The numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

HeLa cell ribosomal protein S6, and the increase in its phosphorylation level that occurs after resuspending cells in fresh medium plus serum, were studied using two-dimensional gel electrophoresis. The maximum level of S6 phosphorylation occurs about 2 h after adding fresh medium and seum to cells that have been allowed to grow to high density; this results in an almost complete shift of the spot representing S6 in two-dimensional polacrylamide gels to a new location. Mixing experiments showed that the differences in the level of phosphorylation occur in vivo and are not an artifact of in vitro sample preparation. This method of stimulating S6 phosphorylation provides a convenient system for studying the functional significance of the phenomenon. Only one other ribosomal protein was detectably phosphorylated using [32P]-labeling and autoradiography of dried two-dimensional gels. The level of phosphorylation of this protein, L14, does not change after serum stimulation.

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The order in which ribosomal proteins become associated with nucleolar pre-rRNA was studied by measuring the kinetics of label accumulation in ribosomal proteins isolated from the cytoplasmic ribosomes, as well as by two-dimensional polyacrylamide gel electrophoresis of protein extracted from ribosome precursor particles. Tentative identifications of early-adding and late-adding groups of proteins have been made.The process by which ribosomes are assembled in growing HeLa cells has been studied by following the appearance of labeled rRNA [1] and the appearance of labeled ribosomal proteins [2,3] in cytoplasmic ribosomes at short times after addition of a labeled precursor to the medium. The rRNAs become labeled only after a lag period, which is about 20 min for the 1 8 4 rRNA and about 40 min for the 28-S rRNA. These lag periods are thought to represent the time between the transcription of the 45-S pre-rRNA and the appearance of the new ribosomal subunits in the cytoplasm (i.e. the minimum time required for rRNA transcription and processing, and for subunit assembly and transfer of the cytoplasm). When accumulation of labeled ribosomal proteins in the cytoplasmic ribosomes was examined, diflerent proteins were found to accumulate different amounts of radioactivity [2 -41. These results suggested that proteins enter the process of ribosome assembly at different times after the transcription of the 45-S pre-rRNA. This conclusion has received support from studies on the protein composition of ribosome precursor particles [5 -81. The experiments described below used a combination of kinetic measurements of label accumulation in the proteins of cytoplasmic ribosomes and direct enumeration of ribosomal precursor particle proteins on twodiinensional gels, to show that many proteins can be classified into early-adding and late-adding groups. MATERIALS AND METHODS Cell Growth und LhelingHeLa cells were grown in spinner culture flasks in Eagle's minimal essential medium supplemented with 10% calf serum (Gibco). The methionine in the medium used for the kinetic experiments was half that normally present (7.5 pg/ml instead of 15 pg/ml). Incorporation of [35S]methionine into acid-precipitable material (Fig. 3 ) was followed by resuspending 6.6 x lo7 exponentially growing HeLa cells in 25 ml Eagle's minimal essential medium supplemented with 5 calf serum. After 15 min, 50 pCi [35S]methionine was added. At the indicated times 1.0-ml samples were removed, filtered through Whatman GF/A filters, and immediately washed twice with unlabeled medium at 0°C. The filters were then heated in 5 % trichloroacetic acid (95 "C for 10 min) and vortexed in a small test-tube. After cooling to 0 'C, this solution and two 5 % trichloroacetic acid washes were filtered through a Millipore filter (0.45 pm pore size). The GF/A filter was combined with the Millipore filter in a scintialltion vial and counted by adding 0.6 ml water and 15 ml Aquasol (New England Nuclear).For the measurements of accumulation ratios (Table I), cells were grown for 3 da...