The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We use in vitro culture of erythroblasts derived from this patient, and separately shRNA mediated depletion of band 3, to investigate the development of a band 3 deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally GFP tagged band 3, we demonstrate the ability to restore expression of band 3 to normal levels and to rescue key secondary protein deficiencies including GPA, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3 deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrate the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation.