HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.     

Isolation of chromaffin cell plasma membranes on polycationic beads

We have tried to define which proteins of chromaffin cell plasma membranes are facing the cytoplasm by surface labelling a selectively oriented membrane preparation.Viable chromaffin cells were isolated by collagenase treatment of bovine adrenals. Plasma membranes from these cells were isolated on polycationic beads by the method of Jacobson and Branton (Jacobson, B.S. and Branton, D. (1977) Science 195, 302–304). The purity and orientation of the membranes were defined by biochemical and morphological criteria. The membranes, with their external side apposed to the bead surface, were enriched about 10-fold with respect to a whole cell homogenate, and contained only small amounts of contaminating organelles. Surface specific iodination of membranes on beads with 1,3,4,6-tetrachloro-3α, 6α-diphenylglycoluril (Iodogen), followed by polyacrylamide gel electrophoresis, allowed the identification of cytoplasmically exposed proteins. A different pattern was observed when intact cells were labelled prior to membrane isolation. The advantages and possible uses of this immobilized membrane preparation are discussed.     

Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins.

1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5'-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside-out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins lableled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic ³²P. We conclude that the phagolysosome is probably oriented in an “inside-out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Transferrin receptor and its recycling in HeLa cells.

The transferrin receptor is a 180 000-dalton protein which can be dissociated to two 90 000-dalton polypeptides under reducing conditions. It can be labelled by lactoperoxidase-catalysed iodination on the cell surface at 0 degree C. Trypsin digestion of labelled cells at 0 degree C can be used to degrade those receptors on the cell surface; they release a 70 000-dalton soluble fragment which binds to transferrin. When cells are labelled at 0 degree C, then warmed to 37 degrees C, the labelled receptors enter the cells and become trypsin resistant. These receptors enter the cells, probably via coated pits, with a half-life of approximately 5 min. Since there is about three times as much receptor inside cells as on the surface, this means that transit through the cell to the cell surface takes approximately 21 min, if all receptors are on the same cycling pathway.     

Resolution of the paradox of red cell shape changes in low and high pH

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.     

PHAGOCYTOSIS OF LATEX BEADS BY ACANTHAMOEBA CASTELLANII (NEFF) : III. Isolation of the Phagocytic Vesicles and Their Membranes

A method is described for the rapid and efficient isolation of phagocytic vesicles from large scale cultures of Acanthamoeba castellanii (Neff) that have been incubated with polystyrene latex beads. Cells were allowed to phagocytose latex beads for 30 min and then were homogenized, and the phagocytic vesicles were isolated by one centrifugation through several layers of sucrose. Identity and purity of the phagocytic vesicles were determined by electron microscopy, chemical analyses, and assays of acid phosphatase, α- and β-glucosidase, and reduced nicotinamide adenine dinucleotide dehydrogenase. When phagocytosis was allowed to occur for longer periods the phagocytic vesicles appeared to fuse with each other and perhaps with digestive vacuoles. The resultant vesicles which contained many beads were heavier than those which consisted of only one bead or a few beads with a closely applied membrane. Ultrasonication ruptured the isolated vesicles, and the membranes could then be isolated in 30–50% yield based on phospholipid analysis. These membranes were essentially free of acid hydrolases and, presumably, other soluble proteins, as was also indicated by their low ratio of protein to phospholipid. The membranes have been prepared both as closed vesicles and as open sheets.     

Active calcium transport and calcium-dependent membrane phosphorylation in human peripheral blood lymphocytes

The characteristics of the calcium pump were investigated in intact human peripheral blood lymphocytes /PBL/ and in inside-out vesicles prepared from their plasma membranes. Intact PBL were loaded with calcium by a short exposure to A23187 ionophore. After the elimination of the ionophore, calcium-loaded PBL produced an ATP-dependent, external lanthanum sensitive, uphill calcium extrusion. Calcium pump in intact PBL was insensitive to ouabain and /until cellular ATP was provided/ to oligomycin and dinitrophenol. Maximum calcium extrusion rate and the alkali cation sensitivity of the process were similar to those in human red cells. Calcium was partially sequestered by PBL, and this calcium could be released by A23187 ionophore only.Inside-out plasma membrane vesicles prepared from hypotonically lysed PBL showed and ATP + Mg²⁺-dependent uphill calcium uptake. This calcium transport was insensitive to ouabain, oligomycin, or dinitrophenol, while blocked by lanthanum and quercetin. Calmodulin significantly stimulated calcium pumping in EDTA-washed vesicles. ATP-dependent and -independent calcium uptake rates, respectively, showed different calcium concentration dependences.When PBL membrane vesicles were phosphorylated by γ ³²P-ATP, a calcium-induced, hydroxylamine-sensitive incorporation of ³²P was found in 120–150 000 molecular weight proteins. Depending on the way of membrane preparation, the molecular weight of the phosphoprotein was shifted. Similarly to that found in red cell membranes, sensitivity to calmodulin stimulation and partial proteolysis of the calcium pump molecule showed an inverse relationship.     

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures.

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.     

Interactions of Vesicular Stomatitis Virus Structural Proteins with HeLa Plasma Membranes

THE processes whereby nucleoprotein core particles of certain animal viruses become enveloped by and bud off from host cell membranes can be studied by preparing membrane^1,2 or “sedimentable”³ fractions from infected cells and examining them for the presence of virus proteins. We find that similar experiments designed to monitor assembly of vesicular stoma-titus virus (VSV) at sites along the plasma membranes of HeLa cells are best interpreted after first investigating the possibility that virus proteins adsorb to plasma membranes during cell fractionation and membrane isolation. In this report, we show that at 0° C the membrane protein of VSV, among other virus proteins, adsorbs to plasma membranes isolated from uninfected HeLa cells. With appropriate pulse-chase experiments, however, we are able to demonstrate the progressive association, in vivo, of VSV core protein with plasma membranes of infected HeLa cells.     

Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.     

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 μU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristics responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.     

Surface peptides on intact Ehrlich ascites tumor cells : Identification by a method not requiring subcellular fractionation

Lactoperoxidase catalysed iodination of tyrosyl residues was used to label the exposed plasma membrane proteins in intact Ehrlich ascites tumor cells. Autoradiography of ¹²⁵I-labeled intact cells revealed that the label was predominantly associated with the plasma membrane. When whole cells were solubilized and subjected to gel electrophoresis, two major labeled peptide classes of 100 000 and 80 000 D along with 4 minor labeled classes were found. An identical labeling pattern was obtained when plasma membranes isolated from labeled cells were solubilized and subjected to gel electrophoresis. These results demonstrate that the number of exposed plasma membrane peptides and their molecular weights can be determined without first isolating the membrane by subcellular fractionation procedures, a standard approach in most studies.     

The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae

Gregarines, which are parasitic protozoa living in invertebrates, possess a cortical structure specific to their vegetative stage: namely two additional cytomembranes are lying just under the plasma membrane. This cortical complex has been isolated by centrifugation on discontinuous sucrose gradients and characterized chemically. Its integrity was tested by electron microscopy. Ghost proteins were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. About 30 polypeptides of mol.wt. 15000–300000 were present in this fraction and four glycoproteins were detected after periodate/Schiff staining. Ten major proteins were labelled after lactoperoxidase-catalysed iodination. The GP₂ glycoprotein (41000–49000 apparent mol.wt.) appears to be a major component of the cell surface. Effects of trypsin and Pronase digestion on ghosts and cells were monitored by gel electrophoresis and by electron microscopy. Ghosts treated with low trypsin or Pronase concentrations (10–25μg/ml) became drastically disorganized; many proteins were vigorously attacked in comparison with those of control ghosts. Variations in proteinase-sensitivity of proteins are pointed out. The GP₃ glycoprotein (130000–160000 apparent mol.wt.) seemed to be the only glycoprotein released from the cell surface by trypsin. Whole cells treated under the same conditions or with higher proteinase concentrations (up to 1mg/ml) do not exhibit morphological modifications of the cell surface; furthermore, no discernible cleavage of membrane proteins was indicated by electrophoretograms. It is postulated that cell-surface proteins are protected by the dense carbohydrate cell coat. By using various different methods (change of ionic strength, detergent, denaturing agent, labelling experiment) it was possible to localize several major proteins within the protozoon cortical membranes.     

Disposition of membrane proteins as affected by changes in the electrochemical gradient across mycoplasma membranes

The effect of ionophores on the exposure of $\text{Acholeplasma}$$\text{laidlawii}$ membrane proteins to the aqueous surroundings was studied by the lactoperoxidase-mediated radio-iodination procedure. The iodination values of intact cells pre-treated with valinomycin and/or carbamylcyanide m-chlorophenylhydrazone (CCCP) were much lower than those of untreated cells. The iodination values of isolated membranes from treated or untreated cells were, however, the same. Our results suggest that membrane proteins are less exposed to the aqueous external surroundings when electrical gradients of ions across the cell membrane are collapsed.     

Membrane isolation on polylysine-coated glass beads. Asymmetry of bound membrane

Erythrocyte membranes isolated on polylysine-coated glass beads exhibit many of the properties of the native membrane. Gel electrophoresis indicates that all major protein components of the membrane are retained during membrane isolation. The membrane integrity and accessibility of selected components was tested using non-penetrating probes. In general, membranes on beads displayed accessibility properties typical of inside-out vesicles. The accessibility of membrane acetylcholinesterase to assay reagents, as well as membrane accessibility to the actions of neuraminidase, trypsin and galactose oxidase-NaB3H4 demonstrated that the protoplasmic surface of membrane isolated on beads was exposed, while the extracellular surface was inaccessible. The differential accessibility of the membrane surfaces demonstrates the feasibility of investigating asymmetry of membranes isolated on cationic glass beads.     

The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli.

The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized [125I]di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination. The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments. Hydrogenase (mol.wt. approx. 63 000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.     

Ultracryotomy for the study of unfixed membranes

Plasma membranes from chromaffin cells of bovine adrenal medullae and from chicken macrophages were isolated on a urografin density gradient, frozen and sectioned without previous chemical fixation. Their receptor binding sites were localized by specific labelling. The sections were then post-fixed in the presence of K2Cr2O7 to produce positive staining of the membrane proteins. Chromaffin cell membranes formed single vesicles. The nicotinic acetylcholine receptor (localized using a monoclonal antibody against its cholinergic binding site) was always found in patches on the surface of vesicles, whose profiles corresponded to thickened bilayers. Macrophage membrane vesicles were agglutinated. The mannose receptor (localized using the ligand, mannosylferritin) was randomly distributed within the electron-dense coat of the agglutinated vesicles or on electron-dense caps involved in agglutination. The binding sites of both receptors were intact, as revealed by their being recognized by a monoclonal antibody against their cholinergic binding sites and by the active binding of the mannosylated ligand which was inhibited by mannan. The distribution of the receptors on the vesicles reflected their distribution on the cell surface.     

ON THE SPATIAL ARRANGEMENT OF PROTEINS IN MICROSOMAL MEMBRANES FROM RAT LIVER

Rat liver rough microsomes were labeled enzymatically with ¹²⁵I using lactoperoxidase and glucose oxidase. In intact microsomes only proteins exposed on the outside face of the microsomal membrane were iodinated. Low concentrations of detergent (0.049% deoxycholate) were used to allow entrance of the iodination system into the vesicles without disassembling the membranes. This led to iodination of the soluble content proteins and to an increased labeling of the membrane proteins. The distribution of radioactivity in microsomal proteins was analyzed after separation by sodium dodecyl sulfate acrylamide gel electrophoresis. Most membrane proteins were labeled when intact microsomes were iodinated. No major membrane proteins were exclusively labeled in the presence of low detergent concentrations or after complete membrane disassembly. Therefore it is unlikely that there are major membrane proteins, other than glycoproteins, present only on the inner membrane face or completely embedded within the microsomal membrane. Microsomal proteins were also labeled by incubating rough microsomes with [³H]-NaBH₄ after reaction with pyridoxal phosphate. Microsomal membranes were permeable to these small molecular weight reagents as shown by the fact that proteins in the vesicular cavity as well as membrane proteins were labeled with this system.