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1159 Journal of Cell Science 112, 1159-1168 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0136 Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51 Gina M. Trimbur, Jennifer L. Goeckeler, Jeffrey L. Brodsky and Charles J. Walsh* Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA *Author for correspondence (e-mail: [email protected]) Accepted 22 January; published on WWW 23 March 1999 SUMMARY BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that comigrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using ‘Far Western’ blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae. INTRODUCTION structures. CMT often seem to arise from these surrounding components, rather than from the basal bodies themselves (see, for example, Fulton, 1971). In the amebo-flagellate Naegleria gruberi two of the potential roles of basal bodies, organization of the flagellar axoneme and the sites of MTOC activity for CMT, are temporally separated. Naegleria amebae lack centrioles as well as basal bodies and CMT while flagellates possess basal bodies, flagella and CMT (Fulton and Dingle, 1971; Walsh, 1984). During the differentiation of amebae into swimming flagellates, basal bodies form 55 to 60 minutes after initiation and about 10 minutes before the flagella become visible on the cell surface (Dingle and Fulton, 1966; Fulton and Dingle, 1971; Walsh, 1984). However it is not until some 20 minutes later, when the flagella have elongated to near their full length, that a large complex of CMT forms from the basal body region (Walsh, 1984). To characterize the non-tubulin components of the Naegleria Basal bodies and centrioles are frequently associated with microtubule organizing centers (MTOCs). In the case of centrioles it seems clear that it is not the centrioles themselves but the surrounding material, the centrosome or pericentriolar material, that is the site of MTOC activity (reviewed by Baluska et al., 1997; Joshi, 1994). The role of basal bodies, if any, in MTOC activity is less well understood. Three kinds of microtubules are usually associated with basal bodies. The doublet microtubules of the flagellar axoneme are direct extensions of the triplet microtubules of the basal body wall while the single central pair microtubules of the axoneme arise from the distal surface of the basal body. In addition many basal bodies are associated with arrays of cytoplasmic microtubules (CMT). However, it is unclear where the MTOC activity lies that gives rise to these CMT. Basal bodies frequently exist as complexes with various accessory Key words: Naegleria, BN46/51, Fibrillarin, Nucleolus, Yeast, FK506 1160 G. M. Trimbur and others basal body complex, monoclonal antibodies (mAbs) were developed against proteins derived from a basal body fraction (Trimbur and Walsh, 1992). The mAb BN5.1 was identified based on its specific reaction to the basal body region. BN5.1 also reacts with the large central nucleolus of both amebae and flagellates. The BN46/51 antigens from both the basal body complex and from nucleoli were isolated, characterized and proved to be identical. When solubilized from nucleoli or from basal body fractions, the antigen was a complex of approximately equal amounts of two subunits with molecular masses of 46 kDa and 51 kDa and pIs of 5.0 and 4.9, respectively (Trimbur and Walsh, 1992). Both subunits were recognized by BN5.1. This protein was named BN46/51. Analysis of the cytoplasmic appearance of BN46/51 during the differentiation demonstrated that BN46/51 is absent when basal bodies form or as the flagellar axoneme elongates, but BN46/51 appears when the cytoplasmic microtubule complex elongates from the basal body region. When flagellates spontaneously revert to amebae with the abrupt loss of the CMT, BN46/51 disappears from the cytoplasm, although the nucleolar concentration of BN46/51 remains constant during the formation of flagellates and the reversion to amebae (Trimbur and Walsh, 1992). BN46/51 is restricted to the granular component (GC) of Naegleria nucleoli (Trimbur and Walsh, 1993), a region where ribosomes are assembled and from which they exit the nucleus (reviewed by Hadjiolov, 1985). BN46/51 is also restricted to the GC-like cortex of nucleolus-like particles (NLPs) which assemble in vitro from extracts of Naegleria nucleoli (Trimbur and Walsh, 1993). Thus BN46/51 is specifically targeted to a subnucleolar region both in vivo and in vitro and BN46/51 has the unusual property of associating with the basal body region only when this region acts as an MTOC. The mechanism of nucleolar targeting is poorly understood (reviewed by Olson, 1990; Shaw and Jordan, 1995). To test whether nucleolar targeting of BN46/51 is intrinsic to the individual subunits or the heterodimer, or requires additional components, we have further characterized the genetic, cellular and biochemical properties of this protein. We describe two cDNA clones, JG4.1 and JG12.1, that represent the mRNAs for the 46 kDa and the 51 kDa subunits of BN46/51. We also measured the in vitro association of BN46/51 with nucleoli of mammalian cells, the binding of BN46/51 to other nucleolar proteins on western blots, and the in vivo targeting of both BN46/51 subunits to yeast nucleoli. We conclude that BN46/51 is a novel nucleolar protein that binds specifically to fibrillarin and other nucleolar proteins in vitro and to the nucleoli of fixed cells, and that the BN46/51 subunits are individually targeted to the nucleolus in vivo. MATERIALS AND METHODS Cell culture and fluorescence microscopy Amebae of Naegleria gruberi strain NB-1 (Fulton, 1970, 1977) were grown on NM agar with a lawn of Klebsiella pneumoniae as previously described (Fulton and Dingle, 1967). Amebae were harvested and washed free of bacteria in ice-cold 2 mM Tris-HCl (pH 7.6). M15 cells (M1536-B3, parietal endoderm-like mouse cell line) (Chung et al., 1977) were maintained in DMEM (Dulbecco’s modified eagle medium, Gibco) supplemented with 10% fetal calf serum (Gibco-heat inactivated), 2 mM glutamine and 0.1 mM βmercaptoethanol at 37°C in a humidified incubator with 5% CO2. Cells were prepared for indirect immunofluorescence by growth on sterile microscope slides. When the cells were 60-70% confluent, the slides were rinsed briefly 3 times with TBS (50 mM Tris-HCl, pH 7.4, 200 mM NaCl) followed by either immersion into ice-cold methanol for 10 minutes or ice-cold SPMG (63 mM sucrose, 25 mM sodium phosphate pH 7.2, 2.5 mM MgCl, and 0.5 mM EGTA) – 1% formaldehyde for 10 minutes followed by immersion in ice-cold methanol for 10 minutes. M15 cells were incubated with antibody as described below. Either fixation method was satisfactory for labeling with the AH6 mAb, although the DE6 mAb labeled nucleoli more consistently when the cells were fixed first in formaldehyde. 3T3 cells grown on coverslips, as above, were lysed by incubation in 80 mM Pipes-KOH, pH 6.8, 1 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100 and 10% glycerol (lysis buffer) for 5 minutes at 30°C (Lieuvin et al., 1994). Following lysis, cells were fixed in −20°C methanol for 6 minutes. Fixed cells were incubated in a Triton X-100 soluble extract of amebae containing BN46/51 (Trimbur and Walsh, 1992) or in buffer for 30 minutes at room temperature in a humid atmosphere. Following 3 washes with lysis buffer, coverslips were incubated in primary antibody for 60 minutes at 37°C, washed 3 times with TBS, incubated for 60 minutes at 37°C in FITC-labeled goat antimouse IgG and washed 3 times in TBS before being mounted in 90% glycerol, 10% 0.1 M Na2CO3 pH 9.6, containing 100 mg/ml of 1,4diazabicyclo[2.2.2]octane (Langanger et al., 1983). Photography was carried out as previously described (Trimbur and Walsh, 1992). Yeast cells were initially grown as described below, fixed in formaldehyde, and the cell wall digested as described (Kilmartin and Adams, 1984). In later experiments cells were fixed in formaldehyde, digested with zymolase, placed on polylysine coated slides, and stained with first and second antibody as described (Pringle et al., 1991). Images in Fig. 10 were collected with a digital camera (Hamamatsu) and graphics were prepared using Photoshop software (Adobe V.4.0.1). Characterization and expression of BN46/51 cDNAs RNA was isolated from mid-log phase amebae (Chomczynski and Sacchi, 1987). Poly(A)+ RNA was isolated using an oligo(dT) column as previously described (Shea and Walsh, 1987). A cDNA library was prepared in the Lambda ZAPII vector using oligo(dT) as a primer and the kit provided by Stratagene. Plaques expressing the antigen recognized by BN5.1 were identified by the plaque lift technique (Sambrook et al., 1989) and purified by three rounds of subcloning. Positive clones were excised into the Bluescript II SK− phagemid (Stratagene) according to the manufacturer’s specifications. Both strands of the cDNAs were sequenced by the dideoxy method (USB corporation). For expression in Saccharomyces cerevisiae, cDNAs were excised with EcoRI and XhoI and subcloned into the identical sites of the GAL1-inducible pYES2 expression vector (Invitrogen). After initial selection on complete medium lacking uracil and containing glucose, cells were grown overnight in complete medium supplemented with either 2% glucose or 2% galactose. Antisense RNA probes were produced by cutting the JG4.1 and JG12.1 clones with HindIII and XhoI to remove approximately 500 nucleotides from their 3′-ends. This removed a region that is identical in both original clones. Antisense probes produced from the resulting constructs designated JG4.1X and JG12.1X would be expected to provide an unambiguous detection of the 45 nucleotide region present in JG12.1 but absent in JG4.1. Antisense probes were synthesized using T7 RNA polymerase and a MAXIscript kit (Ambion). Total RNA was isolated and RNase protection assays were performed (Ambion). RNA fragment sizes were determined using labeled fragments produced with an Ambion Century Marker Plus kit. Nucleolar targeting of BN46/51 1161 Isolation of nucleoli, preparation of nucleolus-like particles (NLPs), preparation of cell extracts and BN46/51 binding assays Nucleoli, nucleolar extracts, NLPs and Triton X-100 extracts of amebae were prepared from Naegleria amebae as previously described (Trimbur and Walsh, 1992, 1993). Yeast cell extracts of BN46/51 expressing cells were prepared as described (Brodsky et al., 1998). In earlier experiments total yeast protein used for analysis of mAbs was isolated from spheroplasts prepared as described (Kilmartin and Adams, 1984) except that the fixation step was omitted. Proteins were fractionated on 7.5% or 10% SDS polyacrylamide gels (PAGE) and transferred to nitrocellulose by electroblotting (Trimbur and Walsh, 1992). Immunoblots were probed with the mAbs BN5.1, AH6 and DE6 (Trimbur and Walsh, 1992) followed by horseradish peroxidase-coupled second antibody and visualized with either 4-chloro-1-naphthol and H2O2 (Trimbur and Walsh, 1992) or using the ECL method (Amersham). For the isolation of nuclei, M15 cells grown to confluence in 25 cm2 flasks were washed twice with ice-cold TBS followed by the addition of 10 ml Rootlet Lyse Buffer (RLB, 30 mM Tris-HCl, pH 8.0, 50% glycerol, 1 mM EGTA, 1 mM DTT, 10 mM ε-amino-ncaproic acid, containing 0.5% Triton X-100, 0.005% PMSF and 0.02 mM leupeptin) (Trimbur and Walsh, 1992) to each flask, as described in part (Muramatsu and Onishi, 1977). Briefly, the flasks were kept on ice for 5 minutes followed by vigorous tapping to detach the cells from the flask. Glycerol was diluted to a final concentration of 25% by the addition of an equal volume of RLB without glycerol (LB) and nuclei were pelleted by centrifugation at 4,080 g for 10 minutes in an HB-4 swinging bucket rotor. The pellet was resuspended in 4 ml of 0.5 M sucrose-LB, the suspension was underlayed with 2 ml of 1 M sucrose-LB and centrifuged as above. The pellet, which contained nuclei with visible nucleoli as judged by phase contrast microscopy (data not shown), was resuspended in 0.2 ml of LB. An equal volume of 2× SDS-sample buffer was added to the nuclear suspension and immediately heated in a boiling water bath for 3 minutes. The resulting viscous solution was vortexed extensively and used as a source for M15 nuclear proteins on immunoblots. Affinity chromatography was carried out as previously described (Trimbur and Walsh, 1992). Columns were prepared using ascites fluids containing either the BN5.1 or DE6 monoclonal antibodies (Trimbur and Walsh, 1993). The antigens recognized by the DE6 mAb were separated from the other components that elute from a DE6 affinity column (see Fig. 5) by addition of 0.1 M Na2CO3, pH 11, followed by elution with 0.1 M Na citrate, pH 2.9, 3 M urea. Components not recognized by DE6 elute at pH 11 while those recognized by DE6 are retained and elute at pH 2.9 (cf. Fig. 8, panel ‘CB’, lane 2, with Fig. 5A, lane 2). A variety of controls demonstrated the specificity of the affinity columns. Most importantly a BN5.1 column failed to bind the components retained by the DE6 column and vice versa, demonstrating the binding specificity of the monoclonal antibody coupled to the resin (Trimbur and Walsh, 1993). ‘Far Western’ binding assays were carried out on western blots containing fractionated NLP proteins or proteins eluted from affinity columns. After staining with 0.2% Ponceau S in 0.3% acetic acid for 5 minutes, blots were cut into strips, blocked for 1 hour in 10% horse serum in TBS and incubated in Triton X-100 soluble extracts of amebae containing BN46/51 for 60 minutes at room temperature. After incubation all strips were washed 4 times for 5 minutes with gentle shaking in TBS-0.5% Tween-20 followed by a rinse in TBS. Following incubation with amebae extracts and washing, strips were incubated with mAb BN5.1, washed, incubated with horseradish peroxidase coupled second antibody, washed, and visualized with 4-chloro-1naphthol and H2O2 as previously described (Trimbur and Walsh, 1992). The mAbs P2G3, P1D10, P2B11, and P1G12, directed against distinct epitopes on fibrillarin, were a gift from Dr Mark Christensen (Christensen and Banker, 1992). Purified Nop1p was a gift from Dr John Woolford (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213). M15 and 3T3 cells were a gift from Marcia Lewis and Dr Albert Chung (Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260). RESULTS Cloning and sequencing BN46/51 cDNAs A cDNA library was constructed in the Lambda ZAP II vector using poly(A)+ RNA from log phase amebae. Recombinant phage were screened for expression of the antigen recognized by BN5.1 using plaque lifts. Three positive lambda clones were ultimately identified. Inserts from the phage were subcloned into the Bluescript II SK− phagemid vector and two cDNAs of 842 bp and 904 bp, designated JG4.1 and JG12.1, were chosen for further analysis. When the JG4.1 or JG12.1 cDNAs were individually expressed in E. coli as fusions with the 4 kDa alpha peptide of β-galactosidase, they each produced a single polypeptide recognized by the BN5.1 mAb (Fig. 1A). Expression of the JG4.1 cDNA in E. coli produced a soluble polypeptide while the JG12.1 cDNA product was found in large inclusion bodies (Fig. 1A, lanes 1 and 3). Expression of the cDNAs in yeast produced polypeptides that co-migrated with the appropriate BN46/51 subunits (Fig. 1B). DNA sequencing showed that the JG4.1 cDNA was identical to the JG12.1 cDNA except that JG4.1 contains three additional nucleotides between positions 7 and 8 and between positions 289 and 290 and lacks 45 nucleotides beginning at position 346 as numbered in the JG12.1 sequence (Fig. 2). Both clones contained one continuous open reading frame beginning with a methionine at nucleotide 10 and terminating 65 nucleotides upstream of the poly(A) tail. The poly(A) tail is proceeded by a typical poly(A) addition site (Wickens, 1990). Conceptual translation produced identical polypeptides except for the addition in JG4.1 of an aspartic acid between positions 94 and 95, and the absence of 15 amino acids corresponding to positions 113 through 127 in JG12.1 (Fig. 2). Fig. 1. Expression of the BN46/51 cDNAs JG4.1 and JG12.1 in E. coli and S. cerevisiae as detected on western blots. (A) Expression in E. coli as fusions with the 4 kDa alpha peptide of β-galactosidase. Cells carrying the JG4.1 or JG12.1 inserts in pBluescript plasmids were induced with 1 mM IPTG for 3 hours. After lysis with lysozyme and sonication, extracts were fractionated into soluble proteins, 8 M urea soluble proteins and 8 M urea insoluble proteins. Approximately 60% of the JG4.1 peptide was found in the soluble fraction (lane 1). Lane 2, Naegleria nucleolar protein containing BN46/51. Lane 3, the JG12.1 peptide was found only in the 8 M urea insoluble fraction, which contained large inclusion bodies. No BN5.1 antigen was present in cells lacking plasmid and very low levels were seen in uninduced cells (data not shown). (B) Expression in S. cerevisiae. Lane 1, Naegleria nucleolar protein containing BN46/51. Lanes 2 and 3, extracts from yeast cells containing the pGAL-JG4.1 expression plasmid; lanes 4 and 5 extracts from yeast cells containing the pGAL-JG 12.1 expression plasmid. Cells were grown in either glucose, lanes 2 and 4, or in galactose, lanes 3 and 5. 1162 G. M. Trimbur and others 1 1 61 18 121 38 181 58 241 78 301 98 361 118 421 138 Fig. 2. The nucleotide and predicted amino acid sequences of the BN46/51 cDNAs JG4.1 and JG12.1. The sequence of the larger subunit cDNA, JG12.1, is presented. The smaller subunit differs in the presence of 3 additional nucleotides between positions 7 and 8 and between positions 289 and 290; as indicated by the triangles above the sequence, and in the absence of 45 nucleotides at positions 346 through 390, as indicated by the boxed region. The JG12.1 cDNA has 4 additional adenosine residues at the 3′ end that are not shown, while the JG4.1 cDNA has only 18 adenosine residues at the 3′ end. The KKE/D and KKX motifs are underlined. The JG4.1 and JG12.1 sequences are deposited in GenBank under accession numbers AF091603 and AF091604, respectively. 481 158 541 178 601 198 661 218 721 238 781 258 841 C AA . . . . . . GCACGAGCAATGTCCAACATTTTCTCATTCTTCGGTCAAGAAATCAAGACTGGTGCTCCA M S N I F S F F G Q E I K T G A P . . . . . . CAAGCCTTCGAAATCCCATTCGGTGAAGTTATTCTCCACTTGTCCACTGTTTCCCTCGCT Q A F E I P F G E V I L H L S T V S L A . . . . . . AAGGACACCCCAAAGGGATCTATCACTAGAGTTTTTGTTCACTCTGTTGATGAAGATGAA K D T P K G S I T R V F V H S V D E D E . . . . . . AAGGAAACCAAGTATGTCATTTGCACACTTGTTGGAAAGGAAAAGGAATCCGTCTCTATT K E T K Y V I C T L V G K E K E S V S I ATG . . . . . . GATTTGAACTTTAGCGAAGATGTTGCTCTCTCCATTGAAACATCCGCTAATGATACCACA D L N F S E D V A L S I E T S A N D T T D . . . . . . GTCCATGTTACTGGTTACATCAACTTGATTAATGAAGATGGTGAAGAAGGTGAGTATGGA V H V T G Y I N L I N E D G E E G E Y G . . . . . . GGTTATTCAGTAATTGACGGTGATGATCTTGAAGATGAAAGTGATGAAGAAGAAAAGGCT G Y S V I D G D D L E D E S D E E E K A . . . . . . AAGCTTTTGAGAAAGATGTTGGAAGAAGATGATGACGAAGATGATGAAGATTTCAAGCCA K L L R K M L E E D D D E D D E D F K P . . . . . . GACTTGAATGAATCTTCTGAATCTGCTAAGCTCGAAGAATTGAGCGACGAAGATGAAATG D L N E S S E S A K L E E L S D E D E M . . . . . . GAAGGCGACGATCTTGATGATGACCAAACTGAAGAAGTTGTTAAGAGAGTTCAAGATCTT E G D D L D D D Q T E E V V K R V Q D L . . . . . . GAAAATAGATTGGGCAGAGAAGCTAACGATGAAGAAATCAAGGAAATTGTTACCAGAGTC E N R L G R E A N D E E I K E I V T R V . . . . . . CAAGCTGGCCAACCTGAACCAGCTCCAAAGAAGAAGGAACAACCAAAGAAGGAGCAACCA Q A G Q P E P A P K K K E Q P K K E Q P . . . . . . AAGAAGCAACCACAACAACAACAACAAAAGCAACAACAACAACCAAAGGAAGATAAGAAG K K Q P Q Q Q Q Q K Q Q Q Q P K E D K K . . . . . . AGAAGCAAGAAGAAGCTCTAGGAAACAATAAGAATAAGAAGAACAAGAAGAACAAGAAGA R S K K K L * . . . . . . AATAAATTAACTTTTTTTATCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA The predicted amino acid sequences are highly acidic with 26% aspartic acid and glutamic acid residues. The predicted molecular mass of the polypeptide encoded by the JG4.1 cDNA is 28.8 kDa and that of the JG12.1 cDNA is 30.1 kDa, both significantly less than the observed molecular mass of the native subunits, the fusion proteins expressed in E. coli, and the polypeptides produced in yeast (Fig. 1). Similar anomalously high molecular mass of acidic nucleolar proteins on SDS gels have been noted in other systems (Benton et al., 1994; Fuller et al., 1989). The predicted JG12.1 amino acid sequence can be divided into three broad regions. The N-terminal region (residues 1-80) is relatively neutral while the central region (residues 81-193) is highly acidic with 47 acidic amino acids and only 6 basic residues. The C-terminal region (residues 194-266) is quite basic with 21 basic amino acids and only 12 acidic residues and may contain the nuclear localization sequence (NLS; residues 229-266). There is a limited similarity to the SV40 NLS between residues 229-238, but this region lacks both the arginine and valine found in the SV40 NLS consensus sequence. Residues 260 through 266 bear some resemblance to the bipartite NLS, but the ten residue spacing between basic groups and the terminal arginine are absent (reviewed by Boulikas, 1993). The C-terminal region of both BN46/51 sequences contains 60 17 120 37 180 57 240 77 300 97 360 117 420 137 480 157 540 177 600 197 660 217 720 237 780 257 840 263 900 two KKE motifs. Similar KKE/D repeats occur 11 times in Nop56p/Nop5p and 13 times in Nop58p (Gautier et al., 1997; Wu et al., 1998). The C-terminal region also contains three KKX repeats as found in the nucleolar proteins Cbf5p (13 copies; Cadwell et al., 1997) and Dbp3p (10 copies; Weaver et al., 1997), Fig. 2. We were unable to identify a putative RNA recognition motif (RRM) (reviewed by Dreyfuss et al., 1988; Kim and Baker, 1993). The absence of residues 116 through 130 in the JG4.1 sequence eliminates five acidic residues but otherwise has little obvious effect on the overall organization of this polypeptide. The BN46/51 subunits have some identity with a family of FK506 binding proteins The predicted amino acid sequence encoded by JG12.1 is 26 to 31% identical to the sequence of a group of FKBPs (FK506 binding proteins). The FKBPs include the Drosophila nuclear 39 kDa FKBP, FKBP39 (31% identity) (Theopold et al., 1995). The quality index of this alignment, as determined using the Bestfit program (Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, Wisc.), was 127, more than ten standard deviations greater than alignments to random arrangements of the JG12.1 amino acid composition. A similar degree of identity, with a quality index of 129, was found between the large BN46/51 subunit and a 59 kDa Nucleolar targeting of BN46/51 1163 FK506 binding protein from Sf9 cells, FKBP46 (Alnemri et al., 1994). In this case the quality index of the match was six standard deviations greater than the quality index of random alignments (103±4.3). For both JG4.1 and JG12.1 there was no similarity with the catalytic (i.e. peptidyl-prolyl cis-trans isomerase) domain of the FKBPs (Fig. 3). A third FK506 binding protein was identified by a FASTA search. The 46 kDa nucleolar protein NPI46p/Fpr3p from yeast (Shan et al., 1994; Benton et al., 1994), is 26% identical to JG12.1 with a quality index of 117, four standard deviations greater than a random alignment. The BN46/51 subunits are coded for by separate mRNAs RNase protection assays using antisense transcripts of both cDNAs were carried out with total RNA from log phase amebae to identify the mRNAs corresponding to BN46/51. Antisense probes were synthesized from subclones lacking the 3′-region common to both JG4.1 and JG12.1 in order to accentuate the expected differences in the sizes of the protected fragments. When antisense RNA from the 5′-region of the JG12.1 clone was used as a probe, two fragments of 440 and 360 nucleotides were present (data not shown). These are close to the 431 and 351 nucleotide fragments expected from separate mRNAs for each polypeptide. Assays with the JG4.1 5′-region antisense probe also produced two protected fragments close to the expected lengths of 420 and 392 nucleotides. These results demonstrate the presence of unique mRNAs encoding each subunit. BN46/51 binds to other nucleolar proteins in vitro BN46/51 is targeted to a distinct morphological subregion of nucleoli and NLPs and this specific targeting requires neither RNA nor DNA (Trimbur and Walsh, 1993). This suggests that BN46/51 forms specific complexes with other nucleolar proteins. This possibility was examined by incubating western blots prepared from SDS gels of NLP proteins with fractions containing solubilized BN46/51; solubilized BN46/51 is a large multimeric complex containing only the 46 and 51 kDa subunits (Trimbur and Walsh, 1992). When these blots were probed with the BN5.1 mAb, BN46/51 associated with bands of 74 kDa and 44 kDa (Fig. 4A, lane 4). Lesser amounts of BN46/51 were also associated with a band at 31 kDa. When blots were incubated with BN46/51 containing extracts in 0.4 M NaCl, a condition that dissociates nucleoli and NLPs and releases the BN46/51 complex (Trimbur and Walsh, 1992; Trimbur and Walsh, 1993), BN46/51 was not associated with other nucleolar proteins (Fig. 4A, lane 5). The 44 kDa and 74 kDa polypeptides which bind BN46/51 on western blots correspond in size to previously identified Naegleria nucleolar antigens present in NLPs (Fig. 4B): antigens recognized by the mAbs DE6 and AH6, respectively (Trimbur and Walsh, 1993). The DE6 mAb recognizes 44 kDa and 31 kDa polypeptides on western blots of Naegleria Fig. 3. Comparison of the predicted amino acid sequence of the Naegleria gruberi (N.g.) JG12.1 cDNA with the amino acid sequence of the Drosophila melanogaster (D.m.) FK506 binding protein FKBP38 (Theopold et al., 1995) and the FKBP from Sf9 cells, FKBP46 (Alnemri et al., 1994). Alignments were carried out using the Pileup program of the GCG package. Identities are indicated in reverse type. 1164 G. M. Trimbur and others Fig. 4. BN46/51 binds to nucleolar proteins. Nucleolar proteins isolated from nucleolus-like particles (NLPs) were fractionated and transferred to nitrocellulose. Each lane of the gel received the same mixture of nucleolar proteins. (A) Incubation with solublized BN46/51 (lanes 2 and 4) or the same BN46/51 extract made 0.4 M NaCl (lane 5*). Some strips were incubated in the Triton X-100 containing buffer alone adjusted to 0.15 M NaCl (lanes 1 and 3). Strips were either probed with the BN5.1 mAb (lanes 3-5) or with buffer (lanes 1 and 2). All strips received horseradish peroxidase labeled second antibody followed by 4-chloro-1-naphthol (lanes 1-5). (B) Western blots directed against nucleolar proteins, AH6 (lane 1), DE6 (lane 2), and BN5.1 (lane 3) are shown. One strip received no primary antibody (lane 4). nucleoli and NLPs (Fig. 4B, lane 2). DE6 binds to the dense fibrillar component (DFC) of Naegleria nucleoli and to the DFC-like component of NLPs (Trimbur and Walsh, 1993). In order to determine if the 44 kDa nucleolar protein that bound BN46/51 was in fact the polypeptide recognized by the DE6 mAb, we prepared a DE6 affinity column. When extracts of Naegleria nucleoli were passed over a DE6 column only five polypeptides were retained (Fig. 5A, lane 2). Two of these polypeptides were the 44 kDa and 31 kDa antigens recognized by DE6 (Fig. 5B, lane 2). Two additional bands in this complex, 74 kDa and 60 kDa, were recognized by the AH6 mAb (Fig. 5B, lane 1), a mAb that binds to both the DFC and the GC of Naegleria nucleoli and NLPs (Trimbur and Walsh, 1993). Thus in extracts of Naegleria nucleoli the 74 kDa and 60 kDa antigens recognized by AH6 are associated with one or both of the antigens recognized by DE6. The five polypeptides retained by the DE6 column were used to evaluate the binding of BN46/51. When western blots were incubated with soluble extracts containing BN46/51, BN46/51 was found associated with the 74 kDa antigen Fig. 5. The antigens recognized by DE6 and the AH6 are associated in a complex in solubilized nucleolar extracts. (A) Isolation of the antigens recognized by DE6 on a DE6 affinity column. A 0.4 M NaCl extract of nucleoli was fractionated on a DE6 affinity column and eluted with 0.1 M Na citrate, pH 2.9, 3 M urea. The salt extract, lane 1, and the bound proteins subsequently eluted, lane 2, were fractionated by 7.5% SDS-PAGE and visualized by Coomassie blue staining. (B) Immunoblot analysis of the proteins eluted from a DE6 affinity column. Proteins retained by the column were eluted, fractionated as in A, transferred to nitrocellulose, and probed with AH6, lane 1, or with DE6, lane 2. recognized by AH6 and the 44 kDa and 31 kDa antigens recognized by DE6 (Fig. 6C, lane 4). The amount of BN46/51 binding to the 44 kDa polypeptide was significantly less in this case than the binding to the 31 kDa polypeptide and the association with all three was greatly reduced in 0.4 M NaCl (Fig. 6C, lane 5). The absence of any other polypeptides and the intense immunoreactivity to the purified antigens (Fig. 5) strongly support the identity of the DE6 and AH6 antigens as the BN46/51 nucleolar-binding proteins. To determine if these interactions were conserved, further characterization of the nucleolar polypeptides which bind BN46/51 was undertaken. Both the DE6 and AH6 mAbs bound to mammalian and yeast nucleoli (Fig. 7). The yeast antigen recognized by DE6 proved to be a 36 kDa polypeptide and DE6 recognized a 32 kDa antigen in mammalian cells (data not shown). The size of the yeast antigen recognized by DE6 suggested that this component might be the yeast homologue of fibrillarin, Nop1p. Fibrillarin (Ochs et al., 1985) is a protein found in the U3 as well as other snoRNPs of both yeast and mammalian nucleoli (reviewed by Woolford, 1991; Henriquez et al., 1990; Schimmang et al., 1989). In fact DE6 cross reacts with purified Nop1p (data not shown) and in the reciprocal experiment, a series of mAbs against fibrillarin (Christensen and Banker, 1992) reacted with the Naegleria 44 kDa antigen purified on a DE6 affinity column, Fig. 8. Thus, BN46/51 binds to Naegleria nucleolar components that have homologues in both yeast and mammalian cells. Nucleolar targeting of BN46/51 1165 bind to yeast and mammalian nucleoli. This experiment was possible because both yeast and mammalian cells fail to bind four mAbs that recognize distinct epitopes on both BN46/51 subunits and thus apparently lack a BN46/51 homologue (Trimbur and Walsh, 1992). Binding to mammalian nucleoli was examined by incubating permeabilized and methanol-fixed mouse 3T3 cells with Naegleria extracts containing solubilized BN46/51. When the 3T3 cells were subsequently probed with mAb BN5.1 and labeled second antibody, BN46/51 was found specifically associated with nucleoli (Fig. 9B). The ability of the BN46/51 subunits to associate with the yeast nucleolus was examined by regulated expression of the JG4.1 and JG12.1 cDNAs in yeast cells. When induced by galactose each polypeptide specifically accumulated in the yeast nucleolus (Fig. 10B and D). The nucleolar staining pattern seen when the BN46/51 subunits were expressed individually in yeast is characteristic of the yeast nucleolus (Fig. 7). Overall, both of the BN46/51 subunits contain the sequences necessary for specific targeting to yeast and mammalian nucleoli. DISCUSSION Fig. 6. BN46/51 binds to the nucleolar proteins DE6 and AH6 isolated on a DE6 affinity column. The proteins co-isolated from a DE6 affinity column, (see Fig. 5A) were concentrated to about 200 µg/ml and fractionated on a 7.5% SDS-PAGE gel. Each lane received 15 µl of the same mixture of eluted proteins except for the molecular mass standards. (A) Total protein by Coomassie Blue staining; lane 1, molecular mass standards; lane 2, eluted protein. (B) Western blot using the AH6, lane 1, and the DE6, lane 2, mAb. (C) BN46/51 binding assay. As in Fig. 4 the asterisk indicates that the extract contained a final concentration of 0.4 M NaCl. BN46/51 binds to nucleoli in vitro and in vivo Given the presence of proteins which bind BN46/51 in yeast and mammalian cells it was of interest to determine if BN46/51 could We have cloned and sequenced cDNAs for the 46 kDa and 51 kDa subunits of the basal-body-binding nucleolar protein BN46/51. Specific nucleolar targeting of both BN46/51 subunits was observed when the cDNAs were expressed in yeast and when incubated with fixed mammalian cells. We have also demonstrated the binding of BN46/51 to the Naegleria homologue of fibrillarin and to an unidentified nucleolar polypeptide of 74 kDa. Although sequence analysis indicates that BN46/51 represents a novel nucleolar protein, the BN46/51 subunits are approximately 30% identical to the nuclear FKBP of Sf9 cells, FKBP46, and the FKBP of Drosophila, FKBP39, and 26% identical to the nucleolar FKBP of yeast, NPI46p/Fpr3p. The Fig. 7. Monoclonal antibodies against Naegleria nucleolus-like particles bind to yeast and mammalian nucleoli. S. cerevisiae (A,B,E,F) were fixed with formaldehyde and incubated with mAb DE6 (A and B) or with mAb AH6 (E and F) followed by incubation with a FITC-conjugated antibody against mouse IgG. The mouse parietal endoderm-like cell line M15 was fixed in formaldehyde, incubated with DE6 (C,D) or AH6 (G,H) and FITC second antibody as described above. Epi-fluorescence (A,C,E,G) phase contrast (B,D,F,H) images are shown. Bar, 10 µm. 1166 G. M. Trimbur and others Fig. 8. The 44 kDa antigen recognized by DE6 is fibrillarin. Total NLP protein (lane 1) or affinity purified antigens recognized by DE6 (lane 2) were fractionated by 10% SDS-PAGE and visualized by Coomassie blue (CB), or transferred to nitrocellulose and incubated with DE6, or four anti-fibrillarin mAbs as indicated above each panel. identity extends over the entire BN46/51 sequence and is not simply a reflection of regions rich is repeated acidic or basic residues. However, there is no similarity between the BN46/51 subunits and the peptidyl-prolyl cis, trans-isomerase domain of the FKBPs. Binding to fibrillarin may provide one mechanism for targeting BN46/51 to the nucleolus. The Naegleria 44 kDa nucleolar protein bound by BN46/51 is highly homologous to fibrillarin based on the presence of at least three common epitopes and the fact that the 44 kDa antigen is restricted to the DFC of nucleoli (Trimbur and Walsh, 1993), the location of fibrillarin (reviewed by Shaw and Jordan, 1995). The common epitopes extend through the highly conserved central region (P1D10, P2B11), and into the C-terminal quarter of fibrillarin (P2G3) (Christensen and Banker, 1992) (Fig. 8). The reaction of P1G12, the mAb directed against the N-terminal peptide of fibrillarin, with the 44 kDa antigen was weak and P1G12 reacted with an additional Naegleria polypeptide of high molecular mass that was not recognized by the other mAbs. However, both limited homology of fibrillarins in the Nterminal region and the presence of P1G12 epitopes common to other nucleolar proteins has been noted (Christensen et al., 1986; Christensen and Banker, 1992). The targeting of the BN46/51 subunits to yeast nucleoli and the fact that BN46/51 binds to the nucleoli of fixed mammalian cells suggest that fibrillarin binding reflects a mechanism to target BN46/51 to the nucleolus. The presence of two KKE/D motifs and three KKX motifs in the C-terminus of the BN46/51 subunits is of particular interest. In yeast (S. cerevisiae), tandem repeats of these motifs are an exclusive property of nucleolar proteins and two of these nucleolar proteins, Nop56p and Nop58p/Nop5p, are found in a complex with the yeast homologue of fibrillarin, Nop1p, a protein that binds BN46/51 (Gautier et al., 1997; Wu et al., 1998). The KKX repeat is essential for nucleolar targeting of Dbp3p (Weaver et al., 1997) but it is not required for nucleolar localization or Nop1p binding of Nop58p/Nop5p (Gautier et Fig. 9. BN46/51 binds to the nucleoli of fixed mammalian cells. Coverslips containing 3T3 cells permeablized in lysis buffer and fixed in methanol were incubated in extracts containing solubilized BN46/51 at room temperature for 30 minutes. The cells were stained with the anti-BN46/51 mAb BN5.1 and visualized with a FITC-conjugated second antibody. (A and C) Phase contrast. (B and D) Epifluorescence. (A and B) Cells received BN5.1 first antibody. (C and D) Cells received no first antibody. As demonstrated previously, BN5.1 does not bind to mammalian cells (Trimbur and Walsh, 1992). al., 1997; Wu et al., 1998). Thus while the parallels are intriguing, determining the possible significance of the KKX motif in targeting BN46/51 to the nucleolus or in binding BN46/51 to fibrillarin will require additional investigation. The binding of BN46/51 to multiple nucleolar components suggests that a network of interactions contributes to nucleolar localization. This concept is supported by the fact that the same proteins bound by BN46/51 on western blots co-purify on affinity columns directed to the antigens recognized by DE6. That BN46/51 purified by affinity chromatography on BN5.1 antibody columns does not retain its ability to bind to nucleoli or western blots (data not shown) may reflect denaturation by extremes of pH during elution, or it may mean that there are additional components in the cell extract that are needed for BN46/51 binding to nucleolar components. BN46/51 was originally identified by its appearance in the basal body region after the cytoskeletal microtubules of Naegleria flagellates assemble. The explanation for the association of BN46/51 with the basal body region may lie in the unusual properties of Naegleria gruberi biology. Naegleria amebae lack centrioles as well as basal bodies (Fulton and Dingle, 1971) and cytoplasmic microtubules (Walsh, 1984). The only microtubules observed in amebae are those found in the mitotic spindle which is enclosed within the nuclear membrane (Walsh, 1984; Schuster, 1975). As in a number of protists and in yeast, the nuclear membrane does not break down during mitosis in Naegleria (Schuster, 1975; Fulton, 1970). Naegleria nuclei contain a large central nucleolus which also does not break down during mitosis (Schuster, 1975; Fulton, 1970). This may reflect the fact that there are no chromosomal copies of the ribosomal RNA genes. Instead, the nucleolus Nucleolar targeting of BN46/51 1167 Fig. 10. BN46/51 subunits expressed in yeast are targeted to the nucleolus. The JG4.1 cDNA (A and B) and the JG12.1 cDNA (C,D,F) were subcloned into the yeast expression vector pYES2 under control of the GAL1 promoter. Cells were grown in either glucose (A and C) or galactose (B,D,E,F). (E) Cells containing vector lacking insert were grown in galactose. (F) Cells grown as in D processed without first antibody. After fixation and staining with the BN46/51 specific mAb BN5.1 and a cy3 labeled second antibody, cells were stained with DAPI to visualize the nuclei. All images were collected under identical conditions. (G) A montage of cells expressing the JG12.1 polypeptide. Shown are cells from a sample of 68 cells which showed visible expression of JG12.1. microtubules do not associate with any identifiable MTOC (Schuster, 1975; Fulton, 1970; Walsh, 1984 and unpublished observations). The ends of the polar microtubules appear to be embedded in the nucleolus (unpublished observations). The nucleolus becomes elongated during anaphase and gives the appearance of being pulled apart as the chromosomes move to the poles of the elongating spindle (Schuster, 1975; Fulton, 1970 and unpublished observations). If, as seems likely, the nucleolar division is driven by the mitotic spindle, then there must be a physical association between the spindle and the nucleolus. Because BN46/51 is associated with the MTOC activity of the basal body region, we believe it may also be associated with the MTOC activity responsible for forming the mitotic spindle. Given the binding of BN46/51 to multiple nucleolar components, its association with the MTOC activity needed for mitotic spindle formation would provide a mechanism for attaching the nucleolus to the spindle and thus assuring the division of the nucleolus during mitosis. The presence of KKX motifs in the BN46/51 subunits, motifs known to bind microtubules (Jiang et al., 1993; Langkopf et al., 1992; Noble et al., 1989), is consistent with this hypothesis. Gautier et al. (1997) have suggested that a similar mechanism may aid division of the yeast nucleolus. Testing this hypothesis will require characterizing the MTOC activity for both the mitotic spindle and the cytoplasmic microtubules that form in Naegleria flagellates. 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