Composition of the ternary protein complex of the red cell membrane cytoskeleton

Abstract

The red cell membrane skeletal network is constructed from actin, spectrin and protein 4.1 in a molar ratio of actin subunits/spectrin heterodimer/protein 4.1 of 2:1:1. This represents saturation of the actin filaments, since incubation with extraneous spectrin and protein 4.1 leads to do binding of additional spectrin, either to the inner surface of ghost membranes or to lipid‐free membrane cytoskeletons. Partial extraction of spectrin from the membrane is accompanied by release of actin under all conditions. Regardless of the proportion of spectrin extracted, the molar ratio of spectrin dimer/actin subunits is constant at 1:2. This is not the result of release or cooperative breakdown of whole lattice junctions from the network, for the number of actin filaments, judged by capacity to nucleate polymerisation of added G‐actin, remains unchanged even when as much as 60% of the total spectrin has been lost. A similar 1:2:1 stoichiometry characterises the complex formed when G‐actin is allowed to polymerise in the presence of varying amounts of spectrin and protein 4.1. When this complex is treated with the depolymerising agent, 1 M guanidine hydrochloride, it breaks down into smaller units of the same stoichiometry. After cross‐linking these can be recovered from a gel‐filtration column. Complexes prepared starting from G‐actin appear to be much more stable than those formed when spectrin and protein 4.1 are bound to F‐actin.

Calculation of a Gap Restoration in the Membrane Skeleton of the Red Blood Cell: Possible Role for Myosin II in Local Repair

“…It consists of a hexagonal network of spectrin tetramers interconnected at their ends by short (ϳ600 Å) actin protofilaments made of 13 protomers. These actin protofilaments associated with rodlike tropomyosin (Fowler, 1996) can bind several spectrin tetramers to form a multiprotein network that is anchored to the membrane and strengthened by ankyrin (Bennett and Stenbuck, 1980) and protein-4.1 (Pekrun et al, 1989;Tchernia et al, 1981;Waugh, 1983).…”

Biochemical Analysis of Potential Sites for Protein 4.1-mediated Anchoring of the Spectrin-Actin Skeleton to the Erythrocyte Membrane

“…Polymerization of spectrin with actin into a two-dimensional network is strongly dependent on protein 4.1, an ϳ78-kDa polypeptide that binds avidly to the ␤ subunit of spectrin (8 -13) and thereby forms a calmodulin-dependent binding site for actin (14). The approximate stoichiometry of this ternary complex, as estimated from the composition of the dense gel that rapidly forms when protein 4.1 is added to a solution of spectrin and actin, is 1:2:1 of spectrin:actin:protein 4.1 (15,16). Not surprisingly, defects in the structure or level of expression of protein 4.1 in erythrocytes result in fragile, abnormally shaped cells (17)(18)(19).…”