Rightside-out Vesicles Research Articles

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H+, Na+ or Li+ to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na+ in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Ectosomes as immunomodulators

Considerable progress has been made in recognizing microvesicles as important mediators of intercellular communication rather than irrelevant cell debris. Microvesicles released by budding directly from the cell membrane surface (i.e., ectocytosis) either spontaneously or in response to various stimuli are called shed vesicles or ectosomes. Ectosomes are rightside-out vesicles with cytosolic content, and they expose phosphatidylserine in the outer leaflet of their membrane. Depending on their cellular origin, ectosomes have been associated with a broad spectrum of biological activities. In the light of recent findings, we now know that ectosomes derived from polymorphonuclear leukocytes, erythrocytes, platelets, and tumor cells have profound effects on the innate immune system, as well as on the induction of the adaptive immunity, globally reprogramming cells such as macrophages or dendritic cells toward an immunosuppressive and possibly tolerogenic phenotype. Although the effects observed in the circulation are mainly procoagulant and pro-inflammatory, ectosomes might be anti-inflammatory/immunosuppressive in local inflammation.

Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton and Human Erythrocyte Membrane by Directly Interacting with Glucose Transporter-1

Dematin and adducin are actin-binding proteins located at the spectrin-actin junctions, also called the junctional complex, in the erythrocyte membrane. Here we propose a new model whereby dematin and adducin link the junctional complex to human erythrocyte plasma membrane. Using a combination of surface labeling, immunoprecipitation, and vesicle proteomics approaches, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the junctional complex to the lipid bilayer. Because homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.

Polymorphonuclear Neutrophil-Derived Ectosomes Interfere with the Maturation of Monocyte-Derived Dendritic Cells

Polymorphonuclear neutrophils (PMNs) are a key component of the innate immune system. Their activation leads to the release of potent antimicrobial agents through degranulation. Simultaneously, PMNs release cell surface-derived microvesicles, so-called ectosomes (PMN-Ect). PMN-Ect are rightside-out vesicles with a diameter of 50-200 nm. They expose phosphatidylserine in the outer leaflet of their membrane and down-modulate monocyte/macrophage-activation in vitro. In this study, we analyzed the effects of PMN-Ect on maturation of human monocyte-derived dendritic cells (MoDCs). Intriguingly, exposing immature MoDCs to PMN-Ect modified their morphology, reduced their phagocytic activity, and increased the release of TGF-beta1. When immature MoDCs were incubated with PMN-Ect and stimulated with the TLR4 ligand LPS, the maturation process was partially inhibited as evidenced by reduced expression of cell surface markers (CD40, CD80, CD83, CD86, and HLA-DP DQ DR), inhibition of cytokine-release (IL-8, IL-10, IL-12, and TNF-alpha), and a reduced capacity to induce T cell proliferation. Together these data provide evidence that PMN-Ect have the ability to modify MoDC maturation and function. PMN-Ect may thus represent an as yet unidentified host-factor influencing MoDC maturation at the site of injury, thereby possibly impacting on downstream MoDC-dependent immunity.

Red Cells Release Microparticles Heterogeneous in Phenotypes, Procoagulant Activities, and Size under Different Conditions.

BACKGROUND. Hemolytic anemias (HA) such as autoimmune hemolytic anemia (AIHA), thalassemia, and sickle cell disease are associated with increased risk for thrombosis. Although exposure of procoagulant phospholipids on the RBC membrane in HA has been implicated, the pathogenesis remains to be elucidated. We recently showed that microparticles released from red cells (RMP) are procoagulant and significantly elevated in HA, suggesting a link of RMP to thrombotic complications. In this study, we report the release of RMP, heterogeneous in size, phenotypes and procoagulant activity by different methods.METHODS. RMP were prepared from washed RBC (2×109/mL) by three methods, exposure toi.10 μM calcium ionophore A23187 (CaIo) for 60 min,ii.20 μM lysophosphatidic acid (LPA) for 60 min, oriii.shear stress induced by a rotating Teflon pestle for 5 min at 160 rpm in a tissue grinder.After removing residual whole cells, the supernatant was centrifuged at 15,000×g for 10 min (large RMP) and the supernatant was further centrifuged at 150,000×g for 30 min (small RMP). The resulting RMP were labeled for flow cytometry using PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). Thrombin generation of RMP was measured by the calibrated automated thrombogram (CAT) system using fluorescent thrombin substrate on a fluorescence plate reader.RESULTS.1.The two-step centrifugation revealed two distinct populations, the large RMP expressing both GlyP and Ulex binding while the smaller RMP expressed only Ulex binding. Electron micrography showed diameters for the large RMP of 200 - 800 nM while the small RMP were 40 - 80 nm.2.Shear stress produced the greatest number of large GlyP + RMP (5.2 ± 1.3 × 106/μL), followed by CaIo (3.8 ± 0.7 × 106/μL), and LPA (2.5 ± 0.6 × 106/μL). However, CaIo produced the greatest number of small GlyP −/Ulex + RMP (4.6 ± 0.9 × 106/μL), followed by LPA (1.4 ±0.3 × 106/μL), and very few by shear tress (0.3 ±0.1 × 106/μL). The methods also gave different AnV binding: CaIo yielded >90% of RMP (large and small) that were positive, whereas RMP induced by LPA or shear stress gave only 45% and 18% positive, respectively. No TF + RMP were detected in any procedure.3.The RMP also differed in thrombin generation. Adjusting concentrations to equal numbers of Ulex + MP, CaIo RMP displayed the strongest activity (375 ± 62 nM) followed by LPA-induced (227 ± 58 nM) and shear-induced (136 ±33 nM).Thrombin generation correlated well with degree of AnV binding.CONCLUSIONS. We demonstrate that RBC release different species of RMP, heterogeneous in size, phenotypes and procoagulant activity. Both CaIo and LPA induced two distinct species of RMP, the larger expressing GlyP and Ulex binding while the smaller phenotype was negative for GlyP, possibly indicating that the small RMP are inside-out vesicles. In contrast, shear stress produced mainly large rightside-out vesicles with both GlyP expression and Ulex binding, and very low AnV binding. Thus, calcium influx appears necessary to release small inside-out RMP, and to induce the membrane flip-flop bringing negatively charged AnV binding sites to the plasma membrane. We postulated that highly procoagulant RMP that bind AnV may contribute to the thrombotic complications of HA and other RBC disorders associated with thrombosis.

Effect of asymmetric distribution of phospholipids in ghost membrane from rat blood on peroxidation induced by ferrous ion

This study used rat erythrocyte ghost membrane to investigate the effect of aminophospholipid distribution in biological membranes on oxidative susceptibility. Aminophospholipids, lipid peroxidation, and carbonyl compounds were quantified; plasma membrane structure was examined using atomic force microscopy (AFM) and SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Inside-out vesicles (IOVs) had significantly more aminophospholipids and greater lipid peroxidation than right-side-out vesicles (ROVs). Spectrin bands in IOVs disappeared obviously than in ROVs as shown in SDS–PAGE. In both systems vesicle protein size increased significantly with oxidation. Proteins aggregated much more in IOVs than ROVs at 48 h. These observations suggest that IOVs were more susceptible to ferrous ion-induced peroxidation than ROVs and that asymmetric phospholipid distribution affects biomembranes’ oxidative susceptibility.

Functional studies of P-glycoprotein in inside-out plasma membrane vesicles derived from murine erythroleukemia cells overexpressing MDR 3. Properties and kinetics of the interaction of vinblastine with P-glycoprotein and evidence for its active mediated transport.

Active [3H]vinblastine (VBL) transport (efflux) was documented for inside-out plasma membrane vesicles from murine erythroleukemia cells (MEL/VCR-6) resistant to vinca alkaloids and overexpressing MDR 3 P-glycoprotein (P-gp) 80-fold. Uptake of [3H]VBL at 37 degrees C by these inside-out vesicles, but not rightside-out vesicles or inside-out vesicles from wild-type cells, was obtained in the form of a rapid, initial phase (0-1 min) and a slower, later phase (> 1 min). The rapidity of each phase correlated with relative P-gp content among different MEL/VCR cell lines. The initial MDR-specific phase was temperature- and pH-dependent (optimum at pH 7), osmotically insensitive, and did not require ATP. The second MDR-specific phase was temperature-dependent, osmotically sensitive, and strictly dependent upon the presence of ATP (Km = 0.37 +/- 0.04 mM). Although other triphosphate nucleotides were partially effective in replacing ATP, the nonhydrolyzable analogue ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was ineffective. This time course appears to represent tandem binding of [3H]VBL by P-gp and its mediated transport, with the latter process representing the rate-limiting step. In support of this conclusion, both binding and transport were inhibited by verapamil, quinidine, and reserpine, all known to be inhibitors of photoaffinity labeling of P-gp, but only transport was inhibited by C219 anti-P-gp antibody or orthovanadate. Although the rate of transport of [3H]VBL was 7-7.5-fold lower than the rate of binding (Vmax = 104 +/- 15 pmol/min/mg protein, Kon = 1.5 - 2 x 10(5) mol-1 s-1) to P-gp, each phase exhibited saturation kinetics and values for apparent Km and KD for each process were approximately the same (215 +/- 35 and 195 +/- 30 nM). Intravesicular accumulation of [3H]VBL was almost completely eliminated by high concentrations of nonradioactive VBL, suggesting that simple diffusion does not contribute appreciably to total accumulation of [3H]VBL in this vesicle system. This could be at least partially explained by the fact that these inside-out vesicles under the conditions employed did not maintain a P-gp mediated pH gradient. However, ATP-dependent, intravesicular accumulation of osmotically sensitive [3H]VBL occurred against a substantial permeant concentration gradient in both a time- and concentration-dependent manner consistent with an active, saturable process.

Characterization of a Proton-Translocating ATPase in a Tonoplast-Vesicle Fraction from Citrus

Summary Tonoplast vesicles were isolated from Valencia Late ( Citrus sinensis (L.) cv. Osbeck) leaves by sucrose cushion density gradient centrifugation. Measurements of marker enzymes for microsomal membranes like latent IDPase (Golgi apparatus), Antimycin-A-insensitive NADPH-cytochrome c reductase (endoplasmi reticulum) and cytochrome-c-oxidase (mitochondria), and measurements of the ATP-hydrolysis activity with inhibitors specific for ATPases of different microsom indicated a good purity of the tonoplast vesicle fraction. The rate of ATP-hydrolysis of the tonoplast H + -ATPase was optimal at pH 7.4. It was stimulated by Cl − with a maximum at 60 mM and was inhibited by 60 mM NO 3 − . The tonoplast H + -ATPase hydrolyzed [MgATP] 2− in preference to other Mg-nucleotides in the sequence ATP > GTP > UTP >- CTP > ADP. The apparent K m for [MgATP] 2− -hydrolysis was 0.38 ± 0.04 mM. The H + -ATPase activity of tonoplast vesicles was sensitive to the anion channel blocker DIDS (I 50 = 15 µM). Even in the absence of Cl − the rate of ATP hydrolysis was inhibited by 30% at 40 M DIDS, which indicates a direct effect of DIDS on the tonoplast H + -ATPase. Quinacrine-fluorescence quenching measurements of tonoplast vesicles showed that the tonoplast H + -ATPase was able to build up an MgATP-dependent electrochemical proton gradient across the vesicle membrane. This gradient broke down in the presence of the protonophore gramicidine A and by the inhibition of the tonoplast H + -ATPase by NO 3 − . Determination of latent H + -ATPase activity with 0.002 % Brij 58 showed that the tonoplast vesicle fraction contained approximately equal amounts of rightside-out vesicles and inside-out vesicles. In the tonoplast vesicle preparation five polypeptides (63, 52, 37, 30, and 16 kDa) cross-reacted with an antiserum against the tonoplast H + -ATPase from leaves of Kalanchoe daigremontiana . Our results indicate that the tonoplast vesicles of Citrus leaves contain an H + -ATPase with similar properties as the tonoplast H + -ATPase from other plant and fungal sources. This characterization of the enzyme is the basis for the investigation of the energetization and regulation of secondary active transport mechanisms of ions and metabolites at the tonoplast in response to environmental stress.

Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.

Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.

10] Preparation of microcapsules from human erythrocytes: Use in transport experiments of glutathione and its S-conjugate

Publisher Summary This chapter presents the procedure for preparation of microcapsules from human erythrocytes for its use in transport experiments of glutathione and S-conjugate. Purification method relies on the observation that glycoproteins are located only on the external surface of erythrocyte membranes. Glycoprotein sugar chains of the erythrocyte membranes extend outward into the plasma. The concanavalin A-cellulose described in this chapter has a binding affinity for these sugars on the external surface of right-sideout vesicles, whereas inside-out vesicles do not interact with concanavalin. For preparation of sealed inside-out microvesicles, homogenized vesicles are added to 1 volume of a 20% suspension of concanavalin A cellulose. After 10 min at room temperature, the concanavalin A-cellulose is poured onto a coarse sintered-glass funnel G3 and washed with 5 volumes of 0.5 mM Tris-HC1, pH 8.0. The vesicles which do not bind to the mesh are concentrated by centrifugation at 26,000 g for 20 rain at 4 °. The sideness of these vesicles is determined by measurement of glyceraldehyde-phosphate dehydrogenase as a marker of the erythrocyte inner surface and acetylcholinesterase as a marker of the erythrocyte outer surface.

Surface charge at the cytoplasmic membrane of murine C1300 neuroblastoma cells

“Inside-out” vesicles (IV, mainly with the cytoplasmic side outermost) were obtained from the plasma membranes of neuroblastoma cells from strain C1300 mice, clone N18. These served as a convenient model for investigating the surface charge of the cytoplasmic side of the cell membrane using microelectrophoretic techniques. Electrophoretic mobility (EM) at a neutral pH and with an external medium of the same ionic strength was found to be 2.7-fold less than that of “right-side-out” vesicles (RV). Processing vesicles with neuraminidase reduced EM in RV but not in IV, while trypsin did so in both. Treatment with phospholipase C produced the same effect in IV but none in RV. Phospholipase D increases the EM of both types of vesicles. Charged groups at the surface of IV are titrated in relation to a pK of 3.5. The EM of IV is dependent on the Ca2+ concentration of the external medium. On the cytoplasmic side of the membrane, Ca2+ forms a 1∶1 complex with negatively charged protein and lipid molecules as well as those lipid molecules found with a neutral pH, in the form of a zwitterion with binding constants of 50, 12, and 25 liter/mole respectively.

Phosphate transport by basolateral plasma membranes of human small intestine

The characteristics of phosphate transport across the human intestinal basolateral membrane were determined using an enriched preparation in which uphill Na+-dependent d-glucose transport could not be demonstrated but adenosine triphosphate-dependent calcium transport was present. The human basolateral membrane vesicles were oriented as follows: 64% inside-out vesicles and 36% rightside-out vesicles or sheets, or both. Phosphate transport showed a 3.4-fold transient “overshoot” phenomena in the presence of an inwardly directed sodium gradient. Computerized Michaelis-Menten kinetics for the net active transport component indicated a Vmax of 65 ± 5 pmol/mg protein per 8 s and a Km of 93 ± 15 μM. The transport process was electroneutral. Gramicidin D and transstimulation studies confirmed the presence of a Na+-phosphate carrier at the basolateral membrane. Adenosine triphosphate enhanced phosphate uptake, indicating an adenosine triphosphate-dependent phosphate transport process. These studies are the first to determine the presence of a phosphate carrier in human intestinal basolateral membrane.

Orientation and Integrity of Plasma Membrane Vesicles Obtained from Carrot Protoplasts

Two fractions enriched in plasma membrane derived from suspension-cultured carrot (Daucus carota L.) cells were examined to determine if they differed from each other either in physical nature or in orientation. Parameters studied included the protein composition of purified membranes derived from trypsinized and nontrypsinized protoplasts as well as from trypsinized purified plasma membranes, the effect of inhibitors and membrane perturbants on ATPase activity, the binding of [acetyl-(14)C]concanavalin A to purified membrane fractions, and the competitive removal of [acetyl-(14)C]concanavalin A from purified membranes derived from [acetyl-(14)C]concanavalin A-labeled protoplasts. One fraction (at density of 1.102 grams per cubic centimeter on Renografin gradients) appears to be a mixed population of ;tightly' sealed vesicles with the majority being rightside-out vesicles of plasma membrane, and the other fraction (density 1.128 grams per cubic centimeter) apparently is a population of predominantly ;leaky' vesicles and/or nonvesicular fragments of plasma membrane, a large portion of which appear to be ;leaky' inside-out vesicles. In addition, it is shown that plasma membrane-enriched fractions can be distinguished from cellular endomembranes on the basis of protein and glycoprotein composition.

A regulatory component of adenylate cyclase is located on the inner surface of human erythrocyte membranes

Summary Human erythrocyte membranes contain the guanine nucleotide regulatory component of adenylate cyclase termed N, which can be assayed by in vitro complementation of adenylate cyclase in membranes of the cyc − variant of the S49 lymphoma cell. We used inside-out (IO) and rightside-out (RO) membrane vesicles to determine the membrane orientation of N: Trypsin inactivates N in IO but not in RO vesicles. Treatment with cholera toxin and NAD + activates N in IO but not in RO vesicles. These experiments provide strong evidence that N is located on the inner surface of the human erythrocyte membrane.

Partition in two-polymer aqueous phases reflects differences between membrane surface properties of erythrocytes, ghosts and membrane vesicles

Partition of cells (defined as the percentage of added cells in the top phase) in dextran-polyethylene glycol aqueous two-phase systems is an extremely sensitive indicator of cell surface properties. By appropriate choice of polymer concentrations and salt composition and concentration one can, to some extent, select the membrane properties (e.g. charge (Walter, H. (1975) in Methods in Cell Biology (Prescott, D.M., ed.), Vol. 9, pp. 25–50, Academic Press, New York), lipid composition (Walter, H, Krob, E.J. and Brooks, D.E. (1976) Biochemistry, 15, 2959–2964)) that determine the partition behavior of cells (or membranes). In the present experiments we have obtained by partition information on separability, alteration and ensuing heterogeneity during preparation of human erythrocytes, ghosts and rightside-out and inside-out vesicles from human erythrocyte membranes. A phase system in which partition is greatly (although not exclusively) determined by membrane charge was used. It was found that: 1. 1. The partition coefficients (and hence the membrane surfaces) of ghosts and of rightside-out vesicles differ from those of the erythrocytes from which they are derived. 2. 2. Rightside-out and inside-out vesicles have different countercurrent distribution patterns with the rightside-out vesicles having the higher partition coefficient (probably reflecting higher membrane charge of the latter). 3. 3. Rightside-out vesicles are highly heterogeneous membrane populations as evidenced by broader than theoretical countercurrent distribution curves which, in some experiments, even split into two populations outright. This heterogeneity is particularly interesting since the enzyme markers used to establish vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol. 2, pp. 245–281, Plenum Press, New York) indicate “pure” rightside-out preparations. 4. 4. Inside-out vesicles could, under no conditions tested, be rendered totally free of rightside-out vesicles according to the enzyme markers used to indicate vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol. 2, pp. 245–281, Plenum Press, New York). A discussion of factors involved in the partition of the vesicles and ghosts together with analogies to the partition behavior of stored or in vitro modified human red blood cells is presented.