ALBERT

Description: 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–β-spectrin and glycophorin C–protein 4.1–β-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–adducin–spectrin 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.

Description: Glycolytic enzymes have been recently shown to exist as multi-enzyme complexes in association with the cytoplasmic domain of band 3 at the inner surface of the human erythrocyte membrane. Because several of the glycolytic enzyme binding sites have been mapped to sequences near the NH2-terminus of band 3 (DDYED and EEYED) that are not conserved in mice (EEVLE and EELEN), the question naturally arose whether the existence of glycolytic enzyme complexes on erythrocyte membranes might be only a product of recent evolution. To test this hypothesis, fresh murine erythrocytes were fixed and stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and carbonic anhydrase II (CA II was used as a control, since it binds to a distant site near the COOH-terminus of band 3). Importantly, analysis of intact murine erythrocytes by confocal microscopy demonstrated that all of the above enzymes are localized to the membrane in oxygenated cells. In contrast, upon deoxygenation of the intact cells, release of the glycolytic enzymes (but not CA II) from the erythrocyte membrane and their uniform redistribution throughout the cytoplasm is observed. Because deoxyhemoglobin has been shown in human erythrocytes to compete with glycolytic enzymes (but not with CA II) for a common binding site at the NH2-terminus of band 3, these data argue that murine band 3, despite its weak homology to human band 3, still constitutes an organization center for glycolytic enzymes on the erythrocyte membrane. To further test this hypothesis, erythrocytes from band 3 knockout mice were similarly examined by confocal microscopy. Not surprisingly, all of the enzymes in all of the cells were evenly distributed throughout the cytoplasm, regardless of the oxygenation state of the cell. Further, immunoblot analyses demonstrated that glycolytic enzyme content of the band 3 knockout erythrocytes was measurably reduced compared to healthy mice, suggesting that the anion transporter may also contribute to enzyme stabilization during the lifetime of the erythrocyte. Finally, to determine whether the integrity of other membrane structures might impact the assembly of glycolytic enzyme complexes on the erythrocyte membrane, α-spectrin deficient mice were also examined for their enzyme distributions. Curiously, 〉 50% of the cells in any field exhibited glycolytic enzyme staining throughout the cytoplasm, with the remainder showing mainly membrane staining. Conceivably, the stabiity of glycolytic enzyme complexes on the membrane may also depend on the integrity of the membrane skeleton. Taken together, these data argue that glycolytic enzymes assemble in an oxygenation-dependent manner into complexes on murine erythrocyte membranes and that the stability of these complexes depends on the presence of band 3 and to a lesser extent α-spectrin. Supported by NIH grant GM24417.

Description: The classical model of the human erythrocyte membrane (RBCM) shows two bridges connecting the lipid bilayer to the membrane skeleton: 1) a bridge attaching the cytoplasmic domain of band 3 (CDB3) to ankyrin, which in turn binds β-spectrin, and 2) a bridge linking the cytoplasmic domain of glycophorin C to protein 4.1, which in turn binds the spectrin-actin junctional complex. Recent data, however, suggest while disruption of the band 3-ankyrin- β-spectrin interaction profoundly alters membrane stability, disruption of the glycophorin C-protein 4.1R linkage has no effect on membrane mechanical properties. In a search for additional bridges between the junctional complex and the bilayer, we discovered that adducin, a component of the spectrin-actin junctional complex, binds to band 3. Evidence for this interaction derives from a number of different experimental strategies. We have been able to show that: i) photoactivation of sulfo-SBED-labeled adducin reconstituted onto the RBCM leads to label transfer to band 3, ii) adducin binds to KI-stripped IOVs with a Kd of 280 nM and this binding is inhibited by an antibody to CDB3 (as well as by unlabeled adducin), iii) IOVs derived from normal erythrocytes retain adducin, whereas similar IOVs prepared from erythrocytes deficient in band 3 retain very little adducin, iv) the tail domain (but not the headpiece domain) of β-adducin binds to KI-stripped IOVs and this binding is competed by both anti-CDB3 and intact adducin, v) GST-labeled β-adducin tail domain can pull down band 3 in co-pelleting studies and this co-precipitation is blocked by anti-CDB3, and vi) the tail domain of β-adducin directly binds CDB3. Because adducin is an important structural component of the junctional complex, these data suggest that the junctional complex is linked to the RBCM via CDB3 and that part of the band 3 population must be located adjacent to the junctional complex. This putative new bridge between the RBCM and the spectrin-actin skeleton may also help explain why β-adducin knockout mice have unstable erythrocyte membranes.