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 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.     

Regulation of cell adhesion to collagen via β1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon

Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by β1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate β1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of β1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase C? bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of β1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cε is an essential regulatory step for the trafficking and activation of β1 integrins and cell spreading on collagen.     

Differential turnover of methyl groups on methyl-accepting membrane proteins of irreversibly sickled erythrocytes

Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.     

Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.     

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.     

The 4.1-like proteins of the bovine lens: spectrin-binding proteins closely related in structure to red blood cell protein 4.1

The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.     

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.     

140 000 Dalton surface glycoprotein : A plasma membrane component of the detergent-resistant cytoskeletal preparations of cultured human fibroblasts

A 140 000 D glycoprotein (140 kD gp), labelled radioactively with surface-specific techniques, remained as the major cell surface glycoprotein in the detergent-resistant cytoskeletal preparations of cultured human fibroblasts. The 140 kD gp was present also in trypsinized cells and was not affected by treatment of the cells either with collagenase, chymotrypsin or thrombin. In density gradient fractionation of whole cells the 140 kD gp was recovered in the plasma membrane fraction together with small amounts of cytoskeletal components. In fractionation of cytoskeletal preparations, on the other hand, the 140 kD gp could not be dissociated from cytoskeletal proteins and together with vimentin it formed the major component of the oligomeric polypeptide complex generated by treating the surface-labelled cytoskeletal preparations with bifunctional cross-Linking reagent, dithiobis succinimidyl propionate (DTPS). Moreover, the 140 kD gp seemed to copurify with vimentin upon reconstitution of intermediate filaments from urea-solubilized cytoskeletal preparations. On the other hand, low ionic-induced degradation of vimentin led to a decrease in the amount of the detergent-resistant 140 kD gp on the cell surface. In electron microscopy, a close apposition between bilayer-like plasma membrane remnants of the adherent cytoskeletons and cytoskeletal elements could be seen. The results indicate that the 140 kD gp is a plasma membrane glycoprotein which closely interacts with the detergent-resistant cytoskeleton of cultured human fibroblast. Possible mechanisms of the association are discussed.     

Mechanisms of cytoskeletal regulation: modulation of aortic endothelial cell protein band 4.1 by the extracellular matrix

The bovine aortic endothelial cell (BAEC) cytoskeleton is a complex structure modulated by many stimuli including release from contact inhibition and various components of the extracellular matrix (ECM). Transduction of information from the ECM to the cell nucleus proceeds via several complex pathways including the cytoskeleton. We have demonstrated the presence of an immunoreactive isoform of the human erythrocyte cytoskeletal protein band 4.1 (4.1) in BAEC. BAEC 4.1 is similar in molecular weight to the erythroid protein by immunoblot analyses and produces a similar pattern of cysteine specific cleavage products consistent with a cluster of cysteine residues previously described in the erythroid molecule. We have also examined the effects of defined ECM proteins on the distributions of cultured BAEC 4.1 and actin filaments (AF) at confluency and following release from contact inhibition. The distribution of 4.1 in BAEC on a plasma fibronectin substrate is complex, having partial codistribution with cytoplasmic AF and a unique perinuclear staining. In contrast, on a collagen type I/III substrate, 4.1 is localized, in part, to peripheral areas of cell-cell contact distinct from the dense peripheral band staining of AF. During migration on this substrate, 4.1 had a filamentous distribution having partial codistribution with AF. Indirect immunofluorescence staining of cross-sections of bovine calf aortae revealed a cortical staining pattern in the aortic endothelial cells with staining noted on the luminal and basolateral aspects of the cells. These data suggest that, in endothelial cells, protein 4.1 is a cortical membrane protein which may function to link actin filaments to other skeletal proteins such as spectrin. These findings also suggest an active role for protein 4.1 in cytoskeletal reorganization events which can occur in response to external stimuli, such as the extracellular matrix or contact with other cells.     

Suppression of adenylyl cyclase-mediated cAMP production by plasma membrane associated cytoskeletal protein 4.1G

It has been shown lately that activity of G protein-coupled receptors (GPCRs) is regulated by an array of proteins binding to carboxy (C)-terminus of GPCRs. Proteins of 4.1 family are subsets of subcortical cytoskeletal proteins and are known to stabilize cellular structures and proteins at the plasma membrane. One of the 4.1 family proteins, 4.1G has been shown to interact with the C-terminus of GPCRs and regulate intracellular distribution of the receptors, including parathyroid hormone (PTH)/PTH-related protein receptor (PTHR). PTHR is coupled to trimeric G proteins G_s and G_q, which activate the adenylyl cyclase/cyclic AMP (cAMP) pathway and phospholipase C pathway, respectively. During the course of investigation of the role of 4.1G on adenylyl cyclase/cAMP signaling pathway, we found that 4.1G suppressed forskolin-induced cAMP production in cells. The cAMP accumulation induced by forskolin was decreased in HEK293 cells overexpressing 4.1G or increased in 4.1G-knockdown cells. Furthermore, PTH -(1-34)-stimulated cAMP production was also suppressed in the presence of exogenously expressed 4.1G despite its activity to increase the distribution of PTHR to the cell surface. In cells overexpressing FERM domain-deleted 4.1G, a mutant form of the protein deficient in plasma membrane distribution, neither forskolin-induced nor PTH -(1-34)-stimulated cAMP production was not altered. The suppression of the forskolin-induced cAMP production was observed even in membrane preparations of 4.1G-overexpressing cells. In 4.1G-knockdown HEK293 cells, plasma membrane distribution of adenylyl cyclase 6, one of the major subtypes of the enzyme in the cells, showed a slight decrease, in spite of the increased production of cAMP in those cells when stimulated by forskolin. Also, cytochalasin D treatment did not cause any influence on forskolin-induced cAMP production in HEK293 cells. These data indicate that plasma membrane-associated 4.1G regulates GPCR-mediated G_s signaling by suppressing adenylyl cyclase-mediated cAMP production.     

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.     

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.     

Protein 4.1 in forebrain postsynaptic density preparations: enrichment of 4.1 gene products and detection of 4.1R binding proteins.

4.1 Proteins are a family of multifunctional cytoskeletal components (4.1R, 4.1G, 4.1N and 4.1B) derived from four related genes, each of which is expressed in the nervous system. Using subcellular fractionation, we have investigated the possibility that 4.1 proteins are components of forebrain postsynaptic densities, cellular compartments enriched in spectrin and actin, whose interaction is regulated by 4.1R. Antibodies to each of 4.1R, 4.1G, 4.1N and 4.1B recognize polypeptides in postsynaptic density preparations. Of these, an 80-kDa 4.1R polypeptide is enriched 11-fold in postsynaptic density preparations relative to brain homogenate. Polypeptides of 150 and 125 kDa represent 4.1B; of these, only the 125 kDa species is enriched (threefold). Antibodies to 4.1N recognize polypeptides of approximately 115, 100, 90 and 65 kDa, each enriched in postsynaptic density preparations relative to brain homogenate. Minor 225 and 200 kDa polypeptides are recognized selectively by specific anti-4.1G antibodies; the 200 kDa species is enriched 2.5-fold. These data indicate that specific isoforms of all four 4.1 proteins are components of postsynaptic densities. Blot overlay analyses indicate that, in addition to spectrin and actin, postsynaptic density polypeptides of 140, 115, 72 and 66 kDa are likely to be 4.1R-interactive. Of these, 72 kDa and 66 kDa polypeptides were identified as neurofilament L and alpha-internexin, respectively. A complex containing 80 kDa 4.1R, alpha-internexin and neurofilament L was immunoprecipitated with anti-4.1R antibodies from brain extract. We conclude that 4.1R interacts with the characteristic intermediate filament proteins of postsynaptic densities, and that the 4.1 proteins have the potential to mediate the interactions of diverse components of postsynaptic densities.     

Vimentin in the Central Nervous System

Intermediate filament proteins were identified by two-dimensional gel electrophoresis in urea extracts of rat optic nerves undergoing Wallerian degeneration and in cytoskeletal preparations of rat brain and spinal cord during postnatal development. The glial fibrillary acidic (GFA) protein and vimentin were the major optic nerve proteins following Wallerian degeneration. Vimentin was a major cytoskeletal component of newborn central nervous system (CNS) and then progressively decreased until it became barely identifiable in mature brain and spinal cord. The decrease of vimentin occurred concomitantly with an increase in GFA protein. A protein with the apparent molecular weight of 61,000 and isoelectric point of 5.6 was identified in both cytoskeletal preparations of brain and spinal cord, and in urea extracts of normal optic nerves. The protein disappeared together with the polypeptides forming the neurofilament triplet in degenerated optic nerves.     

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.     

Composition and expression of spectrin-based membrane skeletons in non-erythroid cells

Cellular differentiation is often accompanied by the expression of specialized plasma membrane proteins which accumulate in discrete regions. The biogenesis of these specialized membrane domains involves the assembly and co-localisation of a spectrin-based membrane skeleton. While the constituents of the membrane skeleton in non-erythroid cells are often immunologically related to erythroid spectrin, ankyrin, and protein 4.1, there are structural and functional differences between the isoforms of these membrane skeleton polypeptides, as well as highly variable patterns of expression during cellular differentiation. We consider this heterogeneity of structure and expression during development in the context of the hypothesis that non-erythroid spectrin, ankyrin, and protein 4.1 are involved in the formation of specialized membrane domains.     

Two different mRNAs are transcribed from a single genomic locus encoding the chicken erythrocyte anion transport proteins (band 3).

The chicken erythrocyte anion transport protein (band 3 of the erythrocyte cytoskeleton) is a central component taking part in two widely divergent functions of erythroid cells; it is a primary determinant of cytoskeletal architecture and responsible for electroneutral Cl-/HCO3- exchange across the plasma membrane. To analyze interesting aspects of the developmental regulation of this gene, we have cloned the cDNA and genomic counterparts of the erythroid-specific anion transport protein. We show that a single genetic locus for band 3 encodes two different erythroid cell-specific mRNAs, with different translational initiation sites, which predict polypeptides of sizes very close to those observed in vivo. In vitro translation and immune precipitation of synthetic mRNA derived from one putative fully encoding cDNA clone demonstrate that this clone gives rise to a protein which is identical in size and antigenicity to bona fide chicken erythroid band 3.