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.     

Integral protein linkage and the bilayer-skeletal separation energy in red blood cells

Stabilization of the lipid bilayer membrane in red blood cells by its association with an underlying membrane-associated cytoskeleton has long been recognized as critical for proper red blood cell function. One of the principal connections between skeleton and bilayer is via linkages between band 3, the integral membrane protein that transports anions across the cell surface, and membrane skeletal elements including ankyrin, adducin, spectrin, and the junctional complex of the skeleton. Here, we use membrane tether formation coupled with fluorescent labeling of membrane components to examine the importance of band 3 in stabilizing the bilayer-skeletal association. In membranes from a patient deficient in band 3, the energy associated with the bilayer skeleton is approximately zero, whereas when band 3 is immobilized by ligation with the monoclonal antibody R10, the energy of association approximately doubles. Fluorescence images of tethers reveal that ∼40% of the band 3 on the normal cell surface can be pulled into the tether, confirming a lateral segregation of membrane components during tether formation. These results validate a critical role for band 3 in stabilizing the bilayer-skeletal association in red cells.     

Identification of Contact Sites between Ankyrin and Band 3 in the Human Erythrocyte Membrane

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.     

Visualization of the hexagonal lattice in the erythrocyte membrane skeleton

The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y-shaped spectrin molecules linking three junctional complexes, three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.     

Analysis of integral membrane protein contributions to the deformability and stability of the human erythrocyte membrane

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.     

Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.     

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.     

The spectrin–ankyrin–4.1–adducin membrane skeleton: adapting eukaryotic cells to the demands of animal life

The cells in animals face unique demands beyond those encountered by their unicellular eukaryotic ancestors. For example, the forces engendered by the movement of animals places stresses on membranes of a different nature than those confronting free-living cells. The integration of cells into tissues, as well as the integration of tissue function into whole animal physiology, requires specialisation of membrane domains and the formation of signalling complexes. With the evolution of mammals, the specialisation of cell types has been taken to an extreme with the advent of the non-nucleated mammalian red blood cell. These and other adaptations to animal life seem to require four proteins—spectrin, ankyrin, 4.1 and adducin—which emerged during eumetazoan evolution. Spectrin, an actin cross-linking protein, was probably the earliest of these, with ankyrin, adducin and 4.1 only appearing as tissues evolved. The interaction of spectrin with ankyrin is probably a prerequisite for the formation of tissues; only with the advent of vertebrates did 4.1 acquires the ability to bind spectrin and actin. The latter activity seems to allow the spectrin complex to regulate the cell surface accumulation of a wide variety of proteins. Functionally, the spectrin–ankyrin–4.1–adducin complex is implicated in the formation of apical and basolateral domains, in aspects of membrane trafficking, in assembly of certain signalling and cell adhesion complexes and in providing stability to otherwise mechanically fragile cell membranes. Defects in this complex are manifest in a variety of hereditary diseases, including deafness, cardiac arrhythmia, spinocerebellar ataxia, as well as hereditary haemolytic anaemias. Some of these proteins also function as tumor suppressors. The spectrin–ankyrin–4.1–adducin complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.     

Rate of rupture and reattachment of the band 3-ankyrin bridge on the human erythrocyte membrane

The principal bridge connecting the erythrocyte membrane to the spectrin-based skeleton is established by band 3 and ankyrin; mutations leading to reduced bridge formation or increased bridge rupture result in morphological and mechanical abnormalities. Because membrane mechanical properties are determined in part by the protein interactions that stabilize the membrane, we have evaluated the rates of rupture and reattachment of band 3-ankyrin bridges under both resting and mechanically stressed conditions. To accomplish this, we have examined the rate of ankyrin displacement from inside-out vesicles by the hexahistidine-tagged cytoplasmic domain of band 3, cdb3-(His)6 and the rate of substitution of cdb3-(His)6 into endogenous band 3-ankyrin bridges in resealed erythrocytes in the presence and absence of shear stress. We demonstrate that 1) exogenous cdb3-(His)6 displaces endogenous ankyrin from IOVs with a half-time and first order rate constant of 42 +/- 14 min and 0.017 +/- 0.0058 min(-1), respectively; 2) exogenous cdb3-(His)6 substitutes endogenous band 3 in its linkage to ankyrin in resealed cells with a half-time and first order rate constant of 12 +/- 3.6 min and 0.060 +/- 0.019 min(-1), respectively; 3) cdb3-(His)6-mediated rupture of the band 3-ankyrin bridge in resealed cells results in decreased membrane mechanical stability, decreased deformability, abnormal morphology, and spontaneous vesiculation of the cells; and 4) the above on/off rates are not significantly accelerated by mechanical shear stress. We conclude that the off rates of the band 3-ankyrin interaction are sufficiently slow to allow sustained erythrocyte deformation without loss of elasticity.     

Fatty acylation of a 55 kDa membrane protein of human erythrocytes.

The major palmitoylated human erythrocyte membrane protein has an M(r) of 55,000. It is distinct from the glucose transporter and is not derived from band 3 or ankyrin. It resists salt extraction suggesting a high affinity for the membrane. Pulse chase experiments demonstrate that palmitoylation is a dynamic process, and it may therefore have regulatory significance in membrane protein-protein or protein-lipid interaction. Slower dynamics of palmitoylation in erythrocytes from patients suffering from chronic myelogenous leukemia, which are less stable than normal erythrocytes, strengthen this view.     

Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G- spectrin, although alpha-spectrin was still present. Beta G-spectrin- deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin- associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E- cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)- ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.     

Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction

Erythrocyte adducin is a membrane skeletal protein that binds to calmodulin, is a major substrate for protein kinase C, and associates preferentially with spectrin-actin complexes. Erythrocyte adducin also promotes association of spectrin with actin, and this activity is inhibited by calmodulin. This study describes the isolation and characterization of a brain peripheral membrane protein closely related to erythrocyte adducin. Brain and erythrocyte adducin have at least 50% antigenic sites in common, each contains a protease-resistant core of Mr = 48,000-48,500, and both proteins are comprised of two partially homologous polypeptides of Mr = 103,000 and 97,000 (erythrocytes) and Mr = 104,000 and 107,000-110,000 (brain). Brain and erythrocyte adducin associate preferentially with spectrin-actin complexes as compared to spectrin or actin alone, and both proteins also promote binding of spectrin to actin. Brain adducin binds calmodulin in a calcium-dependent manner, although the Kd of 1.3 microM is weaker by 5-6-fold than the Kd of erythrocyte adducin for calmodulin. Brain adducin is a substrate for protein kinase C in vitro and can accept up to 2 mol of phosphate/mol of protein. Adducin provides a potential mechanism in cells for mediating site-directed assembly of additional spectrin molecules and possibly other proteins at the spectrin-actin junction. Brain tissue contains 12 pmol of adducin/mg of membrane protein, which is the most of any tissue examined other than erythrocytes, which have 50 pmol/mg. The presence of high amounts of adducin in brain suggests some role for this protein in specialized activities of nerve cells.     

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.     

Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.     

Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37° C, 60 min) or glucose-starved (37° C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14- 15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under nonstarving conditions.     

Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions.

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37 C, 60 min) or glucose-starved (37 C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14-15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under non-starving conditions.     

Proteolysis of ankyrin and of band 3 protein in chemically induced cell fusion. Ca2+ is not mandatory for fusion.

Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.     

Immunochemical characterization of interactions between circulating autologous immunoglobulin G and normal human erythrocyte membrane proteins

Autologous immunoglobulin G present during electrophoresis of human erythrocyte membrane proteins influenced the electrophoretic mobility of some of the proteins. Different types of non-ionic detergents were used for solubilization of the membranes and together with experiments using dimyristoylphosphatidylcholine-derived erythrocyte membrane vesicles this indicated that IgG binds to spectrin, ankyrin, and band 3 protein. The binding was independent on proteolysis and not due to unspecific protein-protein interactions. Immunoblotting experiments also showed binding to polypeptide bands in the spectrin and ankyrin regions and demonstrated the presence of erythrocyte-associated IgG. The reactivity may be due to natural autoantibodies involved in the clearance of cellular debris in vivo. Whether the observations are of relevance for the putative immune-mediated clearance of old erythrocytes from the circulation remains to be established.