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The methylation of HeLa cell ribosomal proteins has been studied by paper electrophoresis separation of the peptides produced after trypsin digestion of individual ribosomal proteins. Methylation in vivo was detected by 3H labeling of peptides from cells that had been incubated with a mixture of [methyl-3H]methionine plus [35S]methionine, the incorporation of the 3H-labeled amino acid methionine being corrected by the 35S uptake. Each methylated peptide thus represented at least one methylation site (one or more methylated amino acids and their adjacent amino acid sequence). Eleven ribosomal proteins were found to be methylated. As many as five methylation sites were present in one ribosomal protein. In several ribosomal proteins some sites were methylated when ribosome synthesis was suppressed with low levels of actinomycin D, while other sites were not. Therefore, some of the sites in a given protein were apparently methylated in mature ribosomes, while other sites were methylated during ribosome processing. The methyl-group-specific labeling of some sites, but not others, was suppressed by cycloheximide, thus suggesting that the methylation of some sites was dependent on protein synthesis. A cell-free system from cultured HeLa cells is described, which reproduces the methylation pattern of ribosomal proteins in vivo in two ways: methylation was observed both in vivo and in vitro (a) in the same ribosomal proteins; and (b) in the same methylation sites of a given ribosomal protein. In addition, our data in vitro suggest that the methylation of some human ribosomal protein sites may involve a methyl donor other than S-adenosylmethionine. [ABSTRACT FROM AUTHOR]

Two-dimensional analysis of the ribosomal proteins from 60-S subunits of HeLa cells revealed a triplet of acidic proteins, L40a, L40b and L40c, of identical molecular weight (13700), which can be separated only on the basis of their charge differences. Two of the spots, L40 b and L40c, become labeled after incubation of the cells with inorganic [32P]phosphate. The electrophoretic behavior and molecular weights of these proteins support the notion that the proteins L40b and L40c, are phosphorylated forms of the protein L40a. The same proteins can be phosphorylated also in vitro by a HeLa protein kinase on 60-S subunits but not on 80-S ribosomes. The inaccessibility of L40 proteins to the phosphorylation in vitro on 80-S ribosomes suggests that they are located in the interface between the 40-S and 60-S subunits. [ABSTRACT FROM AUTHOR]