Inverted membrane vesicles

D-Lactate (14C)-methylamine, (14C)-thiocyanate Inverted membrane vesicles of Escherichia coli R Dung and Chen (1991)... 

Muller and Blobel (1984a,b) have partially purified a soluble factor required for protein secretion from an in vitro translocation system derived from E. coli. As it sediments at about 12 S, the authors have suggested that it is a complex of smaller molecules. It does not contain 6 S RNA (see below), but may contain some other small RNA. Its function is unknown. There is also evidence of at least one protein at the cytoplasmic surface of the E. coli membrane that is involved in translocation Treatment of inverted membrane vesicles with protease renders the membrane inactive for subsequent translocation (Rhoads et al., 1984 Chen et al., 1985). 

Non-bilayer-forming lipids are also required for protein translocation across the membrane of E. coli. The only non-bilayer-forming lipid in E. coli mutants lacking PE is CL. Protein translocation into inverted membrane vesicles prepared from PE-lacking cells (now enriched in CL) is reduced with divalent cation-depletion but can be enhanced by inclusion of Mg or Ca [ 1 ]. Protein translocation in the absence of divalent cations was restored by incorporation of non-bilayer PE (18 1 acyl chains) but not by bilayer-prone PE (14 0 acyl chains). These results indicate that lipids with a tendency to form non-bilayer stmctures provide a necessary environment for translocation of proteins across the membrane. 

In rat liver most of the cellular mono(ADP-ribosylated) proteins are associated with the mitochondrial fraction (1). Two mono(ADP-ribosyl)ating systems have been described in mitochondria, one in die soluble (matrix) fraction (2, 3), the other in submitochondrial particles (SMP, inverted inner membrane vesicles) (3, 4). The ADP-ribosylated matrix protein has a molecular mass of 100 kDa and appears to consist of two major subunits of equal mass. In SMP of both rat liver (4) and beef heart (3), there is one major acceptor protein for mono(ADP-ribose), which migrates with an apparent molecular mass of 30 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Mono(ADP-ribosylation) of the acceptor protein of beef heart SMP was suggested to occur non-enzymicaUy (3). In rat liver SMP, ADP-ribosylation of the 30 kDa protein most probably occurs at an arginine residue, and is readily reversible in the presence of ATP (4). The characteristics of this ADP-ribosylation reaction, i.e. protein specificity and sensitivity to ATP, together widi the observation that intramitochondrial hydrolysis of NAD(P)+ is accompanied by release of Ca + from mitochondria suggests a functional link between mitochondrial protein ADP-ribosylation and Ca2+ release (5,6). 

Bennett, V., and Cuatrecasas, P., 1973, Preparation of inverted plasma membrane vesicles from isolated adipocytes, Biochim. Biophys. Acta 311 362. 

The same type of narrow resonance was observed during fusion of membrane vesicles (Verkleij et ai, 1979, 1980). This was interpreted in terms of a structure allowing rapid isotropic averaging (Cullis et ai, 1978). A variety of possibilities can lead to such averaging—small vesicles, micelles, inverted micelles, even cubic or rhombic phases. In some cases the narrow resonances were correlated with the appearance of lipidic particles in freeze-fracture electron micrographs (Verkleij et ai, 1979 de Kniijff et ai, 1979) and interpreted as intramembrane inverted micelles. 

Lyotropic lamellar (La) liquid crystals (LC), in a form of vesicle or planar membrane, are important for membrane research to elucidate both functional and structural aspects of membrane proteins. Membrane proteins so far investigated are receptors, substrate carriers, energy-transducting proteins, channels, and ion-motivated ATPases [1-11], The L liquid crystals have also been proved useful in the two-dimensional crystallization of membrane proteins[12, 13], in the fabrication of protein micro-arrays[14], and biomolecular devices[15]. Usefulness of an inverted cubic LC in the three-dimensional crystallization of membrane proteins has also been recognized[16]. 

Fig. 5 The snake PLA2 neurotoxin is depicted here as a snake, which binds to an active zone, i.e., a synaptic vesicle (SV) release site, and hydrolyses the phospholipids of the external layer of the presynaptic membrane (green) with formation of the inverted-cone shaped lysophospholipid (yellow) and the cone-shaped fatty acid (dark blue). Fatty acids rapidly equilibrate by trans-bilayer movement among the two layers of the presynaptic membrane. In such a way lysophospholipids, which induce a positive curvature of the membrane, are present in trans and fatty acid, which induce a negative curvature, are present also in cis, with respect to the fusion site. This membrane conformation facilitates the transition from a hemifusion intermediate to a pore. Thus, the action of the toxin promotes exocytosis of neurotransmitter (NT) (from the left to the right panel) and, for the same membrane topological reason, it inhibits the opposite process, i.e., the fission of the synaptic vesicle.

In addition to the transfer of electrons, two protons are bound on fumarate reduction (see reaction 1) and two protons are liberated on menaquinol oxidation (see reaction 2). The protons consumed on fumarate reduction are undoubtably bound from the cytoplasm (see Fig. 8a). The experimental results on intact bacteria, with inverted vesicles or liposomes containing W. succinogenes QFR (Kroger et al., 2002 Biel et al., 2002), suggest that the oxidation of menaquinol by fumarate as catalyzed by W. succinogenes QFR is an electroneutral process. The protons formed by menaquinol oxidation have therefore been assumed to be released to the cytoplasmic side of the membrane where they balance the protons consumed by fumarate reduction. 

The following data indicate that CH3-H4MPT H-S-CoM methyltransferase is the site of primary Na translocation (see Figs. 6 and 12) (i) the enzyme has been partially purified from Methanosarcina barkeri and Methanobacterium thermoautotrophicum and found to be tightly membrane bound [69b] (ii) inverted vesicles of the methanogenic strain G61 catalyzed methyl transfer from CH3-H4MPT to H-S-CoM. This reaction was stimulated by Na ions and was coupled with the accumulation of Na" into the vesicles. Na uptake was inhibited by Na ionophores rather than by protonophores indicating primary Na translocation [168]. 

The major necessary strategy of eukaryotes to reconcile a reductive cell s cytoplasm with an oxidizing environment was the development of new membranes and compartments separated from the cytoplasm inside cells and where oxidizing chemistry could take place (Table 21.5) [6, 7]. (Such a compartment outside the cytoplasm is also the periplasmic space between the internal and the external membranes, seen in aerobic bacteria.) Vesicles inside cells probably originated from invagination of the new external membranes, incidentally inverting all their pumps so that their contents became similar to the external environment relative to the cytoplasm. For example, they have high concentrations of Ca, Na , Mn, and... 

Bilayers, Vesicles, Liposomes, Biological Cell Membranes and Inverted Micelles... 

HPTS fluorescence has been employed in the study of proton diffusion within inverted micelles [24], in liposomes [4a], in apomyoglobin and the inter-membranal hydration layers of multi-lamellar vesicles [4b]. The simplest case for analysis involves the HPTS molecule in the center of a sphere (inverted micelle, liposome) whose walls are impermeable to protons on the timescale of the experiment. This outer wall is therefore described by an additional reflective boundary condition. Inside such a sphere, even a single proton/anion pair ultimately reaches an equilibrium situation The long-time tail approaches a plateau, rather than decaying to zero. The smaller the radius of the sphere, the higher the expected asymptotic plateau. 

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