increased phospholipase treatment, other polymorphs are observed, often co-existing within the same crystal. For example, two distinct (but closely related) types of lattices occur in the field corresponding to the CCF of Figure 2: a “contracted” version of the parallelogram lattice (a=13 run, b=10 run, θ=99°), and a near-rectangular lattice (a=8.5 run, b=5 nm). The pattern of maxima in this CCF suggests that a third, near-hexagonal lattice (a=4.5 nm) may also be present. The correlation averages of Figures 3B-D were computed from polycrystalline fields, using peak coordinates in regions of CCFs corresponding to each of the three lattice types.

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Faculty Opinions recommendation of Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1011234.178620 ◽

2003 ◽

Author(s):

Sharad Kumar

Keyword(s):

Outer Membrane ◽

Mitochondrial Outer Membrane

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Faculty Opinions recommendation of Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726084556.793520120 ◽

2016 ◽

Author(s):

Benoit Kornmann

Keyword(s):

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Large Ring

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Faculty Opinions recommendation of Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726084556.793520063 ◽

2016 ◽

Author(s):

Christopher Janetopoulos

Keyword(s):

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Large Ring

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

AJP Cell Physiology ◽

10.1152/ajpcell.00183.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. C1109-C1117 ◽

Cited By ~ 59

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.

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