Methylation of ribosomal proteins in HeLa cells.

Methylated amino acids from both 40 and 60S subunit proteins of HeLa cytoplasmic ribosome were analyzed. It was observed that methylation of ribosomal proteins occurs in both subunits with N^G,N^G-dimethylarginine as the major methylated amino acid. The presence of N^G,N^G-dimethylarginine has been identified by high-voltage paper electrophoresis, by paper chromatography, and by amino acid analysis. In addition, both ribosomal subunits contain methylated lysines with ?-N-trimethyllysine being the predominant one, followed by ?-N-dimethyllysine. Little, if any ?-N-monomethyllysine was detected in either subunit. The cytoplasmic 60S ribosomal subunit contains much more ?-N-trimethyllysine compared to the 40S ribosomal subunit. The possible biological significance of methylation was discussed.     

Uptake of ribosomal proteins by isolate HeLa nuclei.

The uptake of radioactive ribosomal proteins by isolated HeLa cell nuclei has been studied. Ribosomal proteins are taken up by nuclei $\text{in vitro}$ more rapidly than are cytosol proteins, suggesting that the uptake is selective. In addition, the ribosomal proteins are found associated with the nucleolus to a greater extent than are the cytosol proteins.     

Relative stoichiometry in ribosomal proteins in HeLa cell nucleoli.

Total protein was released from isolated HeLa cell nucleoli by guanidine hydrochloride, purified by cesium chloride density gradient centrifugation, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Conditions of electrophoresis restricted attention to proteins that are positively charged at pH 8.6. Most of the major nucleolar protein spots co-electrophoresed with ribosomal proteins; the majority of ribosomal proteins from both the large and small ribosomal subunits were represented. Several proteins found in association with polysomes but not on ribosomal subunits and several proteins unique to the nucleolus were also identified in these nucleolar protein patterns. In order to determine whether the ribosomal proteins found in the nucleolus represented sizable pools of ribosomal proteins, or merely ribosomal proteins contained in the preribosomal particles, [35S]methionine-labeled nucleoli were mixed with [3H]methionine-labeled polysomes. From analysis of isotopic ratios in individual protein spots it was possible to determine the stoidchiometry of individual ribosomal proteins in the nucleolus relative to their complement on cytoplasmic ribosomes. All but a few proteins exhibited relative nucleolar stoichiometry values of approximately one, indicating that there are not significant pools of most ribosomal proteins in isolated nucleoli.     

Distribution of newly formed ribosomal proteins in HeLa cell fractions

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.     

Exchange of ribosomal proteins among the ribosomes of Escherichia coli.

Summary The exchange of ribosomal proteins among ribosomes of E. coli has been measured, using a density label technique. As expected most of the proteins do not exhange appreciably. However a substantial fraction of each of proteins S1, S2, S21, L7/L12, L9, L10, L11, L26 and L33 is found to exchange, but exchange of S1, S2, L7/L12, L10, L11 and L26 is found to occur in vitro after lysis of the cells, and therefore it is not possible to say whether or not these proteins also exchange in vivo. In contrast S21, L9 and L33 do not exchange after lysis of the cells and we therefore conclude that these proteins exchange in vivo. The maximum level of exchange of S21, L9 and L33 is attained so rapidly that we were unable to show whether or not it was dependent on protein synthesis.     

Ribosomal proteins: XLIII. In vivo assembly of Escherichia coli ribosomal proteins

Isolation of ribosomal precursors from Escherichia coli K12 is described. The RNA and protein content of the precursor particles was determined.One physiologically stable precursor was found for the 30 S subunit. The assembly scheme is as follows: p16 S RNA + 9 proteins → p30 S (“21 S” precursor) p30 S + 12 proteins → 30 S subunit where p is precursor.Each of the two precursors for the 50 S subunit, P₁50 S and p₂50 S (“32 S” and “43 S” precursors, respectively), contains p5 S + p23 S RNA's in a 1:1 molar ratio. The assembly scheme is as follows: p23 S RNA + p5 S RNA + 16 or 17 proteins → p₁50 S

In contrast to the p₂50 S precursor the p₁50 S precursor is not similar to any core particles, which were obtained by treating 50 S subunits with different concentrations of LiCl or CsCl.The precursors p30 S and p₂50 S can be converted into active 30 S and 50 S sub-units, respectively, by incubation at 42 °C in the presence of ribosomal proteins and under RNA methylating conditions.     

In vivo stability, maturation and relative differential synthesis rates of individual ribosomal proteins in Escherichia coli B/r

The relative differential synthesis rates² of individual ribosomal proteins (r-proteins) were determined for Escherichia coli B/r growing in succinate medium (growth rate, μ = 0.65 doublings per hour), glucose medium (μ = 1.36) and glucose-amino acids medium (μ = 1.90). These differential synthesis rates were found to increase co-ordinately with increasing bacterial growth rates; this implies that ribosomes from bacteria growing at different rates are homogeneous with respect to their protein composition (i.e. the stoichiometric amounts of the different r-proteins per ribosome are constant and independent of the bacterial growth rate). Following incorporation into ribosomes, the bulk of the r-proteins were found to be as stable as total protein. Only two r-proteins, S6 and S21, were less stable than total protein; their decay half-lives, measured in succinate and glucose-amino acids cultures, were estimated to be approximately 500 minutes. In addition, post-translational modification of proteins S18, L6 and L11 was observed and the possible relations between modification and in vivo ribosome assembly are discussed. Finally, evidence is presented suggesting that the coordinate production of r-proteins may result, in part, from a mechanism that degrades excess r-proteins that are not rapidly incorporated into ribosomal particles.     

Scanning independent ribosomal initiation of the Sendai virus Y proteins in vitro and in vivo.

The Sendai virus P/C mRNA contains five ribosomal initiation sites between positions 81 and 201 from the 5' end. One of these sites initiates in the P open reading frame (ORF) (ATG/104), whereas four initiate in the C ORF (ACG/81 and ATGs/114, 183, 201), to give a nested set of C proteins (C', C, Y1, Y2). Introduction of new ATGs or physically breaking the mRNA upstream of these natural sites was used in vitro to prevent ribosomal scanning downstream. The results suggest that a minority of the ribosomes which initiate C (ATG/114) and all of those which initiate Y1 and Y2 (ATGs/183 and 201) do so by a scanning independent mechanism. When the leaky ACG/81 site is changed to a non-leaky ATG site in in vivo experiments, ribosomal initiation at Y is again not diminished, whereas that at C as well as at P becomes undetectable. Ribosomal initiation at Y appears to be scanning independent in vitro and in vivo. That at C is partly independent in vitro, but completely dependent in vivo. These results are discussed in terms of a model of internal initiation at Y.     

Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross- reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross- reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Changes in the conformation and stability of 5 S RNA upon the binding of ribosomal proteins.

The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.     

Degradation of abnormal proteins in HeLa cells.

The experiments show that abnoramal proteins are degraded faster than normal ones in HeLa cells. Among the fragmentary proteins made in the presence of puromycin, those with low molecular weight are least stable. Proteins made after incubation with 5-fluorouracil or in the presence of some amino acid analogues are also unstable. Breakdown of proteins made in the presence or absence of puromycin is nearly unaffected by cycloheximide and is independent of pH between 7 and 8.