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BN46/51, a New Nucleolar Protein, Binds to the Basal Body Region in Naegleria Gruberi Flagellates

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BN46/51, a New Nucleolar Protein, Binds to the Basal Body Region in Naegleria Gruberi Flagellates Journal of Cell Science 103, 167-181 (1992) 167 Printed in Great Britain © The Company of Biologists Limited 1992 BN46/51, a new nucleolar protein, binds to the basal body region in Naegleria gruberi flagellates GIN A M. TRIMBUR and CHARLES J. WALSH* Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA •Author for correspondence Summary Indirect immunofluorescence with the monoclonal anti- body region is resistant to extraction, even with 2 M body, BN5.1, labels the nucleolus of amebae of the NaCl. Solubilized BN46/51 exists as a heterogeneous amebo-flagellate Naegleria gruberi. When amebae dif- multimer that elutes on gel filtration with a peak at 400 ferentiate into flagellates, BN5.1 binds to nucleoli and to to 500 kDa and sediments on sucrose gradients at 5.5 S. the basal body region. The BN5.1 antigen is not present The multimers consist of only the 46 kDa and the 51 kDa in basal bodies when basal bodies form at about 60 min subunits in equal amounts as judged by glutaraldehyde after initiation of the differentiation or when flagella cross-linking and by chromatography on BN5.1 affinity form at about 70 min. The BN5.1 antigen is first columns. Nucleolar BN46/51 is associated with the dense detectable in the basal body region at 85 min after fibrillar and granular components of the nucleolus. initiation, a time when the basal body region acts as a However, it does not resemble any previously described microtubule organizing center for the formation of the nucleolar protein. Neither BN5.1, nor three other mAbs microtubule cytoskeleton (MTCS) of flagellates. When that recognize additional epitopes on both the 46 kDa flagellates revert spontaneously to amebae, the BN5.1 and 51 kDa subunits of BN46/51, binds to nucleoli from antigen is lost from the basal body region coincident with Saccharomyces cerevisiae or mammalian cells. BN5.1 the loss of the MTCS. The BN5.1 antigen, composed of does not bind to the nucleoli of Dictyostelium discoideum approximately equal amounts of two subunits of 46 kDa or Euglena gracilis. Thus BN46/51 is an unusual and and 51 kDa, both of which carry the BN5.1 epitope, has perhaps unique nucleolar component whose presence in been named BN46/51. BN46/51 in the basal body region the basal body region presents a challenge to our comigrates with the nucleolar antigen by two-dimen- understanding of the cytoskeleton. sional gel electrophoresis. Approximately 75% of the nucleolar BN46/51 is solubilized by extracton with 0.4 M Key words: nucleolar antigen, basal body, cytoskeleton, NaCl. However, the antigen associated with the basal Naegleria. Introduction evidence, however, favors the DFC as the site of transcription. rDNA has been localized to the DFC by Nucleoli are the sites of rRNA transcription, rRNA Wachtler et al. (1989, 1992). By pulse-chase labelling, processing and the assembly of ribosomal subunits nascent rRNA transcripts were found first in the DFC (Scheer and Benavente, 1990; Sommerville, 1986; (Deltour and Mosen, 1987; Goessens, 1976). Topo- Warner, 1990). The nucleolus is composed of three isomerase I and RNA polymerase I have both been generally recognized morphological components: the localized in the DFC (Raska et al., 1989; reviewed by fibrillar centers (FC), the dense fibrillar component Jordan, 1991). Fibrillarin (Ochs et al., 1985), a (DFC), and the granular component (GC) (Jordan, component of the U3 snRNP involved in the first 1984; Goessens, 1984; Warner, 1990). However, despite preribosomal RNA processing step (Kass et al., 1990), extensive investigation, the functional significance of is localized to the DFC (Scheer and Benavente, 1990). these morphological components is unclear. Most of the In contrast it is generally agreed that the GC represents discussion has been focused on the function of the FC ribosomal subunits in various stages of assembly (Hiigle and DFC components. For example, the presence of et al., 1985; Warner, 1990). RNA polymerase I (Scheer and Rose, 1984) and rRNA Nuclei of the amebo-flagellate Naegleria gruberi have genes in the FC of nucleoli (Knibiehler et al., 1977; a single large nucleolus (Fulton, 1970; Schuster, 1975). Jordan and Rawlins, 1990) suggests that the FC is the Despite its large size, the Naegleria nucleolus is site of transcription of the rRNA genes. Other composed of the three characteristic morphological 168 G. M. Trimbur and C. J. Walsh components. It is rapidly labeled by RNA precursors vortexed in the presence of non-ionic detergents (Walsh and and it is enriched in 28 S rRNA (Walsh and Fulton, Fulton, 1973). In contrast, nucleoli are quite resistant and 1973) as are nucleoli in other cells. However, the remain intact. The lysate was centrifuged in an IEC Clinical Naegleria nucleolus does differ from most nucleoli in centrifuge at a setting of 6 for 1 min to pellet cysts and unlysed cells. (As noted by Fulton (1970), expression of these brief the organization of the rRNA genes. Instead of a centrifugations as g forces is meaningless because the tandem array of rRNA genes at one or more chromo- centrifuge is accelerating during this time; they are, however, somal locations, the Naegleria rRNA genes are found reproducible.) Nucleoli were pelleted by centrifugation of the exclusively on approximately 4000 copies of a 17 kbp resulting supernatant at 10,400 g for 2 min. Additional plasmid (Clark and Cross, 1987, 1988). Another nucleoli were collected by centrifuging the supematent a unusual feature of the Naegleria nucleolus is that it does second time as above. When flagellates were used, the final not disperse during mitosis (Fulton, 1970; Schuster, supernatant was saved for the isolation of flagellar rootletyfoa- 1975). Mitosis in Naegleria is closed and the nucleolus sal body complexes, described below. divides as the chromosomes separate on the intra- The nucleolar pellets were pooled by resuspension in 4 ml nuclear spindle. of 1 M sucrose-RLB, overlayed on a 1.62 M sucrose-RLB pad and centrifuged for 13 min at 10,400 g. The pellet was This report describes the identification and character- resuspended in 1 ml of SPMG (63 mM sucrose, 25 mM sodium ization of a novel protein, BN46/51, found in the DFC phosphate, pH 7.2, 2.5 mM MgCI, and 0.5 mM EGTA) and and GC of Naegleria nucleoli. BN46/51 is unusual in nucleoli were counted in a hemocytometer. The nucleoli were that it is also associated with the basal body region of pelleted by centrifugation at 15,000g for 1 min. Nucleoli were Naegleria flagellates. Amebae of N. gruberi can solubilized by resuspension in 0.4 M NaCI-SPMG at 4 x 108 synchronously and rapidly differentiate into swimming nucleoli/ml for 30 min on ice. This extract was centrifuged for flagellates when washed free of their food source 10 min at 15,000 g. The supernatant was saved and used as a (Fulton, 1970; Fulton, 1977; Fulton and Dingle, 1967). source of soluble BN46/51. The differentiation results in the de novo assembly of When nucleoli were prepared for immunofluorescence, basal bodies (Dingle and Fulton, 1966; Fulton, 1970; nucleoli were resuspended in SPMG and an equal volume of Fulton and Dingle, 1971), flagellar axonemes (Dingle fixative (MgCF) was added as described below. Nucleolar fractions contained few if any rootlet/basal body complexes and Fulton, 1966), flagellar rootlets (Dingle and Fulton, when observed by phase-contrast microscopy. 1966; Larson and Dingle, 1981a,b), and a microtubule Rootlet/basal body complexes were prepared from the final cytoskeleton (MTCS) (Walsh, 1984). BN46/51 is associ- nucleolar-depleted supernatant by addition of 1 M KI in RLB ated with the nucleolus in amebae, and appears in the without glycerol to make the extract 0.1 M KI as described by basal body region when the MTCS assembles in Larson and Dingle (1981a). This extract was centrifuged at flagellates. This unexpected observation raises a num- 16,300 g for 10 min. The pellet was resuspended in 1 ml of 0.1 ber of interesting questions about the role of BN46/51 in M KC1-RLB without glycerol, and centrifuged at 15,000 g. the cell. As a first step in examining these questions we The pellet was rinsed with 1 ml of SPMG and resuspended in 9 9 have undertaken a characterization of BN46/51. SPMG at 1 x 10 to 2 x 10 cell equivalents/ml. Rootlet/basal body fractions contained no nucleoli as judged by phase- contrast microscopy. Materials and methods Antigen preparation, monoclonal antibody and ascites production Growth and differentiation of Naegleria gruberi Rootlet/basal body complexes from 2 X 109 flagellates were Naegleria gruberi strain NB-1 (Fulton, 1970; Fulton and solubilized in Laemmli sample buffer (Laemmli, 1970) by Dingle, 1967) was grown on NM or PM agar with Klebsiella heating in boiling water for 2 min and electrophoresed on pneumoniae as previously described (Fulton and Dingle, 7.5% SDS-polyacrylamide gels (Laemmli, 1970). Gels were 1967). Amebae were harvested and washed free of bacteria in stained for 5 min with Coomassie Blue and destained in ice-cold 2 mM Tris-HCl (pH 7.6 at 20°C). Differentiation was deionized water. Proteins in the size range of 46 to 48 kDa initiated in this same buffer at 25°C with 15 ml in 125 ml were cut out of the gel and the gel piece was minced in TBS Erlenmeyer flasks or 25 ml in 250 ml Erlenmeyer flasks on a (50 mM Tris-HCl, pH 7.4, 200 mM NaCl). Complete Fruend's reciprocating shaker as described by Fulton and Dingle adjuvant was added at a 3:1 ratio and the mixture emulsified. (1967).
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