IDENTIFICATION OF THE MEMBRANE ATTACHMENT SITES FOR PROTEIN-4.1 IN THE HUMAN ERYTHROCYTE

The nature of the membrane attachment site(s) for protein 4.1 in the h uman erythrocyte membrane has yet to be fully elucidated. In this pape r we show that the major attachment site is glycophorin (GP) C/D, and that purified protein 4.1 can bind to two distinct sites on glycophori n C/D. One of these interactions is direct, involving residues 82-98 o n glycophorin C (61-77 on glycophorin D), while the other interaction is mediated by p55. We have localized the binding site for p55 on glyc ophorin C to residues 112-128 (glycophorin D 91-107). We also provide evidence that band 3 is an additional, minor, protein 4.1 binding site . The binding sites for band 3, glycophorin C/D, and p55 are all locat ed within the 30-kDa domain of protein 4.1. We estimate that the relat ive utilization of the three sites in normal membranes comprises 40% t o p55, 40% to GPC/D, and 20% to band 3. The same region of protein 4.1 binds GPC/D and band 3, while the p55 binding site is distinct. The i nteractions involving protein 4.1 with p55 and p55 with GPC/D are of h igh affinity (nM), while those involving GPC/D and band 3 are 100 fold lower (mu M). These results suggest that the most significant interac tions between protein 4.1 and the membrane are those involving p55.