Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system.

We have used defined subcellular fractions to reconstitute in a cell-free system vesicle fusions occurring in the endocytic pathway. The endosomal fractions were prepared by immuno-isolation using as antigen an epitope located on a foreign protein, the transmembrane glycoprotein G (G-protein) of vesicular stomatitis virus. The G-protein was first implanted in the cell plasma membrane and subsequently endocytosed for 15 to 30 min at 37 degrees C. The endosomal fractions were immuno-isolated on a solid support using as antigen the cytoplasmic domain of the G-protein in combination with a specific monoclonal antibody. For comparative studies the plasma membrane was immuno-isolated from cells in the absence of G internalization with a monoclonal antibody against the exoplasmic domain of the G-protein. The immuno-isolated endosomal vesicles contained 70% of horseradish peroxidase internalized in the endosome fluid phase, exhibited an acidic luminal pH as shown by acridine orange fluorescence and differed in their protein composition from the immuno-isolated plasma membrane fraction. The fusion of endocytic vesicles originating from different stages of the pathway was studied in a cell-free assay using both a bio-chemical and a morphological detection system. These well defined endosomal vesicles were immuno-isolated with the G-protein on the solid support and provided the recipient compartment of the fusion (acceptor). They were mixed with a post-nuclear supernatant containing endosomes loaded with exogenous lactoperoxidase (donor) at 37 degrees C. Fusion delivered the donor peroxidase to the lumen of acceptor vesicles permitting fusion-specific iodination of the G-protein itself. The fusion of vesicles required ATP and was detected only with an endosomal fraction prepared after internalization of the G-protein for 15 min at 37 degrees C but not with a plasma membrane or with an endosomal fraction prepared after 30 min G-protein internalization.     

The behavior of the plasma membrane following osmotic contraction of isolated protoplasts: Implications in freezing injury

Summary Following osmotic contraction of isolated rye protoplast (Secale cereale L. cv. Puma) that results in nearly a 50% reduction in volume, the plasma membrane was smooth, with no folding or pleating. Instead, deletion of plasma membrane occurred and numerous cytoplasmic vesicles were observed. As a result, the area of the plasma membrane was reduced by approximately 40%. Thin sections revealed that the cytoplasmic vesicles were membrane bound and not merely voids in the cytoplasm. High resolution video microscopy revealed the extent of vesiculation showing large clusters of cytoplasmic vesicles following osmotic contraction. Labeling the plasma membrane with fluorescein-Con-A prior to hypertonic contraction suggested that the cytoplasmic vesicles were derived from the plasma membrane. Freeze-fracture particle density on both the protoplasmic (PFp) and exoplasmic face (EFp) of the plasma membrane remained unchanged following contraction, which is consistent with a unit-membrane deletion into cytoplasmic vesicles. Upon partial re-expansion of the protoplasts, thin sections showed that the vesicles remained in the cytoplasm. These results using osmotic manipulation confirm earlier observations of isolated protoplasts at the light microscope level. Upon contraction plasma membrane is deleted into cytoplasmic vesicles, which are not readily reincorporated into the plasma membrane upon expansion. Lysis occurs before the original volume and surface area are regained.Department of Agronomy Series Paper no. 1456.     

Single-Molecule Imaging of the H-Ras Membrane-Anchor Reveals Domains in the Cytoplasmic Leaflet of the Cell Membrane

In the last decade evidence has accumulated that small domains of 50–700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30–40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.     

Immuno-isolation of a plasma membrane fraction from the Fao cell.

A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.     

Peptide-specific antibody for the melibiose carrier of Escherichia coli localizes the carboxyl terminus to the cytoplasmic face of the membrane

The synthetic decapeptide NH2-Cys-Val-Gly-Ala-Val-Ser-Asp-Val-Lys-Ala-COOH (designated MBct10), which corresponds to the carboxyl terminus of the melibiose carrier of Escherichia coli, was synthesized and used to raise antibodies in a rabbit. Anti-MBct10 antibodies recognizes the normal melibiose carrier but not a truncated carrier lacking 14 carboxyl-terminal amino acids. Thus the antibodies are specific for the carboxyl terminus of the carrier and not for other domains of the protein. When right-side-out and inside-out membrane vesicles were probed with anti-MBct10 serum, only the inside-out vesicles bound antibody. The carboxyl terminus of the melibiose carrier protein is therefore exposed on the cytoplasmic surface of the membrane. The co-localization of both NH2- and carboxyl termini to the cytoplasmic surface dictates that the protein cross the membrane an even number of times. These data together with hydrophobicity analysis support a topological model for the melibiose carrier with 10 or 12 transmembrane domains.     

Caveolae and sorting in the trans-Golgi network of epithelial cells.

VIP21 is a 21 kDa membrane protein present in TGN-derived transport vesicles isolated from the epithelial MDCK cell line. The membrane topology and subcellular localization of VIP21 were studied using antibodies against the N- and C-terminal domains. The protein was found to have a structure with little or no exposure to the exoplasmic side of the membrane. VIP21 was localized to the TGN, consistent with its presence in TGN-derived transport vesicles. Unexpectedly, it was also very abundant in the non-clathrin-coated plasma membrane invaginations called caveolae. We have previously proposed that VIP21 is associated with glycosphingolipid-enriched membrane domains in the TGN which may be involved in the sorting of proteins into vesicles directed to the apical plasma membrane. Caveolae are specialized lipid structures with similarities to the glycolipid microdomains in the TGN. The presence of VIP21 in both locations suggests that the mechanisms governing inclusion of proteins into caveolar plasma membrane domains are related to the processes of protein and lipid sorting at the TGN. This connection is confirmed by the recent finding that the amino acid sequence of VIP21 is almost identical to that of caveolin, a protein previously localized to caveolae.     

Basolateral sorting signals differ in their ability to redirect apical proteins to the basolateral cell surface

Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.     

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells.

Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.     

Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies

We have prepared polyclonal antibodies to the cytoplasmic portion of the envelope glycoprotein G of vesicular stomatitis virus (VSV) by using synthetic peptides corresponding to either the 22 or 11 ultimate carboxy-terminal residues of the G as immunogens. When antibodies to the 22 residue peptide are microinjected into monolayer baby hamster kidney cells before or shortly after infection with wild-type VSV, G protein accumulates in large intracellular patches and little G is observed in the Golgi complex or at the cell surface. In contrast, when antibodies to the 11 residue peptide are injected, no such patches are observed and G protein is seen colocalized with the injected antibody at the endoplasmic reticulum, in the Golgi complex, in transport vesicles, and at the plasma membrane. Microinjection of these antibodies does not disturb the pathway or kinetics of G-protein transport. In cells infected with a temperature-sensitive mutant of VSV, 045, the glycoprotein accumulates in the endoplasmic reticulum at 39.8 degrees C, but rapidly moves through the Golgi apparatus and then to the cell surface after a temperature shift-down to 32 degrees C. Using rhodamine-coupled antibodies to the 11 residue peptide, a microscope stage equipped for precise temperature control, and a silicon intensifier target video camera, we can visualize by video light microscopy the synchronized exocytotic transport of the G protein directly in the living cell.     

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.     

Lipid domains in the exoplasmic and cytoplasmic leaflet of the human erythrocyte membrane: a spin label approach

The existence of different lipid domains in the monolayers of the human erythrocyte membrane was investigated at 4 °C by employing spin-labelled phospholipid analogues. Spectra of analogues located exclusively either in the exoplasmic or in the cytoplasmic leaflet of erythrocyte membranes were recorded. Spectra were simulated by variation of order parameter describing the average amplitude of motion of the long molecular axis of the nitrogen 2pπ orbital of the spin label and of the respective correlation times. For both leaflets at least three components were required to fit the experimental spectra, differing mainly in the order parameter. While the parameters of each component are not very different between both membrane halves, the relative contribution of each component to the spectrum is different between the exoplasmic and cytoplasmic leaflet. The order parameter of the most fluid component, presumably resembling the lipid bulk phase, is smaller in the cytoplasmic leaflet in comparison to the exoplasmic one. The lateral coexistence of different lipid domains in the human red blood cell membrane is concluded. The molecular nature of those domains is discussed. Received: 6 November 1998 / Revised version: 25 January 1999 / Accepted: 29 January 1999     

Isolation of domains of the plasma membrane of hepatocytes

Several recent studies have demonstrated the ability of techniques based on immunoadsorption to selectively isolate specialized subregions of membranes, termed domains, which are derived from a larger more complex parent membrane like the plasma membrane. The immunoadsorbent is directed against a specific antigen that resides exclusively or predominantly in the membrane domain to be isolated. Thus, a monospecific antibody to the domain-specific antigen is required. In the present study we developed a method employing a modified immunoblotting strategy which could utilize polyspecific antibodies to isolate membrane vesicles derived from a specific membrane domain of the hepatocyte plasma membrane. We also used specific cell surface labeling of the hepatocyte plasma membrane by lactoperoxidase-catalyzed iodination at 4 degrees C and preparation of different sized vesicles by sonication to facilitate isolation of the specific domain. For this study, polyspecific antisera were raised in goats against a membrane fraction, denoted N2u, which is enriched in bile canalicular proteins. This antiserum recognizes, among other antigens, a 110,000 Mr polypeptide previously shown to be localized in the bile canaliculus (J. Cook et al. (1983) J. Cell. Biol. 97, 1823-1833). A monospecific antiserum was raised in rabbits against the rat hepatocyte asialoglycoprotein receptor, a sinusoidal domain-specific set of glycoproteins whose major form has a Mr of 43,000. These antisera were each coupled indirectly to different pieces of nitrocellulose by the immunoblotting protocol and were used to isolate membrane vesicles from a crude extract of liver plasma membrane prepared by sonication. The ratio of iodinated asialoglycoprotein receptor to the 110,000 Mr polypeptide in vesicles isolated by the affinity nitrocellulose immunoadsorbent method indicate a 10- to 15-fold enrichment of sinusoidal-derived vesicles relative to bile canalicular-derived membrane vesicles. These results show that the affinity nitrocellulose immunoadsorbent method can be used to isolate domain-specific vesicles. Further, the affinity immunoadsorbent method described here for the isolation of domains of the plasma membrane is an integrative one allowing isolation of vesicles present in relatively small concentration in crude cell extracts and it requires minimal ultracentrifugation time.     

Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi.

Membrane vesicles can be obtained from epimastigote forms of Trypansoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with 131I by the lactoperoxidase catalyzed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein=1.5--2.0; orcinol : protein-0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.     

Plasma membrane vesicles isolated from epimastigote forms of trypanosoma cruzi

Membrane vesicles can be obtained from epimastigote forms of Trypanosoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5–10 times richer in protein-bound iodine when they are prepared from cells previously labeled with ¹³¹I by the lactoperoxidase catalysed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane.Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein = 1.5–2.0; orcinol : protein = 0.07 and absence of diphenylamine reaction. Vesicles contain 0.2–0.5% and 0.3–1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and a 30–50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.     

Vesiculation of platelet plasma membranes. Dilauroylglycerophosphocholine-induced shedding of a platelet plasma membrane fraction enriched in acetylcholinesterase activity

Incubation of washed rabbit platelets with suspensions of dilauroylglycerophosphocholine resulted in the shedding of vesicles without causing any appreciable leakage of cytoplasmic marker (lactate dehydrogenase) or organelle marker ([¹⁴C]serotonin). The response was dependent on incubation time, concentration of dilauroylglycerophosphocholine and reaction temperature. Vesicles were separated from platelets and exogenous dilauroylglycerophosphocholine by a series of centrifugation steps. An average diameter of vesicles was 100–200 nm on scanning electron microscopy. Vesicles were enriched 5-fold in plasma membrane marker enzyme, acetylcholinesterase, whereas specific activities of lactate dehydrogenase and intracellular membrane marker enzyme, NADH-cytochrome c reductase were decreased in vesicles. Protein analysis by SDS-polyacrylamide gel electrophoresis showed that actin and actin-binding protein were present, while myosin was barely detectable in vesicles. Vesicles contained all phospholipid species of intact platelets and cholesterol but almost 50% of phospholipids in vesicles was dilauroylglycerophosphocholine. The phospholipid to protein ratio in vesicles was about 6.5-times higher than in intact platelets.     

Subcellular location of the major protein antigens of paramyxoviruses revealed by immunoperoxidase cytochemistry

The major structural proteins of Newcastle disease virus and Sendai virus were localized in infected BHK-21 and MDBK cells by ultrastructural immunoperoxidase cytochemistry using antibodies against the individual viral protein antigens. The intracellular glycoproteins were strictly membrane bound, being localized in the rough endoplasmic reticulum (RER), perinuclear spaces, smooth membrane vesicles, and presumed Golgi apparatus. The nucleocapsid proteins were detected exclusively in membrane free cytosol and accumulated there, forming inclusions. The membrane (M) protein was found both in cytosol and on RER. The viral proteins on RER exhibited a distinct site specificity; the glycoproteins were facing the lumen of RER whereas M protein was present at the outer cytoplasmic surface. All the viral proteins were detectable at the plasma membrane where virus assembly takes place. However, their modes of distribution differed remarkably. The glycoproteins were spread widely over the entire cell surface including the areas of virus budding and those of normal morphology, whereas M protein was localized in restricted areas of the membrane, frequently forming a patch of virus specific membrane. The presence of nucleocapsids was confined to the virus particles budding from the plasma membrane. These results complement and extend the earlier morphological and biochemical data on the assembly or morphogenesis of paramyxoviruses.     

Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused

We have used thin sectioning and conical electron tomography to determine the three-dimensional structure of synaptic vesicles that were associated (docked) at release sites of the presynaptic membrane, called active-zones. Vesicles docked at the active zone occupied a strategic location: they formed regions of contact with the plasma membrane on one side and with that of one or more vesicles located deeper within the presynaptic terminal on the other side. The region of contact with the active zone measured approximately 15 nm in diameter ( approximately 2% of the vesicle's surface) and contained a smaller approximately 6 nm region where the proximal leaflets merged (hemi-fused). Hemi-fusion was only observed on the side of vesicles in contact with the active zone; at the side of contact between neighboring vesicles, the membranes were not hemi-fused. Approximately three-fourths of the docked vesicles contained hemi-fused regions. Vesicles fully fused to the active zone (exhibiting pores that appeared as interruptions of a single membrane) were less frequently observed ( approximately 1 of 10 hemi-fused vesicles). In conclusion, our observations in cortical synapses strengthen the hypothesis that hemi-fusion is a stable intermediary that precedes full fusion and release.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Induction of serotonin release from mast cells by lymphocyte activators is dependent upon implantation of lymphocyte plasma membrane components.

Plasma membranes (PM) isolated from mouse splenic lymphocytes were successfully fused to rat basophilic leukemia (RBL) cells using intact Sendai virus virions (SV). A two-step procedure was used in which SV were first fused with the PM to create PM + SV vesicles; the vesicles formed were then incubated with the RBL cells. Insertion of lymphocyte PM into the RBL cell's membrane endows a high rate of serotonin secretion upon stimulation of the implanted RBL cells with antimouse Ig antibodies or with concanavalin-A. The results of the present work clearly suggest that activation signals can be delivered via implantation of foreign membrane preparation containing specific receptors, thus rendering the target cells susceptible to stimulation by specific reagents.