Mapping the Functional Domains of Nucleolar Protein B23*

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.

Ribosome assembly is a multistep process that utilizes numerous proteins and small nucleolar RNAs (1,2). One candidate for a ribosome assembly factor is an abundant protein called B23 (also known as nucleophosmin/NPM (3), NO38 (4), or numatrin (5)) whose location, abundance, and multiple activities suggest that it plays a major role in ribosome biogenesis. This is supported by the ability of protein B23 to bind nucleic acids (6,7) and by its association with maturing preribosomal ribonucleoprotein particles (4,8,9). Treatment of cells with drugs that inhibit preribosomal RNA processing or synthesis (10,11) causes translocation of B23 to the nucleoplasm, which further suggests its presence in nascent preribosomal particles. Finally, protein B23 possesses intrinsic ribonuclease activity that has been implicated in the processing of preribosomal RNA in the internal transcribed spacer region 2 region (12,13).
Protein B23 interacts with other nucleolar proteins, including nucleolin (14), protein p120 (15), and the HIV-1 Rev protein (16). Its ability to shuttle between the nucleus and cytoplasm (17), bind nuclear localization signal containing peptides (18), and stimulate import of proteins into the nucleus (18) suggested a role in nuclear import. The latter activity might be explained by its ability to act as a molecular chaperone (19). In normal cells, this activity may aid in the transport of ribosomal or other nucleolar proteins from their site of synthesis into the nucleus or nucleolus. Alternatively, protein B23 could serve as a chaperone in preventing aggregation of proteins in the very crowded environment of the nucleolus during ribosome assembly. Under native conditions protein B23 probably exists as a hexamer and or larger oligomer (4,20). Because many chaperones are oligomers (21,22), the chaperone activity of protein B23 could be related to its oligomerization properties.
Although the activities of protein B23 are commonly found in many proteins, it lacks similarities to any sequence motifs normally correlated with these activities. Specific functions are often contained in discrete structural regions or domains of multifunctional proteins. In such cases, functional studies can be facilitated by characterization of the activities residing in individual domains. In protein B23 there are distinctive sequence motifs along the polypeptide chain, which suggest the presence of functional domains, i.e. the N-terminal region has relatively high density of hydrophobic residues, the central region contains two highly acidic segments, and the C-terminal third of the molecule carries a net positive charge. Thus, it might be possible to characterize segments of the molecule responsible for particular activities and thereby identify novel structural-functional motifs. In this study we generated a series of N-and C-terminal deletion mutants of protein B23 to facilitate the dissection of the molecule into possible functional domains. It was found that different activities reside in mainly independent but slightly overlapping segments of the polypeptide chain.
Expression and Purification of His-tagged Recombinant Proteins-The recombinant plasmids were transformed into M15 bacterial cells and grown in 1-liter volumes of LB medium. The cells were grown to an A 600 of 0.6 and induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside for 3 h. A few of the mutants including full-length B23, ⌬N35, ⌬N90, ⌬N139, and ⌬C35 grew poorly in LB medium and were grown in terrific broth instead. Cells were collected by centrifugation and kept at Ϫ20°C overnight. Harvested cells were resuspended in buffer B (8 M urea, 0.01 M Tris, 0.1 M NaH 2 PO 4 , pH 8.0) and mixed gently on a rocker for 45 min. The homogenate was then spun at 15,000 rpm for 20 min. To the supernatant, 1 ml of pre-equlilibrated Ni 2ϩ -nitrilotriacetic acid superflow resin was added and mixed gently for 2 h. After three washes with buffer C (8 M urea, 0.01 M Tris, 0.1 M NaH 2 PO 4 , pH 6.3) tagged peptide was eluted with elution buffer (buffer C ϩ 250 mM imidazole).
All of the eluates were tested for protein purity using SDS-PAGE and were dialyzed against a modified H1 buffer (50 mM Tris, 0.1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.9) and concentrated to 5-10 mg/ml of protein using Amicon centriprep concentrators with the desired cut-off range. Protein concentrations were calculated using the Bio-Rad protein assay (23).
Expression and Purification of the GST-tagged Recombinant Proteins-The recombinant plasmids were transformed into the bacterial strain BL21. The protocol used for protein expression and purification is as described in Smith and Johnson (24). Purified proteins (GST alone, GST-⌬N255, GST-⌬N240) were dialyzed against H1 buffer (described above) and were tested for purity using SDS-PAGE.
Nucleic Acid Filter Binding Assay-Plasmid pGEM-4Z was labeled using random priming (Megaprime DNA labeling kit) and 50 Ci of [␣-32 P]dCTP (NEN Life Science Products) as per the manufacturer's instructions (Amersham Pharmacia Biotech). Binding to DNA by protein B23 and its mutants was measured using a nitrocellulose filter binding assay as described previously (25). The reaction mixture, which included labeled DNA and mutant proteins (0 -60 M), was incubated in a TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA) at 37°C for 15 min. The reactions were stopped by filtration through a prewetted nitrocellulose filter (Schleicher & Scheull; 0.45 M), followed by washes with 1 ml of TBE buffer. The amount of radioactivity retained on the filter was quantified using a Molecular Dynamics PhosphorImager.
In Vitro Transcription of Rat rDNA Plasmid-The substrate used for ribonuclease digestions was obtained by in vitro transcription of the rDNA plasmid (pXKDF15) as described previously (13). The plasmid contained 1.3 kilobases of the 5Ј external transcribed spacer region sequence with positions ϩ638 to ϩ1880 from the transcription start site. Plasmid pXKDF15 was linearized with XhoI and in vitro transcription was performed using a bacteriophage T7 RNA polymerase (26). Transcripts were uniformly labeled with 32 P by the addition of 50 Ci of [␣-32 P]UTP (NEN Life Science Products). Synthesized transcripts were treated with DNase I and proteinase K, followed by a phenol extraction. To the supernatant, sodium acetate (pH 5.5) was added to a final concentration of 0.3 M, and the mixture was ethanol precipitated at Ϫ70°C. Transcripts were washed with 70% EtOH, dried under vacuum, and resuspended in 10 mM Tris-HCl (pH 7.5).
Perchloric Acid Precipitation Ribonuclease Assay-The perchloric acid precipitation assay used was a modification of the method used by Eichler and Eales (27). Reaction mixtures (20 l) containing radiolabeled RNA (40 g/ml) and protein (50 M concentration) in a buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM MgCl 2 , and RNasin at a final concentration of 0.5 unit/l were digested at 37°C for 15 min. The assays were initiated by addition of proteins and terminated by the addition of 15 l of 2.5 mg/ml yeast RNA, 7 l of uranyl acetate, and 80 l of 10% perchloric acid. Reactions were placed on ice for 20 min, followed by centrifugation at maximum speed for 10 min. 100-l aliquots of the supernatant were taken, and the amount of nonprecipitable nucleotides was determined by liquid scintillation counting. The control in this assay was a catfish T cell receptor ␤ protein cloned into pQE30 vector that was processed in a manner identical to protein B23 and its mutants to account for endogenous ribonuclease activity carried through the purification process.
Molecular Chaperone Assay-The chaperone activity of protein B23 and its mutants was measured using bovine liver rhodanese (Sigma) as a substrate. The anti-aggregation effect was measured using a turbidity assay (28) essentially as described previously (19). Briefly, reaction mixtures contained ice-cold rhodanese solution at a concentration of 300 M in 20 mM Tris-HCl (pH 7.4) with or without added protein B23 or mutants thereof. Aggregation was monitored by spectrophotometrically recording the absorbance at 360 nm at 1 min intervals after addition of the sample to the cuvette held at 65°C. The relative activity was calculated from the ratio of the absorbance of the reaction mixture containing the mutant protein with the absorbance of the sample containing protein B23.1 (100%) at the 15-min time point with a substrate to protein molar ratio of 1:0.5. Under the latter conditions there was a linear relationship between reduction in turbidity and protein concentration.
Size Exclusion Chromatography-Purified proteins (30 l of a 100 M concentration protein solution in 20 mM sodium phosphate buffer (pH 7) were loaded onto a Superose 200 column (Amersham Pharmacia Biotech) equilibrated with sodium phosphate buffer (pH 7). The column was run using a Varian high performance liquid chromatography system at a flow rate of 0.6 ml/min at room temperature, and the elution was monitored at an absorbance of 280 nm. Molecular weight standards were purchased from Sigma, and the column was calibrated using the following protein markers: blue dextran (2,000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), ␤-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (9.6 kDa).

RESULTS
Protein B23.1 has several distinctive segments in its primary structure. These include a nonpolar N-terminal domain, two highly acidic regions in the central portion, and a C-terminal region that is basic (Fig. 1A). To determine whether these segments correlate with functional domains of the protein, we generated a series of N-and C-terminal His-and GST-tagged deletion mutants (Fig. 1A). The purified mutant proteins were analyzed by SDS-PAGE. All of these fusion proteins were efficiently expressed at their predicted sizes, without any significant degradation (Fig. 1, B and C).
Aromatic and Basic Segments within the C-terminal 76 Residues Are Required for Nucleic Acid Binding-Previous studies showed that protein B23 binds both DNA and RNA and that the C-terminal end is essential for this activity (6). To determine which parts of the C-terminal sequence are required for nucleic acid binding, we initially assayed the series of Histagged N-terminal deletion mutants. Nitrocellulose filter binding assays were performed with double-stranded DNA (plasmid pGEM) uniformly labeled with 32 P. The N-terminal deletion mutants ⌬N35, ⌬N185, and ⌬N216 had DNA binding curves very similar to that of the full-length B23 ( Fig. 2A). Likewise, mutant proteins ⌬N90, ⌬N119, and ⌬N139 also had normal DNA binding curves (data not shown). These results clearly indicated that the C-terminal 76 amino acids (⌬N216) are sufficient for nucleic acid binding activity. The C-terminal deletion mutants ⌬C35, ⌬C132, ⌬C161, and ⌬C192 were also inactive in DNA binding ( Fig. 2A), further supporting the importance of the C-terminal end for this activity.
To further dissect the nucleic acid-binding domain, more deletion constructs were tested. The GST fusion tag was used to facilitate expression of the small mutant proteins (GST-⌬N255, GST-⌬N240). The ⌬N255 mutant contains the 37 amino acids that are unique to isoform B23.1 and essential for DNA binding activity as shown with ⌬C35 (see above). However, ⌬N255 did not bind DNA, indicating that this segment alone is not sufficient for activity (Fig. 2B). The mutant ⌬N240, which lacks the basic amino acid cluster at the N-terminal end of the 76-residue segment (Fig. 1A), is also devoid of DNA binding activity. Thus, both ends of the 76-residue C-terminal domain are critical for nucleic acid binding.
The Ribonuclease Activity Requires Segments from the Central and the C-terminal Regions-To determine the location of the B23 endoribonuclease activity in the polypeptide chain, the 5Ј external transcribed spacer region region of preribosomal RNA was used as a nonspecific RNA substrate (13). The ribonuclease activity of the individual deletion mutants was assessed using the perchloric acid precipitation assay with 32 Plabeled RNA. Preliminary studies were performed with varying protein and constant substrate concentrations; this provided information on the linear range of protein and substrate concentrations that could be used for initial rate assays. A control protein not known to have any ribonuclease activity, expressed and purified under conditions identical to those used for the B23 mutants, was used to assess the background ribonuclease level of bacterially produced protein. Fig. 3 shows that a high level of ribonuclease activity is maintained even after deletion of the first 139 amino acids from the N-terminal end of the protein. Interestingly, deletion of the N-terminal 139 amino acids (⌬N139) including the first acidic domain enhances the ribonuclease activity significantly. Conversely, deletion of the C-terminal 35 amino acids (⌬C35) decreases the ribonuclease activity of the protein almost 2 fold, whereas deletion of 132 amino acids from the C-terminal end of the protein (⌬C132) reduces the activity by about 3-4-fold. Mutant proteins with substantial deletions in either the N-or C-terminal ends (⌬N185, ⌬N216, ⌬C161, and ⌬C192) show little or no activity. These results suggest that the central portion of protein B23 and the C-terminal end play crucial roles in maintaining the ribonuclease activity.
The Molecular Chaperone Activity Requires Nonpolar and Acidic Segments in the N-terminal Half of Protein B23-Protein B23 has been shown to have molecular chaperone activity toward substrates typically used in anti-aggregation assays (19). Light scattering studies performed with rhodanese at a concentration of 300 M showed that when the temperature was raised from 4 to 65°C, the protein aggregated and achieved maximum turbidity in 30 min (Fig. 4A). However, the aggregation was almost completely suppressed by adding B23 in a 1:1 molar ratio. The aggregation as measured by turbidity decreased in a linear manner as concentrations of added protein B23 were increased (data not shown). To determine the segments of the polypeptide chain that contribute to the molecular chaperone activity, aggregation assays were performed with the mutants using a substrate to protein molar ratio of 1:0.5 to account for both positive and negative effects.
As portions of the N-terminal region of B23 were deleted, there were moderate reductions in chaperone activity, i.e. the anti-aggregation effect of ⌬N35 and ⌬N90 relative to the fulllength protein was reduced to 84 and 66%, respectively (Fig.  4B). However, deletion of an additional 30 amino acids (⌬N119) reduced the activity to approximately 10% of the control. The remaining N-terminal deletion mutant proteins (⌬N139, ⌬N185, and ⌬N216) had no anti-aggregation activity (Fig. 4B). Upon analysis of the C-terminal mutant proteins, ⌬C35 showed 100% activity, indicating that the C-terminal 35 amino acids did not contribute to the chaperone activity. However, deletion of larger portions of the C-terminal end decreased the antiaggregation effect, with mutant proteins ⌬C132, ⌬C161, and ⌬C192 having 80, 57, and 30% of the control activity, respectively. Similar studies were performed using liver alcohol dehydrogenase and citrate synthase as substrates with results generally similar to those obtained with rhodanese (data not shown).
It has been suggested that molecular chaperones suppress aggregation by making appropriately placed hydrophobic surfaces available to the denaturing protein substrates (29 -31). The N-terminal region of protein B23 is relatively rich in nonpolar amino acids. The substantially decreased anti-aggregation effect seen after removal of the N-terminal region (⌬N119) suggests that the chaperone activity is dependent on this nonpolar region. However, deletion of the acidic regions also results in loss of activity, indicating that the N-terminal region is not sufficient for maintaining full chaperone activity. In other words, both of these segments of the protein seem to be important for the chaperone activity.
The Molecular Chaperone Activity Correlates with the Oligomerization State of Protein B23-Several laboratories have previously observed that protein B23 is capable of oligomeriza- tion and probably exists as a hexamer or larger oligomer in the cell (20,32,33). Gel filtration chromatography was used to assess the oligomeric states of the mutant proteins to determine the possible relationship of this property with chaperone activity. Examples of gel filtration elution profiles using fulllength B23, ⌬N139, and ⌬N90 are shown in Fig. 5A. All of these proteins elute primarily as single peaks; protein B23 has an apparent molecular mass of 350 kDa, which approximates a decameric complex. Similarly, ⌬N139 has an apparent molecular mass of 24 kDa compared to a theoretical monomer molecular mass of 20,130 Da, suggesting that this mutant protein mainly exists as a monomer. Conversely, protein ⌬N90 forms a very large complex that elutes near the void volume. Although it was not possible to estimate the molecular mass of this complex, it is clearly larger than 700 kDa. Other mutant proteins that aggregate into larger complexes are ⌬N35, ⌬C132, and ⌬C161.
Table I provides estimates of molecular weights and oligomerization states of the constructs based on the data presented in Fig. 5B. The data indicate that the N-terminal deletion mutants ⌬N35 and ⌬N90 form very large oligomers, whereas ⌬N119 elutes as a trimer and the remaining N-terminal mutants ⌬N139, ⌬N185, and ⌬N216 elute primarily as monomers. The C-terminal mutants ⌬C35, ⌬C132, and ⌬C161 elute as oligomers, whereas ⌬C192 is a mixture of large oligomeric complexes and monomers. These studies indicate that the N-terminal third of the molecule is essential for oligomerization.
Analysis of these data suggests a link between oligomerization state and chaperone activity. The effect is pronounced with the N-terminal mutants ⌬N119, ⌬N139, ⌬N185, and ⌬N216 that clearly exist as monomers and do not possess any chaperone activity. However, ⌬N35 and ⌬N90 are oligomers and show about 60 -80% retention of chaperone activity. The C-terminal mutants exist mainly as oligomers and show varying degrees of activity. These studies suggest that the chaperone activity requires the ability to oligomerize for maximal activity. The N-terminal region seems to play a dual role in the chaperone activity and oligomerization of protein B23. DISCUSSION The current studies show that the molecular chaperone, ribonuclease, and nucleic acid binding activities of protein B23 reside in nearly independent but partially overlapping segments of the polypeptide chain (Fig. 6). Two adjacent segments in the polypeptide chain are important for the molecular chaperone activity. The first of these is the nonpolar region in the N-terminal end; deletion of this region results in nearly complete abolition of chaperone activity, suggesting that the nonpolar residues play a crucial role in the chaperone activity. The second important segment is the acidic region in the center of the molecule. Removal of this part of the molecule also results in proteins with greatly reduced chaperone activities. Thus, both charge-charge and hydrophobic interactions seem to be essential for the chaperone activity of protein B23. The same combination of interactions is important for the activity of members of the small heat shock class of chaperones, e.g. ␣B-crystallin (29).
Molecular chaperones are divided into several groups on the basis of similarity of structural characteristics and/or similarity of functions. Although there is little or no sequence homology between B23 and the small heat shock proteins, there are other interesting similarities (22). The monomeric sizes of these proteins are 15-40 kDa, but they exist as large oligomeric complexes of up to 50 subunits within cells, with molecular masses ranging from 280 kDa to 2 MDa. Their secondary structures are predominantly ␤-sheet (40 -50%) with some ␣-helix (10 -20%). These proteins have sequence homology with each other in the C-terminal halves (including nonpolar residues), and also contain conserved, flexible, and solvent-exposed C-terminal extensions. Protein B23 shares several features in common with the small heat shock proteins/␣B-crystallins including (a) the dependence on both nonpolar and charged regions for activity, (b) secondary structures composed mostly of ␤-sheets and ␤-turns (34), and (c) a tendency to oligomerize (20).
There is mounting evidence for a relationship between oligomerization state and chaperone activity (35,36). Although protein B23 is known to oligomerize (20,32,33), this is the first study to correlate its oligomerization with molecular chaperone activity. Clearly, no chaperone activity was retained in mutant proteins that were monomeric; however, it cannot be ruled out that these mutants simply lacked a substrate-binding site because this seems to reside in the N-terminal third of the molecule.
The mutagenesis studies provided clues about the general sequence requirements within the 76-residue nucleic acid-binding segment. Deletion of either end of this segment results in complete loss of nucleic acid binding activity. The C-terminal 37-residue segment, which is required but is not sufficient for activity, is relatively rich in aromatic amino acids. In the C-terminal end of this sequence there are five aromatic residues: FINYVKNCFRMTDQEAIQDLWQWRKSL. The placement of the aromatic residues, especially the two tryptophans is highly conserved in analogous proteins including starfish nucleolar protein ANO39 (37), sea urchin mitosis apparatus protein p62 (38), Xenopus NO38 (4), and B23 from chickens (39) and humans (3). The requirement for the two tryptophans is reinforced by experiments in which they were replaced by leucines; the resulting mutant protein did not bind DNA. 2 The spacing of the basic residues in the N-terminal end of the 76-residue segment is also highly conserved; this portion is also necessary but not sufficient for activity. Thus, the aromatic and basic side chains at the two ends of the nucleic acid-binding domain seem to act in combination to serve this function. The N-terminal end of this segment also contains the two putative cdc2 phosphorylation sites (40), which could regulate nucleic acid binding during various stages of the cell cycle.
Finally, analysis of the two isoforms of protein B23 for ribonuclease activity reveals that although these mutants differ only in their C-terminal end, the shorter form shows a significant decrease in activity, suggesting that the C-terminal 35 amino acids are important for substrate binding. This is not surprising because this region is essential for nucleic acid binding activity. Because the shorter isoform possesses a relatively high level of activity, it follows that the catalytic site is in another part of the molecule. Interestingly, deletion of the first acidic domain causes a substantial increase in ribonuclease activity; this effect may be due to exposure of the region between the two acidic segments. It is possible that this increase in activity upon deletion of the acidic domain is due to a decrease in electrostatic repulsion, making RNA-protein interactions more favorable. Alternatively, the shift to the monomeric state resulting in decreased steric hindrance and more rapid diffusion could cause this enhancement. Because deletion of the short region between the acidic segments results in the complete loss of ribonuclease activity, it seems likely that it contains the catalytic site. Curiously, deletion of the N-terminal end of the molecule, which contains all of the histidine residues, does not abolish activity. Although histidine residues are part of the catalytic sites in many ribonucleases (41), this is clearly not the case in the B23 ribonuclease. Determining the catalytic mechanism of the B23 ribonuclease may be facilitated by a crystallographic structure of the protein along with its bound substrate.
What is the advantage to the organism of having a protein with multiple activities that are seemingly unrelated in the same polypeptide chain? The C-terminal nucleic acid-binding domain of B23 seems to be involved with recognition at various levels. First, this region has been shown to be essential for nucleolar localization in the Xenopus version of the protein (42) and in a similar protein from sea urchin (43). Because of its association with preribosomal particles in the nucleolus, the most likely mode of recognition is through RNA binding. Because other parts of the B23 molecule are also important for nucleolar targeting (33), this may be a cooperative process involving interactions with other proteins. The recognition process could target proteins bound to protein B23 to specific sites during the ribosome assembly process. The C-terminal domain could also be important in substrate recognition for the ribonuclease activity of the protein. Preferential cleavage of a narrow region of the pre-rRNA transcript by the B23 ribonuclease (13) could possibly utilize the C-terminal end of B23 for recognition.
The catalytic region of the ribonuclease seems to reside in 2   analytical gel filtration chromatography The molecular size of the mutants was estimated from a plot of V e /V o versus molecular weight constructed from standard proteins of known molecular weight (Fig. 5 a Because these samples eluted out of the linear range of the standards, no effort was made to determine their molecular weights. FIG. 6. Summary of activities of protein B23 N-and C-terminal deletion mutants. The patterns in the scheme are as defined in the legend to Fig. 1A. The chaperone and ribonuclease activities are relative to those of B23.1 (100%). For DNA binding, ϩ represents binding activity comparable with B23.1, and Ϫ represents minimal to no binding affinity. the center of the molecule and overlaps with the chaperonecontaining segment. It is conceivable that the binding of other proteins, e.g. ribosomal proteins, to the chaperone region of B23 could alter the catalytic site and regulate the ribonuclease activity to provide temporal regulation of steps in ribosome biogenesis. Thus, seemingly independent activities could be related to each other through targeting and regulatory processes. Confirmation of this hypothesis will require development of more satisfactory systems for studying ribosome biogenesis.