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

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Abstract
Spectrin, the major protein of the subcortical actin network in erythrocytes, contains two non-identical subunits, alpha and beta (refs 1, 2). Spectrin is indirectly associated with the transmembrane anion transporter through the binding of beta-spectrin to an extrinsic protein, ankyrin. In chicken embryo erythroid cells, alpha-spectrin is synthesized in a threefold excess relative to beta-spectrin, although the two subunits are assembled in equimolar amounts. 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 alpha- than beta-spectrin, 95% of the beta-spectrin and equimolar amounts of alpha-spectrin bind post-translationally to spectrin-depleted rabbit red blood cell membranes, and the excess alpha-spectrin remains unbound. This alpha-spectrin cannot bind spectrin-depleted plasma membranes subsequently added to the lysate. The assembly of alpha-spectrin is, therefore, limited by the availability of beta-spectrin, and both subunits assemble post-translationally.
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