Tropomyosin modulates erythrocyte membrane stability

Abstract

The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a beta-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex.

Figure 1

Effect of magnesium depletion on membrane stability and on membrane protein retention. (A) Membrane stability, expressed as the rate of decline in ektacytometric deformability index (DI), increases (decay curve shifted toward right) with increasing concentrations of MgCl₂ maintained during preparation of the ghosts. (B) Retention of TM by ghosts examined in panel A. (C) Components of junction complexes of ghosts prepared in the absence or in the presence of 2 mM MgCl₂. The panels show immunoblots after SDS-PAGE of total protein from ghosts, prepared with and without MgCl₂ as indicated, probed with antibodies against 4.1R, adducin, protein 4.9, capZ, tropomodulin, and TM.

Figure 2

Reversibility of the effects of magnesium depletion. (A) Membrane fragmentation profiles of (1) ghosts prepared in the presence of 2 mM MgCl₂; (2) ghosts prepared in the absence of MgCl₂; (3) TM-depleted ghosts after addition of purified erythroid TM; and (4) TM-depleted ghosts after addition of a membrane extract containing TM. Note the partial restoration of membrane stability by purified erythrocyte TM and almost complete restoration when the extract was added. (B) Rebinding of erythroid TM to membrane preparations shown in panel A documented by immunoblotting with anti-TM antibody. (C) Membrane stability of ghosts prepared (1) in the presence of MgCl₂; (2) in the absence of MgCl₂; and (3) after addition of purified muscle TM, showing that this fails to increase membrane stability. (D) Failure of rebinding of muscle TM to membrane preparations shown in panel C documented by immunoblotting with anti-TM antibody. (Lane 4) Purified muscle TM used for membrane reconstitution.

Figure 3

Incorporation of polypeptide 1-301 of β-spectrin into membrane skeletal junctions in situ and effect of the incorporation on membrane stability. (A) GST-tagged polypeptide 1-301 was introduced into TM-depleted ghosts. Isolated membrane skeletons from the resealed ghosts were analyzed by electrophoresis in 10% SDS-PAGE. (B) Effect of polypeptide 1-301 on membrane stability of TM-depleted ghosts. Membrane stability, expressed as the rate of decline in DI, diminishes (decay curve shifted toward left) with increasing concentration of peptide. (C) Binding of the peptide to ghost membranes, prepared in the absence (●) and presence (○) of magnesium ions. The curves are calculated best-fits for a single set of independent spectrin-4.1R–binding sites, assuming a site concentration of 1.2 μM. The resulting values of K_a are 3.9× 10⁴ M⁻¹ (± 0.6 × 10⁴M⁻¹) (broken line) and 2.0 × 10⁴ M⁻¹ (± 0.2× 10⁴ M⁻¹) (full line). (D) Effect of incorporated peptide on shear resistance of ghosts prepared in the absence (●) or presence (○) of 2 mM MgCl₂, showing reduced shear resistance in the former. Membrane stability was assessed from DI decay curves. The relative stability is defined as the ratio of time under shear for a 50% drop in signal to that for TM-containing ghosts at zero peptide concentration.