ABSTRACT
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-beta-spectrin and glycophorin C-protein 4.1-beta-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.
Subject(s)
Calmodulin-Binding Proteins/physiology , Cell Membrane/metabolism , Cytoskeleton/metabolism , Erythrocyte Membrane/metabolism , Actins/metabolism , Biotinylation , Calmodulin-Binding Proteins/metabolism , Cytoplasm/metabolism , Erythrocytes/metabolism , Glutathione Transferase/metabolism , Humans , Lipid Bilayers/metabolism , Models, Biological , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Surface Plasmon ResonanceABSTRACT
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.
