Biochemical characterization of complex formation by human erythrocyte spectrin, protein 4.1, and actin

Ternary complex formation between the major human erythrocyte membrane skeletal proteins spectrin, protein 4.1, and actin was quantified by measuring cosedimentation of spectrin and band 4.1 with F-actin. Complex formation was dependent upon the concentration of spectrin and band 4.1, each of which promoted the binding of the other to F-actin. Simultaneous measurement of the concentrations of spectrin and band 4.1 in the sedimentable complex showed that a single molecule of band 4.1 was sufficient to promote the binding of a spectrin dimer to F-actin. However, the molar ratio of band 4.1/spectrin in the complex was not fixed, ranging from approximately 0.6 to 2.2 as the relative concentration of added spectrin to band 4.1 was decreased. A mole ratio of 0.6 band 4.1/spectrin suggests that a single molecule of band 4.1 can promote the binding of more than one spectrin dimer to an actin filament. Saturation binding studies showed that in the presence of band 4.1 every actin monomer in a filament could bind at least one molecule of spectrin, yielding ternary complexes with spectrin/actin mole ratios as high as 1.4. Electron microscopy of such complexes showed them to consist of actin filaments heavily decorated with spectrin dimers. Ternary complex formation was not affected by alteration in Mg2+ or Ca2+ concentration but was markedly inhibited by KCl above 100 mM and nearly abolished by 10 mM 2,3-diphosphoglycerate or 10 mM adenosine 5'-triphosphate. Our data are used to refine the molecular model of the red cell membrane skeleton.     

Interactions among red cell membrane proteins

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.     

The role of band 4.1 in the association of actin with erythrocyte membranes

Spectrin stimulates the association of F-actin with erythrocyte inside-out vesicles. Although inside-out vesicles are nearly devoid of two of the three major cytoskeletal proteins, spectrin and actin, they retain nearly all of the cytoskeletal protein designated band 4.1. Inside-out vesicles which have been substantially depleted of band 4.1 by extraction in 1 M KCl, 0.4 M urea and then reconstituted with spectrin show a markedly diminished ability to bind actin by comparison with vesicles containing normal amounts of band 4.1. This diminution is not due to an impaired ability of the vesicles to bind spectrin. Addition of purified band 4.1 to vesicles either before or after they have been reconstituted with spectrin restores their actin binding capacity to near normal levels as does addition of a spectrin-band 4.1 complex prepared by sucrose gradient centrifugation. Band 4.1 bound to vesicles in the absence of added spectrin has no effect on actin binding. Our results suggest that a spectrin band 4.1 complex is responsible for binding actin to erythrocyte membranes.     

Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin.Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.     

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.     

Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins

Protein 4.1R is the prototypical member of a protein family that includes 4.1G, 4.1B, and 4.1N. 4.1R plays a crucial role in maintaining membrane mechanical integrity by binding cooperatively to spectrin and actin through its spectrin-actin-binding (SAB) domain. While the binary interaction between 4.1R and spectrin has been well characterized, the actin binding site in 4.1R remains unidentified. Moreover, little is known about the interaction of 4.1R homologues with spectrin and actin. In the present study, we showed that the 8 aa motif (LKKNFMES) within the 10 kDa spectrin-actin-binding domain of 4.1R plays a critical role in binding of 4.1R to actin. Recombinant 4.1R SAB domain peptides with mutations in this motif showed a marked decrease in their ability to form ternary complexes with spectrin and actin. Binary protein-protein interaction studies revealed that this decrease resulted from the inability of mutant SAB peptides to bind to actin filaments while affinity for spectrin was unchanged. We also documented that the 14 C-terminal residues of the 21 amino acid cassette encoded by exon 16 in conjunction with residues 27-43 encoded by exon 17 constituted a fully functional minimal spectrin-binding motif. Finally, we showed that 4.1N SAB domain was unable to form a ternary complex with spectrin and actin, while 4.1G and 4.1B SAB domains were able to form such a complex but less efficiently than 4.1R SAB. This was due to a decrease in the ability of 4.1G and 4.1B SAB domain to interact with actin but not with spectrin. These data enabled us to propose a model for the 4.1R-spectrin-actin ternary complex which may serve as a general paradigm for regulation of spectrin-based cytoskeleton interaction in various cell types.     

The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

Modulation of actin polymerization by the spectrin-band 4.1 complex

The effect of human erythrocyte spectrin dimer and band 4.1 on the polymerization of actin was studied by two independent methods: by following the increase in fluorescence of actin covalently conjugated to N-pyrenyl-iodoacetamide (pyrenylactin) and by following the increase in light scattered by actin polymers. Both techniques indicated that the complex of spectrin dimer and band 4.1, but neither spectrin nor band 4.1 alone, stimulates the rate of nucleation (decreases the lag phase of polymerization) and stabilizes oligomers of F-actin. While the band 4.1-spectrin complex, but not spectrin alone, had no immediate effect on the rate of polymerization after the lag phase, it eventually decreases the rate of actin filament growth when the molecular ratio of actin-spectrin-band 4.1 approaches the physiological range. The complex has no detectable effect on the critical actin concentration and does not significantly alter the apparent order of the nucleation reaction.     

Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.     

Structural unit of the erythrocyte cytoskeleton. Isolation and electron microscopic examination

We isolated a protein complex containing major cytoskeletal components from the Triton shell of bovine erythrocytes. This protein complex, which we called the 26-S complex, consisted of three major components, spectrin, band-4.1 protein and actin, and one minor component, band-4.9 protein. The molar ratio of spectrin heterodimer:band 4.1:actin was determined by sodium dodecyl sulfate (SDS) gel electrophoresis to be about 1:2:2, approximately the same as that for the Triton shell. By electron microscopic examinations of rotary-shadowed specimens, it was revealed that the 26-S complex had a "spider-like" morphology with a central core and several spectrin heterodimers radiating from it. The number of spectrin arms in the complex was not constant but was in the range between 3 and 6. The complexes with five spectrin heterodimers were the most numerous. The results showed that the 26-S complex contained on the average five spectrin heterodimers, ten band-4.1 polypeptides and ten actin monomers. As judged from the formation of oligomeric 26-S complexes through spectrin arms, the central core of the complex presumably contains band 4.1 and actin. Supporting this conclusion, the central core acted as a nucleus for actin polymerization when the 26-S complex was mixed with G-actin under an actin-polymerizing condition. The 26-S complex could form large aggregates under a certain condition that spectrin was promoted to associate from dimer to tetramer. We conclude that the 26-S complex is the structural unit of the erythrocyte cytoskeleton.     

Modulation of red cell band 4.1 function by cAMP-dependent kinase and protein kinase C phosphorylation

Human erythrocyte protein 4.1 is phosphorylated in vivo by several protein kinases including protein kinase C and cAMP-dependent kinase. We have used cAMP-dependent kinase purified from red cells and protein kinase C purified from brain to test the effects of phosphorylation on band 4.1 function. In solution, each kinase catalyzed the incorporation of 1-4 mol of PO4/mol of band 4.1. Phosphorylation of band 4.1 by each kinase resulted in a significant (50-80%) reduction in the ability of band 4.1 to promote spectrin binding to F-actin. Direct measurement of spectrin-band 4.1 binding showed that phosphorylation by each kinase also caused dramatic reduction in this association. Phosphorylation of band 4.1 by each kinase for increasing time periods enabled us to demonstrate an approximately linear inverse relationship between PO4 incorporation into band 4.1 and spectrin binding. These results show that phosphorylation of band 4.1 by cAMP-dependent kinase and protein kinase C may be central to the regulation of red cell cytoskeletal organization and membrane mechanical properties.     

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.     

Hereditary spherocytosis of man. Defective cytoskeletal interactions in the erythrocyte membrane.

Hereditary spherocytosis (HS) is an inherited abnormality of red cell shape and results from defective interactions amongst the components of the cytoskeleton. It is known that spectrin/actin dissociates in low ionic strength media from ghosts and cytoskeletons at a rate which is slower for HS than normal preparations. Hybridization experiments have established that this behaviour is not due to a defective spectrin or actin but resides in a spectrin-binding component of the membrane [Hill, Sawyer, Howlett & Wiley (1981) Biochem. J. 201, 259-266]. In the present study erythrocyte shells have been examined in low ionic strength media and a similar difference in the rate of solubilization has been revealed. Since band 4.1 (but not band 2.1) is a common component of cytoskeletons and shells it is possible that 4.1 may be abnormal in the HS condition. The interaction of band 4.1 with spectrin/actin was examined by low shear falling ball viscometry. The addition of a mixture of band 2.1 and 4.1 to a solution of actin and spectrin tetramer increased the viscosity due to cross-linking of the cytoskeletal elements by band 4.1. When band 2.1/4.1 mixtures were derived from five HS families the viscosity was increased to a greater extent than in the normal controls. This difference was not a result of alterations in the calcium dependence of the spectrin/actin-band 4.1 interaction. The results imply that band 4.1 may be defective in the HS condition.     

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.     

Ubiquitination of spectrin regulates the erythrocyte spectrin-protein-4.1-actin ternary complex dissociation: implications for the sickle cell membrane skeleton.

It has been demonstrated by our laboratory that the irreversibly sickled cell (ISC) spectrin-4.1-actin complex dissociates slowly as compared to ternary complexes formed out of control (AA) and reversibly sickle cell (RSCs) core skeletons. These studies indicated that the molecular basis for the inability of irreversibly sickled cells (ISCs) to change shape is a skeleton that disassembles, and therefore reassembles, very slowly. The present study is based on the following observations: a) alpha-spectrin repeats 20 and 21 contain ubiquitination sites, and b) The spectrin repeats beta-1 and beta-2 are in direct contact with spectrin repeats alpha-20 and alpha-21 during spectrin heterodimer formation, and contain the protein 4.1 binding domain. We demonstrate here that alpha-spectrin ubiquitination at repeats 20 and 21 increases the dissociation of the spectrin-protein-4.1-actin ternary complex thereby regulating protein 4.1's ability to stimulate the spectrin-actin interaction. Performing in vitro ternary complex dissociation assays with AA control and sickle cell SS spectrin (isolated from high-density sickle cells), we further demonstrate that reduced ubiquitination of alpha-spectrin is, in part, responsible for the locked membrane skeleton in sickle cell disease.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.     

Influence of calmodulin on the human red cell membrane skeleton

The calcium receptor calmodulin interacts with components of the human red cell membrane skeleton as well as with the membrane. Under physiological salt conditions, calmodulin has a calcium-dependent affinity for spectrin, one of the major components of the membrane skeleton. It is apparent from our results that calmodulin inhibits the ability of erythrocyte spectrin (when preincubated with filamentous actin) to create nucleation centers and thereby to seed actin polymerization. The gelation of filamentous actin induced by spectrin tetramers is also inhibited by calmodulin. The inhibition is calcium dependent and decreases with increasing pH, similar to the binding of calmodulin to spectrin. Direct binding studies using aqueous two-phase partition indicate that calmodulin interferes with the binding of actin to spectrin. Even in the presence of protein 4.1, which is believed to stabilize the ternary complex, calmodulin has an inhibitory effect. Since calmodulin also inhibits the corresponding activities of brain spectrin (fodrin), it appears likely that calmodulin may modulate the organization of cytoskeletons containing actin and spectrin or spectrin analogues.     

Radiolabel-transfer cross-linking demonstrates that protein 4.1 binds to the N-terminal region of beta spectrin and to actin in binary interactions

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)- succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of beta spectrin within a sequence homologous to the actin-binding region of alpha actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.     

Spectrin-actin associations studied by electron microscopy of shadowed preparations

By shadowing specimens dried onto mica sheets we have obtained clear images of actin crosslinked by spectrin, an actin-binding protein found in erythrocytes. We conclude that spectrin dimers possess a single binding site for F actin. Tetramers formed by head-to-head association of two dimers possess two actin binding sites, one at each tail. Polymerizing G actin in the presence of spectrin tetramers or mixing preformed F actin with spectrin tetramer plus band 4.1 results in an extensively crosslinked network of actin filaments. When G actin is polymerized in the presence of spectrin at spectrin:actin mole ratios close to that present on the erythrocyte membrane, large amorphous protein networks are formed. These networks are clusters of spectrin around 25 nm diameter structures which may be actin protofilaments. These networks are similar to the cytoskeletal network seen after erythrocyte membranes are extracted with detergent, and may represent the first in vitro assembly of a cytoskeletal complex resembling that of the native cell both biochemically and structurally.