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 Degradation of transplanted mitochondrial proteins by hepatocyte monolayers

1983 ◽  
Vol 216 (1) ◽  
pp. 151-161 ◽  
Author(s):  
P J Evans ◽  
R J Mayer
Keyword(s):  
Acid Phosphatase ◽  
Monoamine Oxidase ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Proteins ◽  
Mitochondrial Outer Membrane ◽  
Lag Period

Reductively [3H]methylated rat mitochondria and mitochondrial-outer-membrane vesicles and mitochondrial-outer-membrane vesicles where monoamine oxidase is irreversibly labelled by [3H]pargyline have been transplanted into hepatocytes by poly(ethylene glycol)-mediated organelle or organelle-vesicle cell fusion. During subsequent culture of hepatocyte monolayers for 4-5 days, under conditions which mimic endogenous catabolic rates in vivo the transplanted organelle proteins retain their degradation characteristics observed in vivo (e.g. mitochondria: average t 1/2 72.5 h; monoamine oxidase: t1/2 55 h). In all cases protein degradation with first-order kinetics is only observed after an initial lag period (i.e. 24-30 h after fusion). Transplantation of fluorescein-conjugated organelles showed that the fluorescent material is rapidly internalized (average t1/2 1-6 h) and uniformly distributed in the cytoplasm. During a subsequent 18-24 h period (which corresponds to the lag period for intracellular destruction of transplanted mitochondrial material) the transplanted material is translocated to assume a perinuclear distribution. The destruction of transplanted mitochondrial proteins is compared with endogenous mitoribosomally synthesized proteins (average t1/2 52.5 h). Percoll fractionation of cell homogenates containing transplanted mitochondrial outer membranes where the enzyme monoamine oxidase is irreversibly labelled with [3H]pargyline shows a distribution of enzyme similar to lysosomal acid phosphatase. After transplantation of reductively methylated 3H-labelled mitochondrial-outer-membrane vesicles the cells were treated with leupeptin to alter lysosomal density. This treatment leads to the predominant association of acid phosphatase with dense structures, whereas the 3H-labelled transplanted material predominantly does not change density. Therefore transplanted mitochondrial-outer-membrane proteins are found in intracellular vesicular structures from which the proteins are donated for destruction, at least in part, by a lysosomal mechanism.

scholarly journals Degradation of transplanted rat liver mitochondrial-outer-membrane proteins in hepatoma cells

1983 ◽  
Vol 216 (1) ◽  
pp. 163-175 ◽  
Author(s):  
S M Russell ◽  
R J Mayer
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Monoamine Oxidase ◽  
Outer Membrane ◽  
Rat Liver ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Cell Protein ◽  
Mitochondrial Outer Membrane

Reductively [3H]methylated 3H mitochondrial-outer-membrane vesicles from rat liver and vesicles where monoamine oxidase has been derivatized irreversibly by [3H]-pargyline have been deliberately miscompartmentalized by heterologous transplantation into hepatoma (HTC) cells by poly(ethylene glycol)-mediated vesicle-cell fusion. Fluorescein-conjugated mitochondrial-outer-membrane vesicles have also been used to show that transplanted material is patched, capped and internalized. Reductively methylated outer-membrane proteins and monoamine oxidase are destroyed at the same rate (t1/2 24 h). Mitochondrial-outer-membrane proteins are not degraded at the same rate as HTC plasma-membrane proteins, endogenous cell protein, or endocytosed protein. Transplanted radiolabelled mitochondrial-outer-membrane proteins accumulate intracellularly in structures that are distinct from plasma membrane and lysosomes. However, when mitochondrial-outer-membrane vesicles derivatized with [14C]sucrose are transplanted, the acid-soluble degradation products accumulate in the lysosomal fraction. [14C]Sucrose-conjugated HTC cell plasma membrane accumulates in intracellular structures that are again distinct from plasma membrane and lysosomes. In contrast with the above observations, homologously transplanted mitochondrial-outer-membrane proteins from rat liver are destroyed in hepatocytes at rates that are remarkably similar (t1/2 60-70 h) to the rates in rat liver in vivo [Evans & Mayer (1982) Biochem. Biophys. Res. Commun. 107, 51-58].

scholarly journals How the colonic environment influences Enterohaemorrhagic E. coli outer membrane vesicle production, and the interaction between outer membrane vesicles with human host cells

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Daniel Yara ◽  
Regis Stentz ◽  
Tom Wileman ◽  
Stephanie Schuller
Keyword(s):  
Outer Membrane ◽  
Bile Salts ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
T84 Cells ◽  
E Coli

Enterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.

scholarly journals Toll-Like Receptors 2 and 4 Modulate Pulmonary Inflammation and Host Factors Mediated by Outer Membrane Vesicles Derived from Acinetobacter baumannii

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Chad R. Marion ◽  
Jaewook Lee ◽  
Lokesh Sharma ◽  
Kyong-Su Park ◽  
Changjin Lee ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Pulmonary Inflammation ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Toll Like Receptors ◽  
Gram Negative ◽  
Content Type ◽  
Intranasal Introduction

ABSTRACT Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.

scholarly journals Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

2020 ◽  
Vol 117 (17) ◽  
pp. 9302-9310 ◽  
Author(s):  
Davinia Salvachúa ◽  
Allison Z. Werner ◽  
Isabel Pardo ◽  
Martyna Michalska ◽  
Brenna A. Black ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Aromatic Compounds ◽  
Value Added ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Pseudomonas Putida Kt2440 ◽  
Rich Media

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

In vivo Kinetic Biodistribution of Nano-Sized Outer Membrane Vesicles Derived from Bacteria

Small ◽  
2014 ◽  
Vol 11 (4) ◽  
pp. 456-461 ◽  
Author(s):  
Su Chul Jang ◽  
Sae Rom Kim ◽  
Yae Jin Yoon ◽  
Kyong-Su Park ◽  
Ji Hyun Kim ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles

scholarly journals Protein Import Channel of the Outer Mitochondrial Membrane: a Highly Stable Tom40-Tom22 Core Structure Differentially Interacts with Preproteins, Small Tom Proteins, and Import Receptors

2001 ◽  
Vol 21 (7) ◽  
pp. 2337-2348 ◽  
Author(s):  
Chris Meisinger ◽  
Michael T. Ryan ◽  
Kerstin Hill ◽  
Kirstin Model ◽  
Joo Hyun Lim ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Stable Complex ◽  
Intermembrane Space ◽  
Import Receptors ◽  
Conductance States ◽  
Coupled Channel

ABSTRACT The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a ∼400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

Application of cryo-electron microscopy for investigation of Bax-induced pores in apoptosis

2017 ◽  
Vol 6 (1) ◽  
pp. 47-55
Author(s):  
Tomomi Kuwana
Keyword(s):  
Electron Microscopy ◽  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Cryo Electron Microscopy

AbstractMitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis, the molecular mechanisms of which have been a subject of intensive study. This process is important for therapeutic intervention in various diseases, such as cancer. Pro-apoptotic Bax and Bak are functionally redundant and structurally homologous. When activated at the mitochondrial outer membrane, they cause the membrane to permeabilize and release apoptogenic proteins from the intermembrane space. To unravel the molecular mechanisms of this unique and important event, we systematically reduced the experimental system. Simple outer membrane vesicles and liposomes recapitulated many features of MOMP. Although conventional transmission electron microscopy could not detect any membrane changes during MOMP in these vesicles, cryo-electron microscopy successfully revealed Bax-induced delicate pores, owing to its ability to preserve native, hydrated membrane structure. The data are consistent with the idea that Bax is unfolded and embedded in the bilayer and deforms the membrane to form a large pore. Together with the biochemical and structure data in the literature, we now have more comprehensive models of the key function of Bax. We hope that new tools, such as lipid nanodiscs, will give us an atomic-level resolution and finally solve Bax structure in the membrane, where it functions.

scholarly journals Outer Membrane Vesicles: In vivo Kinetic Biodistribution of Nano-Sized Outer Membrane Vesicles Derived from Bacteria (Small 4/2015)

Small ◽  
2015 ◽  
Vol 11 (4) ◽  
pp. 386-386
Author(s):  
Su Chul Jang ◽  
Sae Rom Kim ◽  
Yae Jin Yoon ◽  
Kyong-Su Park ◽  
Ji Hyun Kim ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles
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