The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3-ankyrin-␤-spectrin and glycophorin C-protein 4.1-␤-spectrin. 1-7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C-protein 4.1 interaction has little effect on membrane stability. 8 We demonstrate the existence of a novel band 3-adducin-spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3-adducinspectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane. (Blood. 2009;114: 1904-1912 IntroductionThe model of the erythrocyte membrane presented in cell biology, hematology, and biochemistry textbooks shows 2 major protein bridges that span between the phospholipid bilayer and the spectrin/actin skeleton. [1][2][3][4][5][6][7] The more prominent bridge, a linkage from the integral membrane protein, band 3, to spectrin via ankyrin, is composed of multiple high-affinity protein-protein interactions. [9][10][11] Defects or deficiencies in either band 3 or ankyrin lead to a decrease in cohesion between the lipid bilayer and membrane skeleton, resulting in loss of membrane surface area and a pathology termed hereditary spherocytosis. [12][13][14] Manual rupture of this bridge by addition of competing fragments of either band 3 or ankyrin, or by addition of competing monoclonal antibodies, or mutation of the ankyrin binding site on band 3 induces spontaneous membrane vesiculation and fragmentation. [14][15][16] Spontaneous mutations in the ankyrin-bridging function in other cells can also lead to serious pathologies. [17][18][19][20] Taken together, these data support the importance of the ankyrin-spectrin bridge in maintaining membrane integrity.The second bridge connecting the membrane bilayer to the spectrinactin skeleton consists of the membrane-spanning protein, glycophorin C (GPC), tethered to spectrin via the adapter protein 4.1. [21][22][23] The complex of cytoskeletal proteins at this nexus (primarily actin, dematin, tropomyosin, adducin, protein 4.1, and tropomodulin) forms a junctional complex from which spectrin tetramers extend radially into a 2-dimensional lattice that provides mechanical stability to the overlying membrane. Based on the finding that GPC-deficient red cells exhibit decreased membrane mechanical stability, it has been inferred that the GPC-protein 4.1 bridge is essential to erythrocyte integrity. 24,25 However...

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The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5 untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.Hereditary spherocytosis (HS; OMIM 182900) is a dominant inherited hemolytic anemia that affects approximately 1/2,500 people of all races worldwide (1, 17, 18). Typically, HS patients have mild symptoms, which can be exacerbated by viral infections (19). These symptoms include elevated reticulocyte counts and smaller, spherical erythrocytes on a blood smear and are accompanied by an abnormal osmotic fragility (13,19,23). The majority of HS mutations have been found in the genes encoding the erythrocyte membrane skeleton proteins ankyrin-1 (ANK-1; ϳ60%) and Band 3 (SLC4A1; ϳ20%) (1,19). Virtually all of the described HS mutations cause a functional deficiency of erythrocyte skeleton proteins, either by premature termination and/or amino acid substitutions in regions critical for the protein-protein interactions that stabilize the erythrocyte membrane skeleton (17, 18). In the 10 to 20% of patients in whom no mutations have been detected in the coding region of the membrane skeleton protein genes, the causative mutations are proposed to be in cis-acting regulatory regions resulting in decreased transcription of mRNA resulting in haploinsufficiency (12,17,18).Support for this hypothesis has come from our previous analysis of a German patient with a severe form of HS (20). The patient was shown to have two mutations in the ANK-1 gene. The first was a 20-bp deletion in exon 6, leading to premature termination, presumably inherited from the father. The second mutation was a deletion of a TG dinucleotide in the 5Ј untranslated region of the ANK-1 gene located at position Ϫ72/73 relative to the ATG initiation codon (12,20) or ϩ 12/13 from the transcriptional start site (TSS) ...