Methylation of membrane proteins in human erythrocytes. Identification and characterization of polypeptides methylated in lysed cells.

An in vitro system was developed for studying protein methylation reactions in human red blood cells. Packed erythrocytes were lysed by freeze-thawing in the presence of S-adenosyl[methyl-3H]methionine. Specific incorporation of base-labile methyl groups into the band 3 anion transport protein and the major sialoglycoprotein (glycophorin, periodic acid-Schiff reagent-1) was demonstrated by dodecyl sulfate gel electrophoresis at pH 2.4, selective extractions with Triton X-100 and lithium diiodosalicylate, and protease sensitivity. Two other unidentified intrinsic membrane proteins with Mr = 96,000 and 23,500 were also methylated. Little radioactivity was incorporated into membrane proteins when membranes were incubated with S-adenosyl-L-[methyl-3H]methionine in the absence of cytosol. No evidence was obtained for incorporation of methyl label into extrinsic proteins including bands 1, 2, 2.1, 4, 5, 6, or in zone 4.5. Proteolytic digestion of intact cells and isolated membranes revealed that one site of methylation on the band 3 polypeptide may be at the inner surface of the membrane near the junction of the cytoplasmic domain and the membrane domain. The rates of hydrolysis of the incorporated methyl groups were characterized over a range of pH values. These rates were compared to those of methyl esterified amino acids and peptides, including aspartic acid beta-methyl ester which has been isolated from proteolytic digests of methylated erythrocyte membranes (Janson, C. A., and Clarke, S. (1980) J. Biol. Chem. 225, 11640-11643). We find that the rates of base-catalyzed hydrolysis of beta-methyl esters of aspartic acid and gamma-methyl esters of glutamic acid are highly sensitive to the presence of substituents on the alpha-carboxyl and alpha-amino groups. The rate of hydrolysis of the membrane-incorporated methyl groups are consistent with those of internal aspartic acid and glutamic acid methyl ester residues.