Side-out Vesicles Research Articles

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.

Inside‐out but not right side‐out plasma membrane vesicles from soybean enlarge when treated with ATP + 2,4‐D as determined by electron microscopy and light scattering: Evidence for involvement of a plasma membrane AAA‐ATPase

The concept that the location of an AAA-ATPase associated with the plant plasma membrane may be indicative of a functional relationship to growth or cell enlargement by analogy with roles in physical membrane displacements as proposed for AAA-ATPases associated with internal membranes was tested. A plant growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans was utilized in a completely cell-free system. The rate of enlargement was accelerated by the synthetic plant growth factor 2,4-dichlorophenoxyacetic acid (2,4-D) in a log dose-dependent manner and was increased approximately 2-fold with the addition of 1 microM 2,4-D plus 100 microM ATP compared to 100 microM ATP alone, 1 microM 2,4-D alone or no additions. The cell-free enlargement was inhibited by AAA-ATPase-specific antisera and by CoCl2, an inhibitor specific for AAA-ATPases. The responsible ATP site appears to be on the inside of the cell, since right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination.

ATP‐ and growth substance‐dependent cell‐free enlargement of plasma membrane vesicles from soybean

Growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans prepared by aqueous two-phase partition and everted by freezing and thawing has been achieved in a cell-free system. In the presence of 100 microM ATP in 40 mM HEPES buffer, pH 7, enlargement of isolated plasma membrane vesicles was accelerated by the synthetic plant growth factor, 2,4-dichlorophenoxyacetic acid (2,4-D), compared to ATP alone, 2,4-D alone or no additions. After 20 min with 1 microM 2,4-D, vesicles increased in diameter, 20% on average. Although vesicle diameters in the presence or absence of 2,4-D overlapped, the means were clearly separated. The 20% increases in diameter corresponded to a doubling of vesicle volume. Both 100 microM ATP and 1 microM 2,4-D were necessary to stimulate the cell-free vesicle enlargement. In the presence of 1 microM 2,4-D, enlargement observed with 100 microM ATP was greater than with either 10 microM ATP or 500 microM ATP alone. In the presence of 100 microM ATP, vesicle enlargement was proportional to the logarithm of 2,4-D concentration. With the growth-inactive 2,4-D analog, 2,3-D, no vesicle enlargement was observed either alone or in the presence of 100 microM ATP. Right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination suggesting that the responsible ATP site was on the inside of the cell.

Involvement of heterotrimeric G protein in signal transduction of extracellular calmodulin in regulatingrbcS expression

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulatingrbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhancedrbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibitedrbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin onrbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum onrbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely inhibited both by heterotrimeric G proteins antagonist pertussis toxin and nonhydrolyzable GTP analogs GMP-PCP. These results provided the evidence that heterotrimeric G proteins may be involved in signal transduction pathways of extracellular calmodulin to regulaterbcS gene expression.

Applications of aqueous two-phase partition to isolation of membranes from plants: A periodic NADH oxidase activity as a marker for right side-out plasma membrane vesicles

Phase separations using standardized mixtures of polyethylene glycol, dextran and potassium phosphate are used widely to prepare highly purified plasma membranes from plants and in the preparation of chloroplast subfractions. Other uses include the removal of right side-out plasma membrane vesicles as contaminants from Golgi apparatus, endoplasmic reticulum and tonoplast (vacuole membrane) fractions and separation of right side-out and inside-out plasma membrane vesicles. The higher degree of separation between plasma membranes into the upper phase and internal membranes into the lower phase is in large measure due to the fact that only plasma membranes are oriented cytoplasmic side in. Most other membranes are oriented cytoplasmic side-out. This property extends to separations of right side-out and inside-out plasma membrane vesicles and to the separation of right side-out and inside-out sub-mitochondrial particles. The inside-out vesicles partition into the lower phase whereas the right side-out vesicles remain in the upper phase. The lack of efficacy of aqueous two-phase partitioning in other types of membrane separations is apparently due to the fact that surface characteristics that may distinguish different internal membranes are not located at the cytosolic membrane surface. At present there are no direct enzymatic markers for right side-out plasma membrane vesicles from plants. Demonstrations of sidedness and estimates of fraction purity are based on measurements of latency of marker enzymes, e.g., ATPases, at the cytosolic surface. This report describes a periodic NADH oxidase as an enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions of vesicles for measurement of activity.

Oxidation of NADH by intact segments of soybean hypocotyls and stimulation by 2,4-D

Intact sections of soybean cut from regions of cell elongation of hypocotyls of etiolated soybean seedlings oxidized externally supplied NADH (NADH is an impermeant substrate). The oxidation of NADH by 1-cm intact sections was stimulated by the plant growth factor 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum concentration of 2,4-D for stimulation was about 1 μM. Stimulations also were given by the naturally occurring 2,4-D analog, indole-3-acetic acid (IAA), but not by the growth inactive 2,4-D analog 2,3-dichlorophenoxyacetic acid (2,3-D). The findings confirm studies comparing inside-out and right side-out vesicles that show the 2,4-D-stimulated NADH oxidase to be located at the external cell surface. Since plant cells are unlikely to encounter NADH at their external cell surface, functions of the oxidase in reactions other than oxidation of NADH are discussed.

NADH oxidase activity present on both the external and internal surfaces of soybean plasma membranes

Soybean plasma membranes exhibit NADH oxidase activities accessible to NADH supplied to either the external or internal membrane surfaces. Activity at the external surface was demonstrated using plasma membranes of right side-out orientation prepared by aqueous two-phase partition and using intact soybean cells grown in culture. Activity at the internal membrane surface was demonstrated using vesicles of inside-out orientation obtained by preparative free-flow electrophoresis or by aqueous two-phase partition following freeze–thaw or Brij detergent treatment to invert some of the right side-out vesicles. The NADH oxidase activity of both membrane surfaces appeared to be stimulated by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Following the freeze–thaw treatment utilized to evert the vesicles, however, the NADH oxidase activity of right side-out vesicles was greatly reduced. A 2,4-D-stimulated activity of these vesicles could be restored by treatment first with reduced glutathione and then with hydrogen peroxide or oxidized glutathione. Upon treatment with 0.1% Triton X-100, both right side-out and inside-out vesicles exhibited approximately the same specific activity and the same 2,4-D-stimulated component of activity equal to the sum of the activities of the right side-out and inside-out vesicles assayed separately. These findings suggest that a significant NADH oxidase activity of the soybean plasma membrane responsive to 2,4-D resides on the external plasma membrane surface. This latter activity is unlikely to function under physiological conditions in the direct oxidation of NADH and alternative functions, such as in protein disulfide–thiol interchange, would be more appropriate.

NADH oxidase activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY 181984) at an external site

NADH oxidase activity from HeLa plasma membranes was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′(4-chlorophenyl)urea (LY181984). With sealed right side-out vesicles, the drug inhibited half maximally at about 30 nM and the inhibition was nearly complete. A closely related but growth-inactive sulfonylurea, N-(4-methylphenylsulfonyl)- N′-(phenyl)urea (LY181985), did not inhibit the activity. With plasma membranes first solubilized with 2% Triton X-100, activity also was inhibited by LY181984 and not by LY181985 but the maximum inhibition at 10 μM LY181984 was only 50%. When sealed right side-out plasma membrane vesicles were frozen and thawed repeatedly to evert some of the vesicles into an inside-out configuration, the NADH oxidase activity again was only about 50% inhibited by 1 μM LY181984. In such preparations, the right side-out vesicles exhibited an electrophoretic mobility greater than that of the inside-out vesicles. Sidedness was confirmed by measurements of ATPase latency and binding of immunogold-labeled concanavalin A. When the two vesicle populations were resolved by preparative free-flow electrophoresis, the active antitumor sulfonylurea LY181984 inhibited only the NADH oxidase activity of the right side-out vesicles. These findings suggested two NADH sites or activity isoforms for the plasma membrane NADH oxidase. One activity, inhibited by LY181984, appeared to be accessible to external NADH only with sealed right side-out vesicles. The other, not inhibited by LY181984, was accessible to NADH only with inside-out vesicles or after membrane disruption by Triton X-100. The findings demonstrate that the NADH oxidation site inhibited as a result of binding the active antitumor sulfonylurea LY181984 is at the external cell surface. Plasma membrane vesicles from HeLa cells are able to oxidize NADH supplied to either membrane surface but only with inside-out vesicles is NADH oxidation sensitive to inhibition by the antitumor sulfonylurea.

Plasmalemma-bound ATPase of potato tubers and its inhibition by Phytophthora infestans-derived elicitors of the hypersensitive reaction

Plasmalemma (PM) vesicles were isolated from tuber disks of potato cv. Kennebec and ATPase activity was characterized. The activity was synergistically stimulated by K + and Na + in the presence of Mg 2+ at a pH optimum of 6.5 and increased by the canalogenic antibiotic, alamethicin, suggesting that the enzyme is located inside the right side-out vesicles. It was, on the other hand, inhibited by diethylstilbestrol, ouabain and orthovanadate. Arachidonic acid (AA), present in potato late blight fungus, Phytophthora infestans and considered as a pure elicitor of the hypersensitive resistance response of potato, markedly decreased the (K + + Na +)-stimulated activity of the ATPase when added to PM preparation. The mycelial homogenate, which has been termed a crude elicitor and which also contains AA, had a negligible effect on the enzyme. Both crude elicitor and AA inhibited the ATPase activity when applied to tuber disks. The inhibition was expressed within 3 h after treatments as a result of one of the earliest actions of elicitors. The effect on the ATPase might be the cause of other metabolic changes that finally manifest themselves in the hypersensitive reaction.

Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane.

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.

Properties of erythrocyte membrane binding and autolytic activation of calcium-activated neutral protease

The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.

Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study

The interaction of amphotericin B with isolated human erythrocyte ghosts was monitored by circular dichroism at 37°C and 15°C. Although different, these spectra were not concentration dependent over a concentration range covering the inducement of K + leakage and hemolysis, which suggests the existence of only one bound amphotericin B species. At 15°C, the spectra indicate that amphotericin B is complexed with membrane cholesterol; the complex formation is saturable but not cooperative. At 37°C new spectra are observed, and their existence is conditioned by the presence of membrane proteins. The binding is cooperative but not saturable. The amphotericin B right side-out vesicles complexation is temperature as well as ionic strength dependent: at high ionic strength it is the same as with ghosts, with the same temperature dependence. At low ionic strength it is characteristic of an interaction with cholesterol, regardless of temperature. In the large unilamellar vesicles reconstituted from the total lipid extracts of erythrocyte membranes, amphotericin B is complexed with cholesterol, regardless of temperature and ionic strength. These results indicate that there are two different modes of amphotericin B complexation with erythrocyte membranes, reversible one in the other, depending on the molecular organization of the membrane and the presence of membrane proteins.

Separation and characteristics of inside-out and right side-out vesicles from a rat cardiac sarcolemma preparation

1. 1. Purified cardiac sarcolemma (SL) vesicles are highly suitable to study various Ca 2+-transport systems present in the SL. 2. 2. We describe in this paper the separation of the Inside-Out (IO) and Right side-Out (RO) oriented vesicle subpopulations from a purified rat heart SL preparation. 3. 3. The isolated subfractions were characterized with respect to the number of beta-adrenergic binding sites and the Ca 2+-uptake and (Ca 2+-Mg 2+)-ATPase activities. 4. 4. It was found that the Ca 2+-uptake and the (Ca 2+-Mg 2+)-ATPase activities reside in the IO fraction and are virtually absent in the RO fraction, confirming that the active Ca 2+-uptake represents the outward directed sarcolemmal Ca 2+-flux.

Characterization of (Ca2+ + Mg2+) adenosine triphosphatase activity and calcium transport in boar sperm plasma membrane vesicles and their relation to phosphorylation of plasma membrane proteins.

Properties of (Ca2+ + Mg2+) adenosine triphosphatase (ATPase) in plasma membranes from boar epididymal spermatozoa are described. Enzyme activity is optimum at high pH and has a high affinity for Ca2+. It is not inhibited by the calmodulin antagonist trifluoperazine (TFP), but it is inhibited by low concentrations of Ca2+. Plasma membrane vesicles obtained by hypotonic lysis of intact sperm [mixed inside-out (IOV) and right side-out (ROV) vesicles] transport 45Ca2+ in the presence of oxalate. Similar to the Ca2+-stimulated Mg ATPase activity, transport is unaffected by TFP, but unlike the ATPase, transport is at an optimum rate near neutral pH and is completely inhibited by p-chloromercurphenylsulfonate (pCMS). When plasma membranes are labeled in the presence and absence of Ca2+ and Mg2+ with [gamma-32P]ATP, differences in the intensity of labeling and lability of bound 32P to alkali and hydroxylamine suggest that two polypeptides between 100-120K may be related to a transport ATPase. The addition of TFP at concentrations which stimulate net Ca2+ uptake in intact cells causes intense labeling of a single neutrally charged protein near 68K. These labeling patterns and the properties of (Ca2+ + Mg2+) ATPase identify particular plasma membrane proteins (PMPs) from the complex surface of these cells that may be involved in Ca2+-dependent functions and support the view that calmodulin is not directly involved in the regulation of ATP-driven Ca2+ efflux from boar spermatozoa.

Use of a concanavalin a polymer to isolate right side-out vesicles of purified plasma membranes from eukariotic cells

We have purified two plasma membrane populations using a Concanavalin A polymer. It was assumed that vesicles retained by the polymer were right side-out, whereas vesicles not retained were inside-out. 5′-nucleotidase and (Na + + K +) stimulated Mg ++ ATPase activities were at least two fold higher in inside-out than in right side-out vesicles, though recovered total activity was about 80 % for both enzymes together. Moreover, Concanavalin A modified 5′-nucleotidase activity of right side-out vesicles according to the dose used.

Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes.

The relationship between active extrusion of Ca(++) from red cell ghosts and active uptake of Ca(++) by isolated red cell membrane fragments was investigated by studying the Ca(++) uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca(++) adenosine triphosphatase (ATPase) and ATP-dependent Ca(++) uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more (45)Ca in the presence of ATP compared to preparation B. Separation of the (45)Ca-labeled membrane vesicles by density gradient centrifugation showed that the (45)Ca label was localized to the inside-out vesicle fraction. In addition, the (45)Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of (45)Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La(+8) (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca(++) by red cell membrane fragments is due to the active transport of Ca(++) into inside-out vesicles.