Reconstitution of membrane proteins. Spontaneous incorporation of integral membrane proteins into preformed bilayers of pure phospholipid

The spontaneous reconstitution of lipid-protein complexes was examined by mixing bacteriorhodopsin or UDP-glucuronosyltransferase with preformed, unilamellar bilayers of pure dimyristoylphosphatidylcholine. Spontaneous insertion of these proteins into vesicles of dimyristoylphosphatidylcholine was facilitated by resonicating the vesicles at 4 degrees C. The property of resonicated vesicles that led to spontaneous reconstitution could be annealed by melting the bilayers, which slowed down reconstitution. The overall process of reconstitution consisted, however, of two steps. There was an initial insertion of proteins into a small portion of vesicles followed by subsequent fusion between protein-free vesicles and vesicles containing lipid-protein complexes. The first step appeared to proceed rapidly in all vesicles in a gel phase, whether or not they were resonicated or whether or not resonicated vesicles were annealed. The rate of the second step was sensitive to these treatments. The membrane proteins also inserted into preformed vesicles in a liquid crystalline phase, but this step was slower than for vesicles in a gel phase. Fusion between protein-free and protein-containing vesicles in a liquid crystalline phase was extremely slow. The data show that the spontaneous insertion of pure membrane proteins into preformed vesicles can be a facile event and that the overall reconstitution of membrane proteins into preformed unilamellar vesicles may be simpler to achieve than has been appreciated.     

Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers

We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.     

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [³⁵S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS PAGE of the treated cells showed incorporation of label into a broad 97–121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97–121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5′-O-(3-thiotriphosphate). The 97–121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5′-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteoglycans.

The results demonstrate that chromaffin vesicle matrix proteins are rapidly and intensely thiophosphorylated in cultured chromaffin cells but not in isolated vesicles. The data suggest that phosphorylation must play an important role in the normal function of these vesicle proteins.     

Interaction between ether glycerophospholipid vesicles and serum proteins in vitro

The effect of blood serum on the stability of small unilamellar vesicles consisting of 1-O-(1'-alkenyl)-2-acyl-sn-glycerophosphocholine (choline plasmalogen) or of the alkylacyl-, dialkyl- and diacyl analogs was evaluated by measuring either release of entrapped calcein or transfer of phospholipids from vesicles to serum high-density lipoproteins. The following order of stability was found: alkenyloleoylGPC greater than dioleoylGPC greater than di-O-octadecenylGPC greater than acyloleoylGPC = egg phosphatidylcholine = alkyloleoylGPC. AlkyloleoylGPC and acyloleoylGPC had aliphatic chain compositions similar to that of alkenyloleoylGPC. From the results obtained it is concluded that stability of vesicles in the presence of serum depends on vesicle size (larger vesicles are more stable) and on the type of bond (ether or ester) in position 2 of glycerol. Dioctadecenyl vesicles are about the same size as alkylacylGPC vesicles, but are significantly more stable in the presence of serum. Thus, it appears that an ester bond in position 2 of glycerol (which is replaced by an ether bond in dioctadecenylglycerol) favors the interaction of phospholipids with serum high-density lipoproteins or lipid-exchange proteins. The addition of cholesterol greatly enhances vesicle stability; among the vesicles used in this study those composed of alkenylacylGPC plus 30 mol% cholesterol were most resistant to disruption by serum. Experiments with sn-1 and sn-3 enantiomers of alkylacylGPC and diacylGPC have shown that interaction of vesicle membranes with serum components is independent of the steric configuration of vesicle phospholipids.     

Interactions of divalent cations or basic proteins with phosphatidylethanolamine vesicles

Small unilamellar vesicles have been prepared from phosphatidylethanolamine by sonication of the lipid in aqueous buffers of low ionic strength and high pH. These vesicles and their interactions with various di- and trivalent cations have been characterized using freeze-fracture electron microscopy. Phosphatidylethanolamine from 4 sources was examined: Hens' yolk phosphatidylethanolamine, human grey matter phosphatidylethanolamine, Escherichia coli phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine. The phosphatidylethanolamine from natural sources formed spherical, uniform 20–40 nm vesicles while dimyristoyl phosphatidylethanolamine formed larger, 70 × 25 nm, disc-shaped vesicles when sonicated above the phase transition temperature. Fusion of the unilamellar egg phosphatidylethanolamine, E. coli phosphatidylethanolamine and human grey matter phosphatidylethanolamine vesicles was induced by dialysis against buffers containing 2.0 nM Ca⁺ or 3.0 mM Mg²⁺. The fusion of the vesicles resulted in the precipitation of the lipid and the formation of multilamellar and, in some cases, hexagonal II structures. Dimyristoyl phosphatidylethanolamine vesicles were precipitated at 55°C by 1.0 mM Ca⁺ or 2.0 mM Mg²⁺. Treatment of the calcium- and magnesium-precipitated vesicles of hen's egg yolk phosphatidylethanolamine, E. coli phosphatidylethanolamine, human grey matter phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine with EDTA resulted in resuspension of the lipid. The specific size and shape of the vesicles formed in this manner depends on the type of phosphatidylethanolamine and ion involved. Dialysis of the Ca⁺- and Mg²⁺-precipitated egg phosphatidylethanolamine vesicles against buffer containing no Ca⁺, Mg²⁺ or EDTA also resulted in dissociation of the precipitate and formation again of a new vesicle population. This evidence indicates that the Ca⁺ and Mg²⁺ are not strongly bound to the phosphatidylethanolamine.Egg phosphatidylethanolamine vesicles would fuse in the presence of many di- and trivalent ions. Egg phosphatidylethanolamine vesicles were precipitated by beryllium, aluminum, chromium, manganese, cobalt, nickel, copper, zinc, strontium, cadmium, barium, lanthanium, mercury and lead. The amount of ion required to precipitate the vesicles and the type of structure resulting from the fusion of the vesicles was found to be unique for each ion.Small unilamellar vesicles prepared from egg phosphatidylethanolamine were reacted with several basic proteins (cytochrome c, basic protein from human myelin, protamine, poly-l-lysine and cationically-modified ferritin). The basic proteins also initiated the fusion of egg phosphatidylethanolamine vesicles but these proteins did not fuse egg phosphatidylcholine vesicles nor did normal ferritin initiate fusion. Human myelin basic protein initiated the fusion of dimyristoyl phosphatidylethanolamine vesicles above and below the phase transition of this lipid.     

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.     

Differential trafficking of transport vesicles contributes to the localization of dendritic proteins

In neurons, transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here, we use a novel pulse-chase system, which allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum to follow movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon, they very rarely moved beyond the axon initial segment but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.     

Entrapment of proteins in phosphatidylcholine vesicles.

The trapping efficiency of globular proteins in four different types of phosphatidylcholine vesicles was systematically studied. Vesicles were generated in a mixture of 125I-labeled proteins of various molecular weights. The trapped proteins were separated from untrapped proteins by gel filtration and ultrafiltration and subsequently analyzed by gel electrophoresis and autoradiography. Entrapment of proteins was demonstrated by their resistance to trypsin digestion. The relative amount of each entrapped protein species was then compared to that of the original protein solution. In multilamellar vesicles and large unilamellar vesicles, proteins of molecular weight up to 97 000 had the same trapping efficiency as sucrose. In small unilamellar vesicles generated by either sonication or ethanol injection, however, the relative trapping efficiency of protein decreased progressively as the molecular weight of the protein became greater. For example, the trapping efficiency of alpha-amylase (Mr 97 000) was only half of that for sucrose. The apparent decrease in trapping efficiency with the protein's molecular weight in small unilamellar vesicles canbe accounted for by the combination of the bound water layer at the vesicle's internal surface and the steric hindrance when protein is captured during vesicle formation.     

Differential localization of membrane receptor chemotaxis proteins in the Caulobacter predivisional cell

The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Both the flagellum and the MCPs are partitioned to only one daughter cell, the swarmer cell, upon division. We report the results of experiments designed to determine the distribution of these MCPs within swarmer cells and predivisional cells. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum.     

Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions.     

Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.     

Unequal distribution of penicillin-binding proteins among inner membrane vesicles of Escherichia coli

Escherichia coli penicillin-binding proteins (PBPs) were associated only with inner membrane vesicles when separated on 30 to 65% or 19 to 49% (wt/wt) sucrose gradients. Fractionation of vesicles through the low-density gradient revealed at least two classes of PBP-inner membrane associations. The first class consisted of PBPs 1 through 4, and the second class consisted of PBPs 5 through 8. These classes were distinguished by the density of vesicles with which they were associated; class 1 PBPs migrated with vesicles of higher density than did class 2 PBPs. Such combinations suggest that PBPs are nonrandomly distributed within the inner membrane, implying potential functional relationships among the PBPs themselves and with particular membrane domains. In addition, in cell lysates and in vesicle fractions, a 60,000-dalton aztreonam-insensitive PBP or protein fragment was observed which could potentially be confused with PBP3.     

Foreword: Translocation of proteins across membranes: systems,conservation and evolutionary origin

The interactions of mouse thymocytes with unilamellar phospholipid vesicles comprised of dimyristyl lecithin (DML), dipalmitoyl lecithin (DPL), dioleoyl lecithin (DOL), and egg yolk lecithin (EYL) were examined in vitro.

In cells treated with [³H]DML or [³H]DPL vesicles, electron microscope (EM) autoradiographic analysis showed most of the radioactive lipids to be confined to the cell surface. Transmission EM studies showed the presence of intact vesicles (DPL) and collapsed or ruptured vesicle fragments (DML) adsorbed to the surfaces of treated cells. In cells treated with DPL vesicles containing a watersoluble dye (6-carboxyfluorescein; 6-CF), most of the fluorescent vesicles were localized at the periphery of the treated cells. Furthermore, substantial fractions of the cell-associated DPL and DML could be released by a mild trypsinization without damaging the cells. These results suggest that the uptake of DML and DPL is primarily due to vesicle-cell adsorption. Such an adsorption process appears to be enhanced at or below the thermotropic-phase transition temperature of the vesicle lipid. Under certain conditions these adherent vesicles also formed patches or caps on the cell surface.

In cells treated with DOL or EYL vesicles, transmission EM and EM autoradiography showed relatively little exogenous vesicle lipid located at the cell surface. Thymocytes incubated (37°C) with [¹⁴C] EYL vesicles containing a trapped marker, [³H]inulin, incor porated both isotopes at identical rates. In separate experiments it was found that this marker was located inside the treated cells. Thymocytes treated with DOL vesicles containing 6-CF exhibited a uniform and diffuse distribution of dye in the internal volume of the cells. Little cell-associated EYL or DOL could be released by trypsinization. Evidence against endocytosis of intact vesicles as a major pathway of vesicle uptake is also presented. These observations, coupled with the demonstration of vesicle-cell lipid exchange as a minor component of vesicle uptake suggest that incorporation of EYL and DOL vesicles by thymocytes is primarily by vesicle-cell fusion.     

Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron.     

Brain clathrin and clathrin-associated proteins.

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.     

Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers

The presence of cholesterol in small unilamellar vesicles (ULV) of dimyristoylphosphatidylcholine (DMPC) catalyzes fusion of the vesicles at temperatures below the upper limit for the gel to liquid-crystalline phase transition of the DMPC. The extent to which ULV grow depends on the concentration of cholesterol in the vesicles and on temperature. Maximum growth occurs at 21 degrees C. It decreases as the temperature is lowered below 21 degrees C. Growth does not occur at temperatures above the phase transition. In addition, the presence of cholesterol in ULV of DMPC catalyzes the insertion of integral membrane proteins into the vesicles. Thus, bacteriorhodopsin from Halobacterium halobrium, UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, and cytochrome oxidase from beef heart mitochondria formed stable lipid-protein complexes spontaneously when added to ULV containing cholesterol at temperatures under which these vesicles would fuse. Incorporation of these proteins into the ULV of DMPC did not occur in the absence of cholesterol or in the presence of cholesterol when the temperature of the system was above that for the phase transition. It appears that cholesterol lowers the energy barrier for fusion of ULV of DMPC and for insertion of integral membrane proteins into these bilayers. Studies with bacteriorhodopsin suggest that the energy barrier for insertion of proteins into ULV containing cholesterol is smaller than the energy barrier for fusion of the ULV with each other.     

Characterization of calpain I-binding proteins in human erythrocyte plasma membrane

The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).     

Membrane proteins exposed on the external side of the intestinal brush-border membrane have fusogenic properties

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.     

Vascular permeability to proteins and peptides in the mouse pineal gland

Summary The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.Part of this study was presented at the EMCELL-76 meeting, Copenhagen, 1976     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.