Restoration of normal membrane stability to unstable protein 4.1-deficient erythrocyte membranes by incorporation of purified protein 4.1

Abstract

Protein 4.1, a principal component of the erythrocyte membrane skeleton, is thought to be important in regulating membrane stability through its interaction with spectrin and actin. A key role for protein 4.1 has been indicated in studies in which deficiency of this protein was shown to result in marked instability of the membrane. In order to obtain direct evidence for the functional role of protein 4.1, we reconstituted protein 4.1-deficient membranes with purified protein 4.1 and showed restoration of membrane stability. Erythrocyte membranes totally and partially deficient in protein 4.1 were reconstituted by exchange hemolysis with various concentrations of purified protein 4.1, and their stability measured using an ektacytometer. Native erythrocyte membranes totally deficient in protein 4.1 were markedly unstable, while those partially deficient had intermediate reductions in membrane stability. Reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability. Near-normal membrane stability could be restored to both totally and partially protein 4.1-deficient membranes. In contrast, the addition of protein 4.1 to resealed membranes did not improve membrane stability. This implies that the added protein 4.1 must have access to the cell interior in order to affect membrane stability. Furthermore, in control experiments, the addition of protein 4.1 to normal membranes did not increase their stability. Also, the addition of purified spectrin and human serum albumin during resealing did not improve stability of protein 4.1-deficient membranes. These results provide direct evidence for the crucial role of protein 4.1 in regulating erythrocyte membrane stability.