Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.     

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.     

Isolation of plasma membrane and tonoplast fractions from spinach leaves by preparative free-flow electrophoresis and effect of photoinduction

A membrane fraction enriched in plasma membrane and tonoplast vesicles was isolated from green leaves of Spinacia oleracea L. and subjected to subfractionation by free-flow electrophoresis. The most electronegative membrane vesicle fraction collected after the free-flow electrophoretic separation was identified as derived from tonoplast, while the least electronegative fraction was identified as derived from plasma membrane. The identification of the fractions was based on membrane morphology, and on the presence or absence of biochemical markers. The plasma membrane fraction was enriched in thick (9–11 nm) membranes which bound N-1-naphthylphthalamic acid (NPA), and reacted with phosphotungstic acid at low pH on thin sections for electron microscopy. The tonoplast fraction was enriched in vesicles with 7–9 nm thick membranes that neither bound NPA nor reacted with phosphotungstic acid at low pH. Both the plasma membrane and the tonoplast fraction were about 90% pure, with a cross-contamination of not more than 2%. Membrane vesicles originating from dictyosomes, endoplasmic reticulum, mitochondria, plastids, or peroxisomes contaminated the plasma membrane and the tonoplast fractions by a few % only. In leaves of photoinduced plants (24 h light period), the plasma membranes were thicker than in control leaves (8 h light, 16 h dark). The plasma membrane fraction obtained from photo-induced leaves by free-flow electrophoresis retained this increase in thickness, showing not only that photoinduction alters plasma membrane structure, but also that this change is stable to isolation.     

Localization of the oxytocin receptor in the plasma membrane of rat myometrium.

The distribution of [3H]oxytocin binding sites among various subcellular fractions of rat myometrium paralleled the distribution of 5'-nucleotidase, a plasma membrane marker enzyme, but not of NADPH-cytochrome c reductase or succinate-cytochrome c reductase, which are endoplasmic reticulum and mitochondrial marker enzymes respectively. [3H]Oxytocin binding to the most enriched plasma membrane fraction showed the degree of selectivity with respect to hormone analogues that is expected for the oxytocin receptor. The binding of oxytocin to this fraction showed an apparent Kd of 1.98 X 10(-9) M and a capacity of 1.28 pmol mg-1. It is concluded that the oxytocin receptor is located on the plasma membrane of the smooth muscle cells of the rat uterus.     

Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley

In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides.     

Effects of fatty acids on superoxide radical generation in leukocytes.

Acetylated ferricytochrome c was employed for the detection of superoxide radicals (O-2) generated both in intact cells and in subcellular fractions of leukocytes. Certain saturated fatty acids, myristate in particular, induced the production of O-2 in leukocytes, suggesting a correlation between the formation of O-2 and the hydrophobic interaction of fatty acids with the leukocyte plasma membrane. As compared with O-2 radical generation from phagocytizing leukocytes a greater stimulation of O-2 formation was observed in cells in which myristate was added. The enhanced activity which generated O-2 in the cell-free system was located in a particulate fraction but not in the cytosol. The rate of O-2 generation in the particulate fraction was higher in the presence of NADPH than in the presence of NADH. The effects of reagents such as KCN, 2,4-dichlorophenol and aminotriazole on the O-2 generation in this fraction are examined and the nature of the O-2 generating system is discussed.     

Inositol 1,4,5-trisphosphate receptors. Localization in epithelial tissue.

Using a polyclonal antiserum raised against the inositol 1,4,5-trisphosphate receptor (IP3R) purified from rat cerebellum, we examined the subcellular distribution of IP3R in canine pancreatic homogenates. IP3R was present primarily in a smooth microsomal fraction (low density), a (high density) rough microsomal (RM) fraction previously shown to consist of highly purified rough endoplasmic reticulum (RER) vesicles, and, to a much lesser extent, in an intermediate density microsomal fraction which did not contain markers for RER or plasma membrane. When the RM fraction was subjected to isopycnic centrifugation on sucrose gradients, IP3R equilibrated at high sucrose densities. When ribosomes were extracted from the RM fraction by treatment with puromycin/high salt, IP3R equilibrated at considerably lighter sucrose densities. This shift in density indicated that IP3R which was present in the RM fraction is associated with the RER. Because of a significant amount of IP3R fractionating into the smooth microsomal fraction (which contains plasma membrane, among other "smooth" membranes) and a considerable amount of IP3R present in the nuclear pellet which is also enriched in plasma membrane, we examined the possibility that IP3R may be present in plasma membrane. Further subfractionation of a crude plasma membrane pellet from rat liver revealed that IP3R coenriched with a plasma membrane marker and strongly suggested an association of IP3R with plasma membrane. The issue of why the same receptor is found in multiple biochemically and morphologically distinct membrane fractions is discussed in terms of the possibility of RER subcompartmentalization and IP3R subtypes. The fractionation pattern of IP3R in pancreas is significantly different from that previously reported for calcium (Ca2+)-binding proteins and an intracellular Ca-ATPase (Nigam, S. K. and Towers, T. (1990) J. Cell Biol. 111, 197-200), raising questions as to links between these latter proteins and IP3 sensitive Ca2+ pools. Nevertheless, although the fractionation patterns are different, all of these proteins are clearly associated with the RER.     

Studies on isolated subcellular components of cat pancreas. I. Isolation and enzymatic characterization.

Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na+K+)-ATPase, and 5'-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membranes enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na+K+)-ATPase.     

Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine.

Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.     

Uptake of extracellular biotin by Escherichia coli biotin prototrophs.

Uptake of exogenous biotin by two Escherichia coli biotin prototroph strains, K-12 and Crookes, appeared to involve incorporation at a fixed number of binding sites located at the cell membrane. Incorporation was characterized as a binding process specific for biotin, not requiring energy, and stimulated by acidic pH. Constant saturation quantities of exogenous biotin were incorporated by these cells, and the amounts, which were titrated, depended on whether the cells were resting or dividing. Resting cells incorporated exogenous biotin amounting to 2% of their total intracellular biotin content. Fifty percent of the exogenous biotin was incorporated into their free biotin fraction, and 50% was incorporated into their bound biotin fraction. On the other hand, dividing cells incorporated exogenous biotin into all of their intracellular sites, 88% going into the intracellular-bound biotin fraction, and 12% going into the free biotin fraction. Calculations suggested that each cell contained approximately 3,000 binding sites for biotin. It was postulated that biotin incorporation sites might have been components of acetyl coenzyme A carboxylase located at or near the membrane.     

Localization in the synaptic junction of the cyclic AMP stimulated intrinsic protein kinase activity of synaptosomal plasma membranes.

Synaptosomal plasma membrane fragments contain a tightly bound protein kinase which can catalyse the phosphorylation of endogenous protein the reaction bein stimulated by cyclic AMP. A fraction enriched in synaptic junctions, which can be isolated from Triton X-100-treated synaptosomal plasma membranes, is also enriched in the cyclic AMP stimulated intrinsic protein kinase. The location of the enzyme in the synaptic junction suggests that cyclic AMP-stimulated phosphorylation may have some role in synaptic transmission.     

Subcellular metabolism of exogenous GM1 ganglioside in normal human fibroblasts

The metabolization of exogenous GM1 in normal human fibroblasts at a subcellular level is investigated in the present paper. For this a GM1 ganglioside, radiolabelled on the sphingosine moiety, was given to the cells and all the formed metabolites analyzed, in a time-course study, in enriched fractions of lysosomes, plasma membrane and microsomes. After feeding the cells, the radioactivity incorporation was relevant in the enriched lysosomal and plasma membrane subfractions whereas it was modest in the enriched microsomal fraction. The kinetic curves obtained for each enriched fraction, following a 3-day chase period, suggested a translocation of exogenous GM1 from the plasma membrane to the lysosomal apparatus and, of GM1 itself together with its metabolites, to the Golgi or endoplasmic reticulum and finally again to the plasma membrane.     

Biosynthesis of biotin from dethiobiotin by the biotin auxotroph Lactobacillus plantarum.

Lactobacillus plantarum requires biotin for growth. We show that in the presence of high levels of the biotin biosynthetic precursor, dethiobiotin, L. plantarum synthesizes biotin and grows in medium with dethiobiotin but without biotin. Lactobacillus casei also grew under similar conditions.     

The respiratory burst of bovine neutrophils. Role of a b type cytochrome and coenzyme specificity

A new method of preparation of bovine polymorphonuclear leukocytes (PMN) is described. The subcellular distribution of cytochrome b in resting and activated bovine PMN was compared to that of the O2-.-generating oxidase (assessed as NADPH cytochrome c reductase inhibited by superoxide dismutase). In resting PMN and in PMN activated by phorbol myristate acetate (PMA), cytochrome b was located into two membrane fractions, one of which was enriched in plasma membrane and cosedimented with alkaline phosphatase, while the other consisted of a denser material cosedimenting with markers of the specific and azurophil granules, i.e. the vitamin-B12-binding protein and myeloperoxidase respectively. During activation of PMN by PMA, 15-20% cytochrome b migrated from dense granules to the plasma membrane. The distribution of the O2-. generating oxidase and cytochrome b in subcellular particles was studied during the course of phagocytosis of PMA-coated latex beads by bovine PMN. At the onset of the respiratory burst, the phagocytic vacuoles arising from internalization of the plasma membrane were enriched in oxidase and alkaline phosphatase, but their specific content of cytochrome b was limited; in contrast, cytochrome b was predominant in denser membrane fractions cosedimenting with myeloperoxidase and the vitamin-B12-binding protein. After a few minutes of phagocytosis, a fraction of light vacuoles, slightly denser than the phagocytic vacuoles, became enriched in O2-.-generating oxidase, cytochrome b, the vitamin-B12-binding protein and myeloperoxidase. These vacuoles probably arose from the fusion of the phagocytic vacuoles with dense granules. In bovine PMN supplemented with glucose and maintained in anaerobiosis, activation by PMA induced slow reduction of cytochrome b (60-70% in 15 min at 37 degrees C). Similar results were obtained with cytoplasts after activation by PMA (30% reduction in 3 min at 37 degrees C). Cytochrome b in a particulate fraction obtained by centrifugation at 100 000 X g of an homogenate of PMA-activated PMN, was slowly reduced upon addition of NADPH under anaerobiosis (less 20% in 20 min at 37 degrees C). No reduction occurred in the 100 000 X g fraction prepared from non-activated PMN. The Soret band of cytochrome b reduced by dithionite was displaced by CO only by 1-2 nm. At subsaturating concentrations, CO had no effect on the rate of O2 uptake by activated bovine PMN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.     

Isolation of the plasma membrane of a trypanosomatid flagellate: General characterization and lipid composition

A technique is described for the isolation of a fraction that contains the plasma membrane of the trypanosomatid flagellate Leptomonas collosoma. This fraction has been investigated by electron microscopy and has been shown to be mostly membranes associated with microtubules, a known plasma membrane marker in this organism. The fraction is enriched in Mg²⁺-dependent ATPase but has a decreased specific activity of succinate dehydrogenase. Lipid has been extracted from whole cells and the isolated plasma membrane fraction. A fraction of the total lipid that is eluted from a silicic acid column by acetone is found to be concentrated in the plasma membrane. Also enriched in the plasma membrane fraction is a 5,7-diene sterol identified as ergosterol. The major phospholipids of the whole cell and the plasma membrane are phosphatidylethanolamine and phosphatidylcholine. Approximately 60% of the fatty acids of the cell and plasma membrane have a carbon chain length of eighteen, and half of this is in the form of the mono-unsaturated fatty acid.     

Trypanosoma cruzi: fusogenic ability of membranes from cultured epimastigotes in interaction with human syncytiotrophoblast

Trypanosoma cruzi epimastigotes cultured in vitro were disrupted by successive freezing and thawing and subsequent sonication. The total homogenate was fractionated by differential centrifugation to obtain an enriched plasma membrane fraction. The proteins of subcellular parasite fractions were labeled with 131I and their binding to membrane fractions from human placenta syncytiotrophoblast was studied. Syncytiotrophoblast fractions enriched in plasma showed higher specific activity for binding an enriched T. cruzi plasma membrane fraction compared with other fractions of syncytiotrophoblast. The properties of this interaction were studied with digestive enzymes (trypsin and phospholipase A2). The results showed that both proteins and lipids could be involved in this interaction. The Ca2+ requirements for the membrane-membrane interaction are different for the two membranes studied. Also the enriched plasma membrane T. cruzi fraction had a higher capacity to induce fusion processes than the other subcellular fractions. The above results indicate that a preferential syncytiotrophoblast-T. cruzi interaction may occur between the two cell surfaces as compared to intracellular membranes and that the parasite surface is able to induce an instability process leading to membrane fusion. These results may have implications in regard to the mechanism of entry of the parasite into cells.     

Isolation and partial characterization of glycolipids and a carbohydrate from the secondary granules and plasma membrane of polymorphonuclear leukocytes

Chloroform/methanol extracts of the secondary granule and plasma membrane fractions of polymorphonuclear leukocytes have been shown to contain both non-polar and polar carbohydrate-containing materials. The ratio of the polar to the non-polar material was much higher in the plasma membrane than the secondary granule fraction. The non-polar material contains at least two ceramide-like glycolipids and accounts for most of the broad band of periodic acid/Schiff-positive material which migrates at the dye front in sodium dodecyl sulfate electrophoretic gels of granule and plasma membrane extracts. The polar material appears to be a single substance containing no fatty acids or sialic acid and is composed of glucose, hexosamine and a carboxylic acid derivative of pentose. Expressed on a per mg of protein basis, the amount of carbohydrate associated with the polar material in the plasma membrane fraction was about five times that of the secondary granule fraction.     

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. II. Internalization of proteins

The distribution of surface proteins during phagocytosis by rabbit peritoneal polymorphonuclear leukocytes was studied to determine whether the proteins of the phagocytic vesicles of these differentiated cells were representative of the entire set of plasma membrane proteins. Phagocytosis of bovine serum albumin-diisodecylphthalate emulsion by lactoperoxidase-iodinated rabbit neutrophils was linear over 15-20 min at a rate of 96 microgram oil/min/mg cell protein. This rate was similar to that of unlabeled cells. Incorporation of cell-associated free iodine by endogenous myeloperoxidase during phagocytosis was inhibited by 1 mM cyanide, which had no effect on the rate of particle uptake. The surface of intact neutrophils contained at least 13 iodinated proteins distinguishable by polyacrylamide gel electrophoresis followed by autoradiography. Isolated phagosomes were deficient in six of these proteins. The plasma membrane fraction of these cells was missing five of these same proteins which, however, were enriched in a dense surface fraction (Willinger, M., and F. R. Frankel. J. Cell Biol. 82: 32-44). When experimental conditions were reversed, and the PMNs were labeled after phagocytosis, these five proteins remained on the cell surface, while at least three of the major proteins found on resting cells were depleted. Incubating the cells with colchicine, which has been shown to affect the distribution of some plasma membrane constituents during phagocytosis, had no effect on the distribution of surface proteins in our system. These results indicate that a nonrandom interiorization of lactoperoxidase-labeled surface proteins of polymorphonuclear leukocytes occurs during phagocytosis.     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.