Concanavalin A-agglutinability of membrane-skeleton-free vesicles and aged cellular remnants derived from human erythrocytes. Is the membrane skeleton required for agglutination?

Vesicles and cell remnants have been obtained by aging of erythrocytes in vitro. The vesicles lacking the membrane skeletal proteins and the remnants known to possess a rigid skeleton have been used to assess the role of membrane skeletal proteins in the process of Con A (concanavalin A)-mediated agglutination of erythrocytes. Both the vesicles and the remnants were found to bind Con A at the same density as did intact cells. The vesicles, isolated from normal as well as from the Con A-agglutinable trypsin- and Pronase-treated cells, failed to agglutinate with Con A. They were, however, well agglutinated by WGA (wheat-germ agglutinin) and RCA [Ricinus communis (castor bean) agglutinin], indicating that the vesicles are not defective in agglutination. Large, cytoskeleton-free, vesicles prepared by another procedure also gave the same results. The aged remnants from trypsin- and Pronase-treated erythrocytes showed significantly decreased agglutination with Con A, but were agglutinated as well as the fresh cells by WGA and RCA. The agglutination with Con A is thus abolished when the membrane skeleton is absent, and reduced when it is rigid, suggesting that the skeleton may play an important role in the agglutination of erythrocytes by Con A.     

Protein 4.1 is involved in a structural thermotropic transition of the red blood cell membrane detected by a spin-labeled stearic acid

Proteins involved in a structural transition in red blood cell membranes detected at 8 +/- 1.5 degrees C by a stearic acid spin-label have been investigated. Calcium loading of red blood cells with ionophore A23187 caused the disappearance of the 8 degrees C transition. Protein 4.1 appears to be the most susceptible protein to Ca2+ treatment. Antibodies specific for spectrin, band 3 (43K cytoplasmic domain), and protein 4.1 have been utilized as specific probes to modify membrane thermotropic properties. The 8 degrees C transition was eliminated by anti-4.1 protein antibodies but was not modified by the other antibodies. To further characterize the protein(s) involved in the transition, ghosts were subjected to sequential extraction of skeletal proteins. The extraction of band 6, spectrin, and actin did not modify the 8 degrees C transition. In contrast, high-salt extraction (1 M KCl) of spectrin-actin-depleted vesicles, a procedure that extracts proteins 2.1 and 4.1, was able to eliminate the 8 degrees C transition. Rebinding of purified protein 4.1 to the high salt extracted vesicles restored the 8 degrees C transition. These results indicate the involvement of protein 4.1 in the transition and suggest a functional membrane association of this protein. The binding of protein 4.1 to the membrane seems to contribute significantly to the thermotropic properties of red blood cells.     

Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton.

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.     

Interactions between protein 4.1 and band 3. An alternative binding site for an element of the membrane skeleton

Protein 4.1 from human erythrocytes formed a complex with band 3 in inside-out erythrocyte membrane vesicles and with soluble peptides derived from the cytoplasmic domain of band 3. Protein 4.1 labeled metabolically with 32P bound saturably to vesicles depleted of endogenous protein 4.1. The soluble cytoplasmic domain of band 3 (43K) competitively displaced approximately 60% of bound 32P-protein 4.1 from reconstituted membrane vesicles. Pretreatment of vesicles with anti-43K similarly inhibited the rebinding of protein 4.1. In solution, 125I-43K formed a complex with protein 4.1 that saturated at 1:1 stoichiometry and migrated as a discrete band when analyzed by nondenaturing polyacrylamide gel electrophoresis. In rate-zonal sedimentation in isotonic salt solutions, protein 4.1 and 43K sedimented as a sharp peak at 4.4 S. In experiments aimed at exploring the role of the protein 4.1-band 3 interaction in the organization of the membrane skeleton, the effect of spectrin was investigated. Spectrin and protein 4.1 formed a complex which co-sedimented in sucrose gradients, but the addition of 43K to preformed spectrin-protein 4.1 complexes resulted in disruption of the complex and co-sedimentation of most of the protein 4.1 with 43K. These results suggest that protein 4.1 can associate with band 3 in the erythrocyte membrane and that this association may modulate the attachment of the membrane skeleton to the membrane.     

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.     

Quantitative agglutination of specific populations of sea urchin embryo cells with concanavalin A

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated 32/64 cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Protein 4.1N is required for the formation of the lateral membrane domain in human bronchial epithelial cells

The membrane skeleton forms a scaffold on the cytoplasmic side of the plasma membrane. The erythrocyte membrane represents an archetype of such structural organization. It has been documented that a similar membrane skeleton also exits in the Golgi complex. It has been previously shown that βII spectrin and ankyrin G are localized at the lateral membrane of human bronchial epithelial cells. Here we show that protein 4.1N is also located at the lateral membrane where it associates E-cadherin, β-catenin and βII spectrin. Importantly, depletion of 4.1N by RNAi in human bronchial epithelial cells resulted in decreased height of lateral membrane, which was reversed following re-expression of mouse 4.1N. Furthermore, although the initial phase of lateral membrane biogenesis proceeded normally in 4.1N-depleted cells, the final height of the lateral membrane of 4.1N-depleted cells was shorter compared to that of control cells. Our findings together with previous findings imply that 4.1N, βII spectrin and ankyrin G are structural components of the lateral membrane skeleton and that this skeleton plays an essential role in the assembly of a fully functional lateral membrane.     

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) destabilizes erythrocyte membrane skeleton

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38-97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of alpha-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.     

Cooperative action between band 3 and glycophorin A in human erythrocytes: immobilization of band 3 induced by antibodies to glycophorin A.

The ability of transmembrane receptor proteins to change their association with the cytoskeleton in response to ligand binding seems to be a key mechanism of signal transduction across membranes. To investigate the molecular features of this mechanism we have used the red cell membrane as a model system to study signal transduction through the integral protein, glycophorin A. In these studies the lateral mobility of integral proteins was measured in situ by fluorescence recovery after photobleaching, and membrane rigidity was characterized by micropipette aspiration technique. We found that binding either a monoclonal antibody or its monovalent Fab to the exoplasmic domain of glycophorin A in normal red cells immobilized the receptor and rigidified the membrane. Further, immobilization and rigidification did not occur when antibodies were bound to Miltenberger V cells containing a mutant form of glycophorin A lacking the cytoplasmic domain. These results imply that the site of the immobilization/rigidification lies within the membrane skeletal structure, not in exofacial receptor crosslinking, and requires the extended cytoplasmic domain of normal glycophorin A. In addition, we found that glycophorin A immobilization and membrane skeletal rigidification were accompanied by immobilization of band 3 receptors. This unexpected result indicates a cooperative coupling between liganded glycophorin A, band 3, and the membrane skeleton. We speculate that cooperation of this type may represent a general mechanism for cytoskeletal linkage and transformation initiated by receptors with short cytoplasmic sequences, such as integrins.     

Separation of the lipid bilayer from the membrane skeleton during discocyte-echinocyte transformation of human erythrocyte ghosts

The membrane skeleton, a protein lattice at the internal side of the red cell membrane, is principally composed of spectrin, actin and proteins 4.1 and 4.9. We have examined negatively stained red cell ghosts and demonstrated, on an ultrastructural level, a separation of the lipid bilayer from the membrane skeleton during echinocytic transformation. The electron micrographs of discoidal red cell ghosts suspended in hypotonic buffer revealed a filamentous reticulum that uniformly laminated the entire submembrane region. transformation of the discoidal ghosts into echinocytic form, as induced by incubation in isotonic buffer, resulted in a disruption of skeletal continuity underlying the surface contour of the membrane spicule. The submembrane reticulum extended into the base and the neck of the spiny processes of the crenated ghosts but was absent at the tip of these projections. In addition, membrane vesicles without a submembrane reticulum were detected either attached to the tips of the spicules or released into the supernatant from the echinocytic ghosts. Protein analysis revealed that the released vesicles were enriched in bands 3, 4.1 and 7 and contained very little of the membrane skeletal proteins, spectrin and actin. The data indicate that during echinocyte formation, parts of the lipid bilayer physically separate from the membrane skeleton, leading to a formation of skeleton-poor lipid vesicles.     

The expression and synthesis of the band 3 protein initiates the formation of a stable membrane skeleton in murine Rauscher-transformed erythroid cells.

While the temporal sequences of the synthesis and assembly of membrane skeletal proteins has been studied during erythroid maturation, relatively little is known about the events which initiate the assembly of membrane skeleton at the early stages of mammalian erythroid commitment. To investigate the early events that initiate the assembly of the membrane skeleton in mammalian erythroid cells, we have studied the synthesis and assembly of membrane skeletal proteins in murine Rauscher erythroleukemia virus-transformed cells. These cells are blocked in differentiation at around the early progenitor (burst forming unit-erythroid, BFUe) cell stage but can be induced to differentiate in vitro. Pulse-labeling studies reveal that Rauscher cells actively synthesize alpha spectrin, beta spectrin, ankyrin and band 4.1 proteins. However, the synthesis of the band 3 protein and its mRNA are barely detectable in these cells. The peripheral membrane skeletal components assemble only transiently in the membrane skeleton and turn over rapidly, resulting in about 20-fold lower steady state levels than are found in mature erythrocytes. Upon induction with erythropoietin and dimethyl sulfoxide, the mRNA level and synthesis of band 3 are increased about 50-fold. In contrast, the synthesis of spectrin, ankyrin and band 4.1 is increased only about 1.5 to 2.0-fold. However, after induction, the fraction of these proteins assembled on the membrane is increased, their half-lives on the membrane are nearly doubled with a concomitant 4 to 5-fold increase in their steady-state levels. These results suggest that the synthesis of peripheral membrane proteins is detected at the earliest stages of erythroid commitment and increases only slightly during further differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyadenylation of nascent RNA during the embryogenesis of Ilyanassa obsoleta.

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated $\text{32}\text{64}$ cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Actin—membrane interactions: Association of G-actin with the red cell membrane

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.     

Relationship between the concanavalin A-agglutinability and deformability of human erythrocytes

Cellular deformability has been proposed in the past as a major determinant of lectin-mediated agglutination of cells. In this paper we have evaluated the correlation between deformability and Con A-agglutinability of human erythrocytes by subjecting them to agents that alter either one of the properties and evaluating the effect on the other property. The following results have been obtained: (i) Treatment with pronase or trypsin, which makes the Con A-nonagglutinable normal red cells highly agglutinable, has practically no effect on deformability; while neuraminidase treatment, with a similar effect on agglutinability, produces a small but statistically significant reduction in deformability. (ii) Diamide treatment, on the other hand, produces a drastic reduction in the deformability of pronase-treated erythrocytes but has no effect on the Con A-agglutinability of the cells. Dinitrophenol also reduces deformability but without altering the agglutinability, (iii) Chlorpromazine, at 2 x 10(-5) M, does not have any effect on the deformability of trypsinized cells, but increases the agglutinability substantially. When the Con A-agglutinability of the cells and their deformability after these treatments are compared, a correlation coefficient r = -0.353 (P greater than 0.1) is obtained. This indicates the lack of any direct correlation between the two parameters, and rules out any significant role of deformability in the determination of Con A-agglutinability of erythrocytes. The agglutination with the lectin is completely reversed by methyl alpha-D-mannoside, the specific inhibitory sugar for Con A, also ruling out any secondary role for deformability in the non-lectin-mediated stabilization of clumps. Upon incubation of normal erythrocytes with Con A. a dose-dependent decrease in deformability is observed, with the deformability index falling to almost 25% of the normal value with 500 microgram/ml Con A. This indicates that Con A binding to its receptor produces changes in the membrane probably by altering properties of the membrane skeleton.     

Spectrin involvement in a 40 degrees C structural transition of the red blood cell membrane

Proteins involved in a structural transition detected in red blood cell membranes at 40 degrees C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40 degrees C transition detected in ghosts by both a stearic acid spin label (16-doxyl stearic) and a sulfhydryl-specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25-30 degrees C and a massive transition with an onset temperature of 48 and 40 degrees C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40 degrees C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti-4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1:1 molar ratio. The involvement in this transition of a localized spectrin domain(s) containing few exposed sulfhydryl groups is proposed.     

Glycophorin A interferes in the agglutination of human erythrocytes by concanavalin A. Explanation of the requirement for enzymic predigestion.

Human erythrocytes become agglutinable with concanavalin A (Con A) after treatment with various proteinases or neuraminidase. The extent of agglutinability achieved with different enzymes is, however, different: Pronase, papain, trypsin, neuraminidase and chymotrypsin enhance the agglutinability in decreasing order, the last being barely effective. The actions of the enzymes on band 3, the Con A receptor, do not correlate with their abilities to increase the agglutinability: Pronase, papain and chymotrypsin cleave the protein, but not trypsin or neuraminidase. No significant differences are found in the number of Con A-binding sites or the affinities for the lectin between the normal and trypsin- or Pronase-treated cells. Thus the receptor does not seem to play a role in determining the Con A-agglutinability of erythrocytes. On the other hand, the cleavage of glycophorins, especially glycophorin A, and the release of sialic acid (in the peptide-bound form) are well-correlated with the enhancement in agglutination after the action of proteinases. The release of sialic acid by graded neuraminidase digestion and the increase in Con A-agglutinability show a correlation coefficient of 0.88. The major inhibitory role of glycophorin A in the process is indicated by the agglutination of En(a) heterozygous erythrocytes; the cells, known to bear about 50% glycophorin A molecules in their membrane, are agglutinated approximately half as well without proteolysis as are the trypsin-treated cells. Possible mechanisms by which glycophorin A could affect Con A-mediated agglutination are discussed.     

Lectin-mediated agglutination of erythrocyte ghost membranes following depletion of membrane protein and intramembrane particles

Profound digestion of unsealed human erythrocyte ghosts with high concentrations of Pronase results in a near complete loss of intramembrane particles while trypsin digestion is less effective. The small vesicles formed by proteolysis are agglutinable by soybean agglutinin (SBA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA), but not concanavalin A (ConA). Densitometer tracings of Pronase-treated vesicles analyzed on SDS-polyacrylamide gels demonstrated no detectable protein or glycoprotein migrating slower than the marking dye. The vesicles showed a loss of 90% Lowry positive material (the remainder may be non-protein chromogens), near depletion of sialyl residues, no significant change in lipid composition, and equal amounts of phospholipid phosphorus compared to an equal volume of ghosts. The lipid material extracted from Pronase-derived vesicles or intact ghosts inhibited hemagglutination with SBA and WGA but not ConA. SBA but not ConA was found to specifically bind to Pronase-derived vesicles while both lectins bound to native ghosts. These observations suggest that neither the integrity of the intramembrane particles nor the presence of membrane glycoprotein appears essential for SBA-, WGA-, and PHA-mediated agglutination. Furthermore, it appears that native membrane glycolipids (and perhaps glycopeptides) can bind SBA, WGA and PHA. The membrane glycolipids may play a larger role than heretofore realized in lectin-mediated agglutination of cells.     

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin- reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.     

Agglunation of a transformed mouse cell line and a variant subline with concanavalin A: Effect of temperature and time of lectin incubation

Colchicine treatment enhanced Con A-mediated agglutination of erythrocytes to LM cells (LM is a “spontaneously” transformed mouse line) incubated for brief periods with Con A at 22° C. Longer incubations with Con A at 22° C rendered colchicine treated cells less agglutinable than untreated cells. Even short incubation times with Con A at higher temperature (37° C) rendered colchicine treated LM cells less agglutinable than their untreated counterparts. Below 15° C, colchicine treated cells remained more agglutinable than untreated cells even after long periods of Con A treatment. Cells of a variant clone (Rl) isolated from LM by negative selection with concanavalin A exhibited increased substratum adhesiveness and an absolute serum requirement. LM and variant cells exhibited a differential reponse to colchicine treatment, the variant subline reguiring longer periods of colchicine treatment to elicit changes in morphology and agglutinability.     

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.