Characterization of Different Reactive Lysines in Bovine Heart Mitochondrial Porin

2002 ◽  
Vol 383 (12) ◽  
pp. 1967-1970 ◽  
Author(s):  
J.A. Al jamal
Keyword(s):  
Outer Membrane ◽  
Fluorescein Isothiocyanate ◽  
Mitochondrial Outer Membrane ◽  
Lipid Phase ◽  
Lipid Bilayer Membranes ◽  
Electrical Measurements ◽  
Elution Pattern ◽  
Outer Membrane Porin ◽  
Lysine Residues ◽  
Substantial Change

Abstract Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channels selectivity and its voltage dependence.

scholarly journals Comparison of the degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane in rat hepatocytes. Implications for the cytomorphological basis of protein catabolism

1984 ◽  
Vol 219 (1) ◽  
pp. 61-72 ◽  
Author(s):  
P J Evans ◽  
R J Mayer
Keyword(s):  
Monoamine Oxidase ◽  
Outer Membrane ◽  
Membrane Vesicles ◽  
Fluorescein Isothiocyanate ◽  
Rat Hepatocytes ◽  
Similar Rate ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Protein Catabolism

The degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane has been compared in rat hepatocyte monolayers. Monoamine oxidase was specifically irreversibly radiolabelled by the suicide inhibitor [3H]pargyline. Hepatocyte monolayers were cultured in conditions in which rates of protein catabolism like those in vivo are maintained [Evans & Mayer (1983) Biochem. J. 216, 151-161]. Incubation of hepatocyte monolayers for 17 h with [3H]pargyline specifically radiolabels mitochondrial monoamine oxidase, as shown by Percoll-gradient fractionation of broken hepatocytes. Monoamine oxidase is degraded at a similar rate to that observed in liver in vivo (t1/2 approx. 63 h). The effects of leupeptin, methylamine and colchicine on the degradation of endogenous radiolabelled enzyme has been studied over prolonged culture periods. Culture of hepatocytes for periods of up to 80 h with inhibitors was not cytotoxic, as demonstrated by measurements of several intrinsic biochemical parameters. Leupeptin, methylamine and colchicine inhibit the degradation of endogenous monoamine oxidase by 60, 38 and 18% respectively. Monoamine oxidase in mitochondrial-outer-membrane vesicles introduced into hepatocytes by poly(ethylene glycol)-mediated vesicle-cell transplantation is degraded at a similar rate (t1/2 55 h) to the endogenous mitochondrial enzyme. Whereas leupeptin inhibits the degradation of endogenous and transplanted enzyme to a similar extent, methylamine and colchicine inhibit the degradation of transplanted enzyme to a much greater extent (85 and 56% respectively). Fluorescence microscopy (with fluorescein isothiocyanate-conjugated mitochondrial outer membrane) shows that transplanted mitochondrial outer membrane undergoes internalization and translocation to a sided perinuclear site, as observed previously with whole mitochondria [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The effects of the inhibitors on the distribution of transplanted membrane material in the cell and inhibition of proteolysis show the importance of cytomorphology for intracellular protein catabolism.

scholarly journals Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin

FEBS Letters ◽  
2010 ◽  
Vol 584 (11) ◽  
pp. 2397-2402 ◽  
Author(s):  
Irina V. Perevoshchikova ◽  
Savva D. Zorov ◽  
Elena A. Kotova ◽  
Dmitry B. Zorov ◽  
Yuri N. Antonenko
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Outer Membrane Porin

scholarly journals A unified model for the biogenesis of mitochondrial outer membrane multi-span proteins

2021 ◽  
Author(s):  
Jialin Zhou ◽  
Martin Jung ◽  
Kai S. Dimmer ◽  
Doron Rapaport
Keyword(s):  
Phosphatidic Acid ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Lipid Phase ◽  
Yeast Mitochondria ◽  
Mitochondrial Import ◽  
Membrane Behavior ◽  
Double Deletion ◽  
Variable Extent ◽  
Import Receptor

The mitochondrial outer membrane (MOM) harbors proteins that traverse the membrane via several helical segments, so-called multi-span proteins. Two contradicting mechanisms were suggested to describe their integration into the MOM. The first proposes that the mitochondrial import (MIM) complex facilitates this process and functions as an insertase, whereas the second suggests that such proteins can integrate into the lipid phase without the assistance of import factors in a process that is enhanced by phosphatidic acid. To resolve this discrepancy and obtain new insights on the biogenesis of these proteins, we addressed this issue using yeast mitochondria and the multi-span protein Om14. Testing different truncation variants, we show that only the full-length protein contains all the required information that assure targeting specificity. Employing a specific insertion assay and several single and double deletion strains, we show that neither the import receptor Tom70 nor any other protein with a cytosolically exposed domain have a crucial contribution to the biogenesis process. We further demonstrate that Mim1 and Porin are required for optimal membrane integration of Om14 but none of them is absolutely required. Unfolding of the newly synthesized protein, its optimal hydrophobicity, as well as higher fluidity of the membrane dramatically enhanced the import capacity of Om14. Collectively, our findings suggest that MOM multi-span proteins can follow different biogenesis pathways in which proteinaceous elements and membrane behavior contribute to a variable extent to the combined efficiency.

scholarly journals Lysine Residues at Positions 234 and 236 in Yeast Porin Are Involved in Its Assembly into the Mitochondrial Outer Membrane

1995 ◽  
Vol 270 (47) ◽  
pp. 28331-28336 ◽  
Author(s):  
Mitchell D. Smith ◽  
Michelle Petrak ◽  
Paul D. Boucher ◽  
Kenneth N. Barton ◽  
Latisha Carter ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Lysine Residues

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

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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