Proteolytic analysis of the topological arrangement of red cell phosphoproteins.

The topology of human erythrocyte membrane phosphoproteins was determined by using protease digestion and selective solubilization. The distribution of 32P among the membrane polypeptides in intact cells differs from the pattern found in isolated ghosts. The following membrane skeleton polypeptides, the nomenclature of Steck [Steck, T.L. (1972a) J. Mol. Biol. 66, 295-305] being used, were phosphorylated: 2, 2.1, 4.1, 4.5b, and 4.9, together with two polypeptides at 105000 and 110000 daltons that are heavily phosphorylated. Among integral proteins, band 3 and glycophorins A and B are phosphorylated. Glycophorin C appears to have little turnover of phosphate. Autoradiograms of trypsin-treated membranes permitted identification of the transmembrane proteolytic fragment of glycophorin A as a dimer of 38000 daltons. The band 5 region contained two phosphoproteins, the peripheral protein 4.9 (48 kdaltons) and PAS-2. Similarly, band 7 resolved into an integral phosphoprotein and a nonphosphorylated protein associated with the membrane skeleton.