β-Spectrin limits α-spectrin assembly on membranes following synthesis in a chicken erythroid cell lysate

Abstract

Spectrin, the major protein of the subcortical actin network in erythrocytes, contains two non-identical subunits, α and β (refs 1, 2). Spectrin is indirectly associated with the transmembrane anion transporter through the binding of β-spectrin to an extrinsic protein, ankyrin^1–6. In chicken embryo erythroid cells, α-spectrin is synthesized in a threefold excess relative to β-spectrin, although the two subunits are assembled in equimolar amounts⁷. To investigate the regulation of assembly of equimolar amounts of spectrin, an in vitro system from chicken embryo erythroid cells has now been developed where synthesis and assembly of spectrin can be uncoupled and studied separately. Following the in vitro translation of threefold more α- than β-spectrin, 95% of the β-spectrin and equimolar amounts of α-spectrin bind post-translationally to spectrin-depleted rabbit red blood cell membranes, and the excess α-spectrin remains unbound. This α-spectrin cannot bind spectrin-depleted plasma membranes subsequently added to the lysate. The assembly of α-spectrin is, therefore, limited by the availability of β-spectrin, and both subunits assemble post-translationally.