Topography of protein kinase C substrates analyzed by membrane splitting

We have used the methods of planar cell and membrane monolayer formation and monolayer splitting to study structural details of the transmembrane signaling process mediated by protein kinase C. We analyzed human red cell membrane proteins phosphorylated by phorbol ester activation of protein kinase C. Planar single membrane preparations, extraction procedures, and gel electrophoresis coupled with silver staining and autoradiography confirmed that two bands in the 100 kDa region, and bands 4.1, and 4.9, were peripheral and phosphorylated by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA also stimulated minor incorporation of [32 P]Pi into most integral membrane proteins, including band 3, glycophorin A, the band 4.5 region (glucose transporter) and band 7. Planar cell and membrane-splitting methods revealed that neither integral nor peripheral phosphorylated polypeptides were cleaved by freeze fracture, that all phosphorylated peripheral proteins partitioned intact with the cytoplasmic side of the membrane, and that the percentages of [32P]Pi-labeled peripheral proteins were the same in split membrane cytoplasmic leaflets as in intact membranes. As a unique approach to examining protein topographies membrane splitting provides strong evidence that the major phosphorylated products of the polyphosphatidylinositide pathway are topographically associated with the cytoplasmic leaflet of the human erythrocyte plasma membrane. We further conclude that TPA-induced phosphorylation of red cell peripheral proteins does not significantly alter their transbilayer partitioning patterns after membrane splitting.     

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.     

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.     

Rotational diffusion of band 3 proteins in membranes from En(a—) and neuraminidase-treated normal human erythrocytes

Recent experiments have demonstrated an association between band 3 and glycophorin A in the human erythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887–1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by studying membranes from En(a—) and neuraminidase-treated erythrocytes. Rotational diffusion was measured by observing flash-induced transient dichroism of eosin-labeled band 3. Although erythrocytes of the rare phenotype En(a—) lack the major sialoglycoprotein, glycophorin A, no significant difference in band 3 rotation at pH 7.4 and 37°C could be detected between En(a—) and normal erythrocyte membranes. Band 3 rotation at pH 7.4 was also insensitive to the enzymatic removal of sialic acid from the surface of normal erythrocytes. Moreover, the existence of an essentially similar temperature-dependent equilibrium between band 3 proteins with different mobilities was observed in normal, En(a—) and neuraminidase-treated erythrocytes. From these results it is concluded that glycophorin A contributes less than 15% to the cross-sectional diameter of the band 3-glycophorin A complex in the plane of the normal membrane. The rotation of the complex at pH 7.4 is not significantly influenced by either steric or electrostatic interactions involving the oligosaccharide moiety of glycophorin A.     

A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology

Silver/Coomassie blue staining of human erythrocyte membrane electrophoretograms permits simultaneous visualization and color differentiation of asialoproteins, sialoglycoproteins and lipids in the same gel. Using this technique evidence is provided that chymotrypsin cleaves glycophorin A as well as band 3. The chymotryptic fragmentation pattern of glycophorin A in situ intact cells was different from that generated by trypsin treatment. Chymotryptic cleavage of band 3 generated two Coomassie blue stained fragments at 62,000 and 38,000 Mr, whereas simultaneous cleavage of glycophorin A dimer and glycophorin A B heterodimer yielded yellow silver stained fragments at 68,000 and 47,000 Mr. Trypsin cleaved glycophorin A dimer (88,000 Mr) and monomer (38,000 Mr) to form membrane associated fragments of Mr = 40,000 and 18,000 respectively.     

Regulation of the glycophorin C-protein 4.1 membrane-to-skeleton bridge and evaluation of its contribution to erythrocyte membrane stability

The band 3-ankyrin-spectrin bridge and the glycophorin C-protein 4.1-spectrin/actin bridge constitute the two major tethers between the erythrocyte membrane and its spectrin skeleton. Although a structural requirement for the band 3-ankyrin bridge is well established, the contribution of the glycophorin C-protein 4.1 bridge to red cell function remains to be defined. In order to explore this latter bridge further, we have identified and/or characterized five stimuli that sever the linkage in intact erythrocytes and have examined the impact of this rupture on membrane mechanical properties. We report here that elevation of cytosolic 2,3-bisphosphoglycerate, an increase in intracellular Ca(2+), removal of cell O(2), a decrease in intracellular pH, and activation of erythrocyte protein kinase C all promote dissociation of protein 4.1 from glycophorin C, leading to reduced retention of glycophorin C in detergent-extracted spectrin/actin skeletons. Significantly, where mechanical studies could be performed, we also observe that rupture of the membrane-to-skeleton bridge has little or no impact on the mechanical properties of the cell, as assayed by ektacytometry and nickel mesh filtration. We, therefore, suggest that, although regulation of the glycophorin C-protein 4.1-spectrin/actin bridge likely occurs physiologically, the role of the tether and the associated regulatory changes remain to be established.     

Methyl acceptors for protein methylase II from human-erythrocyte membrane.

Membrane proteins from human erythrocytes were methylated with purified protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC.2.1.1.24). The methylated proteins were analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Monomeric and dimeric glycophorin A (NaIO4/Schiff-2 and NaIO4/Schiff-1 positive bands) and 'band 4.5' were identified as two major classes of methyl-acceptor polypeptides for protein methylase II. In rabbit erythrocyte membrane where glycophorin A is absent, 'band 4.5' was the only major methyl-acceptor protein component. Extracted and purified glycophorin A from human erythrocytes was also found to be an excellent substrate for protein methylase II with a Km of 35.7 microM. The role of erythrocyte membrane protein methylation is discussed with regard to membrane function.     

31P nuclear magnetic resonance studies of the phospholipid-protein interface in cell membranes.

Both native and recombined membrane systems from the human erythrocyte membrane and the rabbit sarcoplasmic reticulum have been studied with 31P Nuclear Magnetic Resonance (NMR). We compare intensities of the anisotropic 31P resonance exhibited by these membranes with the intensity expected from the known phospholipid content of the membranous sample. In a recombinant with human erythrocyte glycophorin, a component of the phospholipid is "missing" from the 31P NMR resonance, apparently due to a severe broadening of the resonance of that component. Approximately 29 phospholipid molecules were found immobilized per glycophorin molecule in the membrane, regardless of the phospholipid:protein ratio. Cholesterol may inhibit the immobilization of phospholipids by glycophorin. Recombinants with band three from the human erythrocyte membrane contain an immobilized phospholipid component, analogous to the results with glycophorin. 31P NMR data from the native sarcoplasmic reticulum membrane also revealed an immobilized phospholipid component whose magnitude is independent of temperature between 30 degrees C and 45 degrees C. Extensive papain proteolysis of the membrane completely digests the Ca++ Mg++ ATPase and removes the immobilization of phospholipids noted in the intact membrane. Limited trypsin cleavage, however, does not completely remove the immobilized component; salt reduces the immobilized component.     

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.     

Species specificity in mouse glycophorin

89, 65, 46 and 29 Kd mouse glycophorin proteins identified during polyacrylamide electrophoresis of mouse erythrocytes have been further characterized. These proteins (1) stain positive with Periodic Acid Schiff reagent after sodium hydroxide treatment; (2) labeled using [125I] in intact cells; (3) co-isolated along with integral membrane proteins in the pellet fraction of sodium hydroxide treated ghosts; and (4) demonstrated a molecular weight downshift after neuraminidase treatment during electrophoresis. We have called them mouse Sialoglycoproteins 1, 2, 3 and 4. Immuno-blot analysis revealed distinct species specificity between human and mouse erythrocyte ghosts, and some cross-reactivity between rat and mouse erythrocyte ghosts.     

Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.     

Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor

Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti-Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations.     

The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions

We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.     

Phosphorylation of glycophorin A in membranes of intact human erythrocytes

The qualitative and quantitative contribution of glycophorin A phosphorylation to the general and specific pattern of membrane protein phosphorylation in intact erythrocytes pre-incubated with 32Pi was examined. Intense 32P-labeled bands at 88,000 and 38,000 Mr were identified as phosphorylated glycophorin A dimer and monomer respectively on the basis of several criteria. Quantitatively, phosphorylated glycophorin A dimer accounted for about 70% of 32P in the band 3 region. This value is at least three times that previously reported. The results of ancillary experiments involving selective extraction of ghosts in acidified chloroform/methanol solvents and electrophoresis in the presence of detergents make it unlikely that the 32P associated with glycophorin A was due to bound polyphosphoinositides.     

Loss of rotational mobility of band 3 proteins in human erythrocyte membranes induced by antibodies to glycophorin A.

The effect of antibodies to glycophorin A on the rotational diffusion of band 3 in human erythrocyte membranes was investigated by transient dichrosim. Three antibodies that recognize different epitopes on the exofacial domain of glycophorin A all strongly reduce the rotational mobility of band 3. The effect is at most only weakly dependent on the distance of the epitope from the membrane surface. The degree of immobilization obtained with two of the antibodies, BRIC14 and R18, is very similar to that produced by antibodies to band 3 itself. Similar results were obtained with membranes stripped of skeletal proteins. Fab fragments and an antibody to glycophorin C had no effect on band 3 rotational mobility. These results rule out a mechanism whereby band 3 rotational immobilization results from enhanced interactions with the membrane skeleton that are mediated by a conformational change in glycophorin A. Rather, they strongly indicate that the antibodies to glycophorin A cross-link existing band 3-glycophorin A complexes that have lifetimes that are long compared with the millisecond time scale of the transient dichroism measurements.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.

This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.     

Glycophorins of human erythroleukemic K562 cells

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.     

Phosphorylation in membranes of intact human erythrocytes.

Studies of phosphorylation in membranes of intact human erythrocytes were performed by incubating erythrocytes in inorganic [32P]phosphate. Analysis of membrane proteins by polyacrylamide gel electrophoresis showed a pattern of phosphorylation similar to that observed when ghost membranes were incubated with [gamma-32P]ATP. Membrane lipid phosphorylation was also similar in intact cells and ghosts. The most heavily phosphorylated lipid, polyphosphoinositide, was closely associated with glycophorin A, the major erythrocyte membrane sialoglycoprotein obtained when the sialoglycoprotein fraction was isolated by the lithium diiodosalicylate-phenol partition procedure. Only 1 molecule of glycophorin A out of every 100 was found to be phosphorylated, and the phosphate exchange occurred specifically in the COOH-terminal intracellular portion of glycophorin A. These studies show that the human erythrocyte can be used as a model for membrane phosphorylation in an intact cell system.