Anchorage of a band 3 population at the erythrocyte cytoplasmic membrane surface: protein rotational diffusion measurements.

Abstract

Direct physical evidence for the linkage of a band 3 population to the cytoskeleton in the erythrocyte ghost membrane is presented. The rotational diffusion of band 3 proteins was measured by observing flash-induced transient dichroism of a covalently bound eosin probe. After proteolytic release of a 40,000-dalton cytoplasmic segment of band 3 by trypsin, a considerable enhancement in the decay of the absorption anisotropy was observed. Analysis of the data indicates that proteolytic cleavage of band 3 produces a mobile band 3 population which has restricted mobility in the unperturbed membrane due to protein-protein interactions involving the cytoplasmic band 3 moiety. Band 2.1 (ankyrin) or 4.1 or both are likefy to be involved in this interaction because a similar effect on band 3 mobility is observed after low-salt/high-salt extraction of these components. Quantitatively, it is estimated that up to 40% of band3 may be linked to the cytoskeleton. Because the ankyrin-band 3 dimer stoichiometry in the membrane is ap proximately 1:5, only about 20% of band 3 dimers can be directly linked to an rin. The remainder could be explained by the existence of higher oligomers of band 3 linked to single ankyrin polypeptides or by linkages involving other components such as band4.1 or 4.2. Elucidation of the interaction between integral and peripheral membrane proteins is important for the understanding of membrane structure and function. The human erythrocyte membrane has often been used as a model system to investigate both the assembly of cytoplasmic structural proteins at the inner membrane surface (the cytoskeleton) and the possible associations of membrane-spanning proteins with this sub-membraneous network (for a recent review, see ref. 1).On the basis of redistribution of membrane components seen in electron micrographs, a direct linkage between integral and peripheral proteins in human erythrocyte membranes was originally suggested (2-4). On the other hand, the rotational diffusion of band 3, the major membrane-spanning protein of the erythrocyte membrane, was found to be essentially independent of the presence or absence of most of spectrin-actin, the quantitatively dominant components of the cytoskeleton (5, 6). Although not ruling out a direct linkage between band 3 and spectrin-actin, this latter finding pointed toward an alternative model in which the bulk of the integral proteins is considered to be trapped within the interstices of a spectrinactin network (5, 7).With the identification of band 2.1 (ankyrin) as the membrane binding site for spectrin (8-10), important progress has been achieved in understanding the interaction between the cytoskeleton and the membrane. Moreover, it was recently shown that ankyrin binds to band 3 in detergent (11), thus immediately raising the question of whether such an interaction also takes place in the intact membrane.Rotational diffusion of band 3 proteins has been measured by using triplet probes (5, 12, 13). In combination with biochemical manipulations of the m...

Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

“…Likewise, the mobile fraction of glycine receptors ranges from ϳ50% in the neuronal cell body to ϳ70% in the processes (54). In addition, the viscosity of the membrane microdomain (55), tethering of proteins with scaffolds/binding partners, and interaction with the cytoskeleton can all contribute to the size of the immobile fraction (56).…”

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

“…The forces could arise from several kinds of interactions within the plasma membrane or between the membrane and the cytoskeleton: (a) The channels might interact directly with the specialized cytoskeleton present in this region of the cell (21). An example of this kind of interaction occurs in erythrocytes in which the diffusion of the band 3 protein is retarded due to interactions with spectrin via the linking protein, ankyrin (38). (b) The channel might interact with other membrane proteins which in turn interact with the cytoskeleton.…”

A molecular defect in two families with hemolytic poikilocytic anemia: reduction of high affinity membrane binding sites for ankyrin.

“…Also, two-dimensional peptide maps of ankyrin-bound band 3 and unbound band 3 appear virtually identical (unpublished data). Some evidence suggests that band 3 exists on the membrane as a tetramer (41,42), and it has been suggested that ankyrin may bind only to a single member of each band 3 tetramer (40). If the binding sites of our patients' cells are unfavorably clustered on the membrane, the sites available for ankyrin binding would appear diminished due to steric effects while the number of potential sites would not be reduced when cleaved from the membrane and measured in solution as in Fig.…”