Protein synthesis in cultured muscle cells: methylation of nascent proteins

Protein methylation was examined in primary cultures of rat leg muscle cells between 7 and 9 days of culture. Methyl¹⁴C]- or ³H]-methionine was introduced into the culture medium and the cells were sampled for radioactive methylated protein residues. Incorporation of the total radioactivity was linear for at least 4 hr after introduction of the methionine label. When labeling was studied for periods between 10–30 min, the methylation of polyribosome-bound, presumably nascent, proteins was unaffected by addition of cycloheximide to the culture medium. The antibiotic, however, inhibited incorporation of methionine, and consequently increased the ratios of the incorporated methylated, to methionine residues and the ratio of ribosome-bound to free radioactivity. The methylated, polyribosome-bound proteins were decreased when puromycin was added to the culture medium. It is proposed that selective methylation of nascent proteins, such as myosin, can begin at the level of polyribosomes and be completed in the cytosol of muscle cells cultured in vitro.