Nucleolar specific acidic phosphoprotein C23 is highly methylated.

Protein C23 (Mr = 110,000; pI, 5.5) is the major phosphoprotein in the nucleolus of Novikoff hepatoma cells and comprises 9.5% of the total nucleolar protein. In addition to being highly phosphorylated (1.2 mol % of phosphoserine), it is also highly methylated. Protein C23 contains 1.3 mol % of NG,NG-dimethylarginine and a trace of NG-monomethylarginine.

The nucleolus of Novikoff hepatoma cells contains two major acidic phosphoproteins, protein C23 ( M , = 110,000; PI, 5.5) and protein B23 ( M , = 37,000, PI 5.5) (1-3). These proteins have been immunochemically localized specifically to the nucleolus (1,4). Protein C23 was also found to be concentrated in the fibrillar centers of nucleoli and present on the nucleolus organizer regions of metaphase chromosomes (1). It has also been proposed that protein C23 is, in part, responsible for silver uptake at the nucleolus organizer regions (3). The presence of protein C23 on the nucleolus organizer regions illustrates its association with ribosomal gene chromatin. This spatial relationship suggests that it is an essential component of the pre-rRNA synthesizing machinery.
Protein C23 has been isolated by preparative polyacrylamide gel electrophoresis (2) and by ion exchange column chromatography (1). The purified protein was found to be highly phosphorylated, it contains 1.2 mol % of phosphoserine (5). The sequence of one acidic tryptic phosphopeptide has been reported (2). A similar protein isolated from mouse ascites sarcoma cell nuclei has been partially characterized (6, 7).
In this report, data is presented which shows that protein C23 is subjected to the post-translational modification of methylation in addition to phosphorylation. Protein C23 con- Nucleoli a n d Protein C2.3-Novikoff hepatoma tissue culture cells (NISI-73) were grown in Dulbecco's minimum essential medium with 5% fetal calf serum and antibiotics. T o label the methylated arginine residues, the cells were incubated for 1 h in methionine-free medium and then for 4 h in medium containing L-[methyl-"Clmethionine.
The cells were harvested and combined with Novikoff hepatoma ascites cells. Nucleoli were isolated as previously described (8). Protein C23 was isolated by DEAE-cellulose and Bio-Rad AG3-X4A column chromatography as reported (1). Polyacrylamide Gel Electrophoresis-The one-dimensional sodium dodecyl sulfate-polyacrylamide gel system used was that of Laemmli (9). The nucleoli were suspended in sodium dodecyl sulfate sample buffer, heated for 3 min a t 100 "C, and an aliquot was applied to the gel (9). The proteins were stained with Coomassie brilliant blue R-250.
Amino Acid Analysis-Purified protein C23 was hydrolyzed for 22 h in uucuo at 110 "C in 6 N HCI. The analyses were performed on a Beckman Model 121 MB amino acid analyzer. Methodology similar to that described by Liu and Chang (10) was used to analyze for the presence of methylated arginines. Acidic and neutral amino acids were eluted from a 25-cm microbore column of AA-10 spherical resin for 50 min with 0.35 N sodium citrate buffer, pH 3.9, at 50 "C. Lysine, histidine, N",N"-dimethylarginine, N'',N";-dimethylarginine, N"monomethylarginine, and arginine were resolved by further elution for 130 min with 0.4 N sodium citrate buffer, pH 5.28, at 50 "C. When the labeled protein C23 hydrolysate was analyzed, 0.5-ml fractions were collected from the analyzer and counted.

Fig. 1 shows one-dimensional polyacrylamide gels of total
Novikoff hepatoma nucleolar proteins (Fig. lA) and purified protein C23 (Fig. 1B). A   determine that protein C23 was 9.5% and protein B23 5.6% of the total stained protein in Novikoff hepatoma nucleoli (data not shown). Proteins C23 and B23 have molecular weights of 110,000 and 37,000 respectively; their PI values are approximately 5 .5 (1-3).
The acid hydrolysate of purified protein C23 was analyzed using a standard program on a Beckman 121 MB amino acid   The mole per cent of NG,NC-dimethylarginine was calculated from the known arginine concentration in the high mobility groups (19) and from the percentage of arginine as NG,NG-dimethylar$nine (13).
analyzer. The composition obtained (Table I) is very similar to that previously reported (2,6). An unusual peak was evident in the basic region of the chromatograph. An extended program was developed as described under "Materials and Methods" to identify it. The basic region of this program is illustrated in Fig. 2. The major unidentified peak comigrated with NG,NG-dimethylarginine and it was labeled when cells were incubated in medium containing L-[ methy2-14C]meth10nine. A trace of NG-monomethylarginine is also visible. NG,N'"-Dimethylarginine migrated slightly slower than NG,NG-dimethylarginine and was not detected in protein C23. No methylated lysine or histidine residues were observed in protein C23.

DISCUSSION
Post-translational methylation of proteins at arginine residues is a common cellular process (11). The enzyme which catalyzes arginine methylation is protein methylase I (Sadenosyl-L-methi0nine:protein (arginine) N-methyltransferase, EC 2.1.1.23) (12). This enzyme can modify arginine residues to NG-mono-, NG,NG-di-, and NG,NG-dimethylarginines. The histones (12), high mobility groups 1 and 2 (13), and heterogenous nuclear RNA binding proteins (14)(15)(16) are nuclear proteins which are known to contain methylarginines. The predominant methylated residue that has been identified in nonhistone chromosomal proteins is NG,NG-dimethylarginine (15, 16). The functional significance of the methylation of nuclear proteins is currently not clear but a direct correlation has been observed between protein methylase I activity levels and the rate of cell proliferation (11).
We report here the presence of NG,NG-dimethylarginine in a high mole per cent (1.3%) in the highly phosphorylated, acidic, ribosomal chromatin-associated protein, protein C23. This protein also contains a trace of NG-monomethylarginine but no NG,N'G-dirnethylarginine was observed. Approximately one-third of the arginine residues in protein C23 are methylated (Table 11). Accordingly, protein C23 is one of the most higNy methylated nuclear proteins thus far detected in higher eukaryotes (Table 11). Only a few heterogenous nuclear ribonucleoprotein proteins contain a comparable amount of NG,NG-dimethylarginine. Unlike protein C23, these proteins are basic and contain a high mole per cent of glycine. Like protein C23, some are phosphorylated (16). It has been sug-general class of RNA-associated proteins (17). Protein C23 has been found in nucleolar preribosomal particles (18). The functional role of methylation in protein C23 may be similar to that of the methylation of the heterogenous nuclear ribonucleoprotein proteins. However, the presence of NG,NG-dimethylarginine in the histones, high mobility groups, and other nonRNA-associated proteins (11) suggests other functional roles exist for this residue.