Localization of Phosphorylated Highly Acidic Regions in the NH2-terminal Half of Nucleolar Protein C23*

110,000), putative nucleolus protein, multiple of To localize the phosphorylated residues (approximately 35 phosphoserines and 1 phosphothreonine) along the polypeptide chain, protein C23 was subjected to cleav- age by N-bromosuccinimide at various molar ratios to tyrosine. At relatively high ratios (e.g. 25:1), a M, 60,000 fragment (60K) was predominant along with several lower molecular weight fragments.

TO whom correspondence should be addressed.

A~p-Glu-Asp-Glu-Glu-Asp-Asp-Asp-Asp-GlU-A~p-A~p-A~p-
interphase cells (Olson et al., 1981) and is found at the nucleolus organizer regions of chromosomes (Lischwe et al., 1981). Protein C23 also preferentially stains with silver on polyacrylamide gels (Lischwe et al., 1979) under the same conditions that portions of the interphase nucleolus  and the nucleolus organizer regions of chromosomes (Howell et al., 1975;Goodpasture and Bloom, 1975) stain with silver. Thus, the protein may play a role in organizing the chromatin and ribonucleoprotein components of the nucleolus.
Organization of the nucleolus by this class of protein would require interaction with various macromolecules; e.g. the highly acidic regions may interact with histones or ribosomal proteins and basic or neutral regions may interact with DNA.
Observations on the partitioning of protein C23 in various nucleolar fractions suggest that this may be the case. For example, a fraction of the protein is extracted with low ionic strength buffers (Rothblum et al., 1977), whereas another fraction is associated with preribosomal ribonucleoprotein particles (Prestayko et al., 1974;Olson et ai., 1974b). More recent studies suggest that protein C23 preferentially binds cloned rDNA sequences upstream from the 18 S coding region.' A nucleolar chromatin-associated phosphoprotein was previously designated C18 (Yeoman et al., 1973;Olson et al., 1975), but data presented in this paper suggest that proteins C18 and C23 are identical and that C18 is a tightly bound form of C23. Thus, protein C23 appears to interact with chromatin as well as with ribonucleoprotein particles, possibly serving as a bridge between the two components.
The phosphorylation of protein C23 in regions which already carry a high negative charge is a puzzling phenomenon.
The added negative charge may be expected to facilitate interactions of these regions with basic regions of other proteins. Since the level of phosphorylation of protein C23 correlates with the synthetic activity of the nucleolus (Kang et aZ., 1975;BaUal et aZ., 1975;Olson et al., 1978), the role of phosphorylation may be a regulatory one. These studies are an initial attempt to cleave protein C23 into structural domains and to locate the phosphorylated acidic regions along the polypeptide chain.

RESULTS AND DISCUSSION
Cleavage of Protein C32 with NBS3--In order to optimize conditions of cleavage, protein C23 was treated with various concentrations of NBS, calculated as ratios to tyrosine. By amino acid composition each molecule of protein C23 was calculated to contain 8 residues of tyrosine and only traces of tryptophan (Tables I and 11) based on M , = 110,000 (Tsutsui et al., 1980). All reactions were carried out at room temperature in 25% acetic acid for 1 h, although preliminary kinetic studies indicated that the reaction was complete in a few minutes. At relatively low ratios of NBS:tyrosine (9:1 or lower) protein C23 was cleaved into four major large fragments estimated to have M, = 88,000,78,000,70,000, and 60,000 ( Fig.  1). In addition, four smaller fragments, 27K, 21K, 19K, and 12K, appeared. At the 9:l ratio the 70K fragment was predominant. At higher NBS:Tyr ratios, starting at approximately 121, the 60K fragment became the predominant fragment ( Fig. 1). This fragment was stable up to a ratio of approximately 25:1, ahove which it also began to disappear. Also appearing at higher NBS concentrations were two other lower molecular weight bands (23K and 13K), while the 27K band disappeared. No bands of intermediate molecular weight (between 27,000 and 60,000) were seen at any of the NBS concentrations. At very high ratios (30:l or above) the 60K as well as lower molecular weight fragments began to disappear.
At least two observations regarding the chemistry of the reaction require comment. First, certain potential cleavage sites appeared more susceptible to cleavage than others. By amino acid analysis, protein C23 was shown to contain 8 tyrosine residues and only traces of tryptophan after hydrolysis with mercaptoethanesulfonic acid. However, sequence studies in progress have revealed the presence of a tryptophan residue on the NH2-terminal side of the 12K fragment.4 If cleavage occurs at all of these 9 sites, a minimum of 10 fragments should be seen. However, under conditions which are optimal for the production of the 60K fragment, only 6 or 7 major fragments were seen. The sum of the molecular weights of these fragments is more than 110,000, implying partial cleavage. It can be seen from Figs. 1 and 2 that higher concentrations of NBS were required to produce the 60K fragment than to generate the extensions of the 60K fragment (70K, 78K, and 88K). Thus, the bond that connects the 60K fragment to the carboxyl-terminal half of the molecule must be more stable (or less susceptible) than the bonds that are cleaved to produce the larger fragments.
The second observation on the NBS cleavage reaction is that much higher than the theoretical values of NBStyrosine were required for extensive cleavage: i.e. more than 20 mol of NBS/mol of tyrosine as opposed to the theoretical value of 3:l (Ramachandran and Witkop, 1967). The high concentrations of NBS required may be due to the NBS reacting with methionine and histidine, the latter of which is not cleaved unless the reaction mixture is heated at pH 3-4 a t 100°C.
Location of Phosphorylated Residues-When protein C23 was analyzed for phosphoserine and phosphothreonine by the p elimination procedure (Richardson et al., 1978), it was found that the protein contained 3.5 mol '% phosphoserine and 0.1 mol % phosphothreonine (Table I). On a residue/110,000 dalton basis, this is approximately 35 residues of phosphoserine and 1 residue of phosphothreonine. Analyses of the partial acid hydrolysate of 32P-labeled protein C23 by thin layer chromatography confirmed that the bulk of the radioactivity    -, not determined. was in phosphoserine. Radioactive phosphotyrosine was not found.
Previous studies (Mamrack et al., 1977) showed that in tryptic digests of protein C23, more than 85% of the 32P radioactivity was in highly acidic peptides. To locate these along the polypeptide chain, "'P-labeled protein C23 was digested with various ratios of NBS:Tyr. Fig. 2a indicates that as the NBS ratio was increased, virtually all the 32P radioactivity moved into the 60K fragment. At lower NBS concentrations some 32P radioactivity was found in the 70K, 78K, and 88K fragments. By densitometric scans of the autoradiograph it was shown that aII of the radioactivity in the original C23 molecule was transferred to the 60K fragment at NBS:Tyr ratios of 24:l (Fig. 2b). Thus, all of the 32P-labeled groups and the acidic regions where they reside are located in approximately one-half of the molecule, i.e. the 60K fragment. Furthermore, since the radioactivity resides only in the large fragments (70K, 78K, and 88K) only at low concentrations of NBS, these fragments are probably extensions of the 60K fragment.
Characteristics of the 60K Fragment-The similarity of the 60K fragment to the parent molecule is illustrated by a comparison of amino acid compositions. Table I indicates that the mole percentages of acidic and basic amino acids are similar in both polypeptides. The number of potentially acidic amino acids greatly exceeds the number of basic amino acids. Therefore, since the isoelectric points are only slightly acidic (approximately 6.4), a large number of the former must be present as glutamine and asparagine.
TO demonstrate that the highly acidic phosphorylated regions can be accommodated in the 60K fragment the residues of the major tryptic phosphopeptides are tabulated in Table  11 along with residue numbers of C23 and 60K. The tryptic  The phosphorylated tryptic peptides were isolated by DEAEcellulose and pH 1.8 paper electrophoresis as described by Mamrack et al. (1977Mamrack et al. ( , 1979

. The compositions of peptides Ca and Cb were taken from Mamrack et al. (1979). Peptides A, B, and D are the remaining major phosphorylated acidic components described by Mamrack et al. (1977).
'Phosphorylated residues were not subtracted from serine and threonine residues.
' The total value for serine is based on the maximal phosphoserine ratios in each of the peptides. See footnote g. phosphopeptides were isolated by DEAE-Sephadex chromatography followed by pH 1.8 paper electrophoresis . In these peptides, which range in length from 15 to 42 residues, 6040% of the amino acids are potentially acidic. In addition, each peptide contains from 1 to 3 phosphoserine residues. The phosphoserine content was based on the number of electrophoretic forms of each peptide, i.e. peptides with identical compositions had different electrophoretic mobilities. Table I1 shows that all 5 acidic phosphopeptides can fit into the 60K fragment, assuming that multiple copies of each are not present. The 5 peptides also have a maximum of 13 phosphoryl groups, or less than half of the phosphoserines in the molecule. Therefore, additional phosphoryl groups must reside in regions other than the highly acidic tryptic peptides.
NH2-terminal Sequences of Protein C23 a n d the 60K Fragment-Protein C23 was subjected to NHz-terminal sequence analysis on the Beckman sequencer. Valine was the only major NHp-terminal amino acid found in the fist cycle. Lysine was found a t positions 2, 5, and 8 to make a relatively basic NHn-terminal sequence: NH2-Val-Lys-Leu-Ala-Lys-Ala-Gly-Lys-Thr-. No conclusive identifications were made beyond residue 9, despite several attempts at automated Edman degradation. Although there was some evidence for Asn and Gly a t positions 10 and 11, respectively, this has not yet been proven. Asparaginyl-glycyl peptide bonds are converted under acidic conditions to cyclic imides or /3-aspartyl peptide bonds which are resistant to Edman degradation (Bornstein and Balian, 1977). Although this bond should be cleavable by hydroxylamine, using this approach has not yet been successful on protein C23. Protein C18 was also subjected to sequencing under the same conditions. The sequence was identical to that of protein C23 for nine cycles, suggesting that they are identical polypeptides. This is further supported by the similarity of amino acid compositions presented in Table I. The isolated 60K fragment also had the same NHz-terminal sequence as protein C23. Thus, the 60K fragment must be placed at the NHp-terminal end of the protein.
Further Cleavage ofthe 60K Fragment-To further localize the phosphorylated acidic regions the 60K fragment was subjected to limited cleavage with submaxillaris protease which cleaves only at arginine (Schenkein et al., 1977). Although the 60K fragment contains numerous arginine residues (Table I) the submaxillaris protease produced two major fragments, 15K and 45K (Fig. 6). NHT-terminal sequence analysis indicated that the 15K fragment had the same NH2-terminal sequence as the parent molecule, thereby placing it at the NHp-terminal end of the molecule.
To determine the distribution of sites of phosphorylation between the two subfragments of the 60K fragment, "'P-labeled material was subjected to submaxillaris protease digestion. After separation by electrophoresis and autoradiography, radioactivity was found in both fragments (Fig. 6). By densitometric scans of the autoradiograph, approximately equal amounts of radioactivity were present in both the 15K and 45K fragments. This indicates that half of the phosphoryl groups in the molecule are located within 15,000 daltons from the NHp terminus of the molecule. The remainder are distributed in an unknown manner throughout the 45K fragment.
Amino acid compositions of the 15K and 45K fragments were also generally similar (Table I) Distribution of radioactivity in NBS fragments from "P-labeled protein C23. Digestion and electrophoresis conditions were essentially the same as those in the legend to Fig. 1. a, autoradiograph of sodium dodecyl sulfate Laemmli-type gel containing 12% polyacrylamide. Numbers at bottom indicate ratios of NBS:tyrosine. 6, plot of data obtained from densitometric scans of autoradiograph in a. The areas of all peaks of radioactivity are presented as percentage of total area. The areas of the intermediate bands (MK, 78K, and 70K) were combined. ment contained somewhat higher percentages of basic amino acids, both fragments were rich in charged amino acids which lends support to the idea that charged residues are rather generally distributed throughout the NH2-terminal half of the C23 polypeptide chain.
Protein C23 has also been found associated with nucleolar preribosomal ribonucleoprotein particles (Prestayko et al., 1974;Olson et al., 1974b). In addition, it has been proposed that the highly acidic regions may interact with histones on nucleosomes to possibly organize nucleolar chromatin . Since this is a large polypeptide chain, it is possible that separate domains of the molecule participate in interactions with several other classes of nucleolar macromolecules. The finding that the phosphorylated acidic regions reside in one end of the molecule lends support to this idea. Another nuclear protein, high mobility group protein 1 has a continuous run of 41 acidic residues in the COOH-terminal Polyacrylamide gel electrophoresis of the 60K fragment and products obtained by digestion with submaxillaris protease. The fractions were run on 15% polyacrylamide Laemmlitype sodium dodecyl sulfate gels. A, 60K fragment obtained by high performance liquid chromatography as in Fig. 36. B, products of digestion of 60K fragment by submaxillaris protease. Lane 2, 15K fragment from high performance liquid chromatography separation of Fig. 5. Lane 3, 45K fragment from high performance liquid chromatography separation of Fig. 5. C, products of digestion of '"I" labeled 60K fragment by submaxillaris protease. Lane 4, autoradiograph of '"P-labeled 60K fragment. Lane 5, autoradiograph of submaxillaris protease digest of 60K fragment.
half of the molecule (Walker et al., 1978). Labhart and KoUer (1982) have proposed that in the amplified rDNA of Xenopus oocytes some factor prevents histones from binding to DNA, thereby promoting transcription. Labhart and Koller further suggest that nucleoplasmin (Krohne and Franke, 1980a, b) which is capable of neutralizing the charge on histones (Laskey et al., 1977) is a candidate for that factor. It seems feasible that protein C23 with its highly negatively charged regions could play such a role in mammalian nucleoli.
These studies show that it is possible to cleave protein C23 into large fragments and purify a number of them, which opens up the possibility of performing functional studies on portions of this molecule. Preliminary studies indicate that some of the NBS-derived fragments retain DNA binding activity.' Thus, it should be possible to use this or similar methods of cleavage and fractionation to determine which regions of the protein interact with various other macromolecules in the nucleolus.