The Cytoskeletal Binding Domain of Band 3 Is Required for Multiprotein Complex Formation and Retention during Erythropoiesis
Authors: Satchwell Timothy J , Hawley Bethan R , Bell Amanda J , Ribeiro Maria Leticia , Toye Ashley M 
Published: October 2019

Abstract
The bicarbonate/chloride exchanger protein band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex required for vertical association between the plasma membrane and the underlying spectrin cytoskeleton. A wealth of knowledge, derived from a host of varied studies including in vitro binding assays, work on mature erythrocytes and in other cellular systems have identified a number of binding partners including ankyrin, adducin and protein 4.2 amongst others. However, studies of the role that band 3 and the establishment of its connectivity with the cytoskeleton play both in assembly of multiprotein complexes during erythropoiesis and in particular in protein retention during enucleation have been understandably limited by the technical challenges associated with study of this protein within its unique native cellular context.
The complete absence of band 3 in human erythrocytes has only been reported once, in a Portuguese patient with severe hereditary spherocytosis and distal renal tubular acidosis resulting from homozygosity for a V488M band 3 mutation (band 3 Coimbra). In this study, we used in vitro culture of erythroblasts derived from this patient as well as shRNA mediated depletion of band 3 to investigate the development of a band 3 deficient erythrocyte membrane and to specifically assess the formation, stability and retention of band 3 dependent protein complexes in the absence of this core protein during erythropoiesis and erythroblast enucleation.
We demonstrate that the mutant band 3 Coimbra protein is expressed at very low but detectable levels during erythropoiesis but does not reach the cell surface and is not rescued by interaction with wild type protein. Failure to traffic to the plasma membrane and rapid degradation during erythropoiesis accounts for the absence of band 3 in Coimbra erythrocytes. The absence of plasma membrane expression of band 3 results in secondary deficiencies of a host of band 3 associated membrane proteins that we quantitatively show result predominantly from reduced plasma membrane expression during erythropoiesis compounded by impaired retention in the nascent reticulocyte membrane during erythroblast enucleation.
In order to explore the importance of the capacity of band 3 to associate with the cytoskeleton for surface expression of this protein and its associated multiprotein complex binding proteins, immature band 3 Coimbra patient erythroblasts were lentivirally transduced with N terminally GFP-tagged wild type band 3 or band 3 mutants with absent or impaired ability to associate with the cytoskeleton. We demonstrate for the first time the ability to restore expression of band 3 to normal levels in this uniquely compromised patient and to rescue key secondary protein deficiencies arising from the absence of band 3 in reticulocytes. Exogenous expression levels of band 3, monitored by GFP intensity, correlate directly with degree of rescue of a variety of band 3 associated proteins.
When expressed in band 3 deficient Coimbra erythroblasts, the band 3 membrane domain, which is unable to associate with the cytoskeleton, exhibits an increased partitioning to the plasma membrane surrounding the extruded nuclei compared to wild type band 3 and fails to rescue reticulocyte membrane retention of band 3 associated proteins. Expression of the kidney isoform of band 3, which is unable to bind ankyrin but retains the binding site for the cytoskeletal accessory protein, protein 4.2 results in partial rescue of the protein 4.2 dependent CD47 only. This demonstrates the importance of band 3 association with the cytoskeleton for efficient retention of band 3 associated proteins during erythroblast enucleation. Interestingly, whilst both exhibit reduced reticulocyte membrane retention relative to wild type, a significant proportion of both band 3 membrane domain and kidney band 3 is retained in the reticulocyte membrane following erythroblast enucleation indicating that cytoskeletal attachment of band 3 is not the sole determinant of partitioning during this complex process.
This study advances our understanding of the mechanisms by which the properties of band 3 influence the sculpting and composition of the erythrocyte membrane and highlights the role of this protein as a core for assembly and stabilisation of key membrane proteins in both the early and late stages of terminal erythroid differentiation.

Disclosures
No relevant conflicts of interest to declare.