Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin-actin association.

Abstract

The integral membrane protein Band 3 of the human erythrocyte, either purified or in a crude Triton X-100 extract of ghosts, was combined with egg lecithin in a cholate solution. During dialysis to remove cholate, lipid bilayer vesicles formed in which Band 3 existed as a dimer and in which intramembrane particles indistinguishable from those in the native membrane were exposed by freeze-fracturing. The recombinant vesicles were stable in both high and low salt concentrations, sedimented at a density that increased in proportion to their protein content, and bound spectrin-actin extracted from erythrocyte ghosts. When spectrin-actin was associated with the vesicles, the behavior of the recombinant intramembrane particles simulated that of the erythrocyte ghost intramembrane particles: they were dispersed at pH 7.6 and aggregated at pH 5-5.5. Thus, some of the characteristics of the native membrane have been reconstituted in the recombinant.Band 3* is one of the major transmembrane polypeptides of the human erythrocyte (2, 3). It may be involved in facilitated anion movements (4, 5) and glucose transport (6-8), and its interactions in and at the surface of the erythrocyte plasma membrane may be the basis of important structural and rheological properties. Its binding characteristics may affect the distribution of other membrane proteins (9, 10); its transmembrane disposition suggests that it may contribute to the appearance of intramembrane particles (2, 11, 12); and its possible interaction with spectrin and erythrocyte actin may influence cell shape, elastic properties, and surface topology (13-15). Now that methods for purifying the Band 3 polypeptides in an apparently undenatured form are available (3, 16), direct verification and analysis of these many functional attributes should be possible.Previous work in this laboratory has implied an interaction between the integral transmembrane protein(s) responsible for the intramembrane particles of the human erythrocyte and the peripheral proteins spectrin and actin. Reasoning that Band 3 polypeptides may form the intramembrane particles, we have inserted purified or partially purified Band 3 into the lipid bilayers of egg yolk lecithin liposomes and evaluated integralperipheral protein interaction by observing particle distribution patterns in the protein-lipid recombinants. Our results demonstrate that Band 3 may form many of the intramembrane particles of the native membrane, show that the Band 3-lipid recombinants can bind spectrin and erythrocyte actin, and suggest that some of the controls that regulate intramembrane particle distribution in the erythrocyte membrane can be reconstituted by binding spectrin-actin to the recombinant. Integral membrane proteins were solubilized and partially purified by incubating a sample of packed ghosts in an equal volume of 1.5% Triton X-100 in 5 mM sodium phosphate buffer, pH 8.0, as previously described (19). The concentration of Triton X-100 in the resultant protein solution was then lowered to its critical micel...

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

“…In recombinant vesicles made from egg phosphatidylcholine, the lateral distribution of the particles was not affected by high ionic strength, low pH or the addition of calcium. This confirms the observations of Yu and Branton [10] and shows that aggregation of the particles under these conditions cannot be attributed to effects on the proteins which form the particles.…”

“…Vesicles showing 80-A particles on the fracture faces were reconstituted by a method described before [12], and large fracture faces were produced by freezing-thawing of the vesicles [12]. In accordance with observations of Yu and Branton [10], recombinant vesicles made from egg phosphatidylcholine did not show changes in the lateral distribution of the particles after incubation in 150 mM NaC1, 10 mM CaC12 or a pH 5.5 buffer (not shown). However, with a mixture of phospholipids resembling the inner monolayer of the erythrocyte membrane [5], the distribution of the particles was found to be affected by the incubation conditions.…”

“…It may be speculated that removal of the extrinsic proteins affects the association of cholesterol with the components in the inner monolayer of the membrane in such a manner that allows the phosphatidylethanolamine to undergo a liquid-crystalline to gel transition. At low temperatures this would cause the exclusion of particles from regions rich in phosphatidylethanolamine * Recombinant vesicles, prepared from a crude Triton X-100 extract from ghosts, show particles on the fracture faces identical to those observed in the erythrocyte membrane [10,12]. Band 3 has been identified as a constituent of these particles by reconstitutions with purified band 3 fractions [10--12].…”

“…Various treatments were shown to cause aggregation of the intramembrane particles, but only after partial removal of spectrin [17]. A similar aggregation of intramembrane particles could be induced in a reconstituted system by including a spectrin-containing protein fraction [10]. It was concluded that precipitation of spectrin was the actual cause of the observed particle aggregation [10,18,19].…”

“…A similar aggregation of intramembrane particles could be induced in a reconstituted system by including a spectrin-containing protein fraction [10]. It was concluded that precipitation of spectrin was the actual cause of the observed particle aggregation [10,18,19]. About the nature of the interaction between spectrin and the intrinsic proteins little is known.…”