BN46/51, a new nucleolar protein, binds to the basal body region in Naegleria gruberi flagellates

Abstract

ABSTRACT Indirect immunofluorescence with the monoclonal antibody, BN5.1, labels the nucleolus of amebae of the amebo-flagellate Naegleria gruberi. When amebae differentiate into flagellates, BN5.1 binds to nucleoli and to the basal body region. The BN5.1 antigen is not present in basal bodies when basal bodies form at about 60 min after initiation of the differentiation or when flagella form at about 70 min. The BN5.1 antigen is first detectable in the basal body region at 85 min after initiation, a time when the basal body region acts as a microtubule organizing center for the formation of the microtubule cytoskeleton (MTCS) of flagellates. When flagellates revert spontaneously to amebae, the BN5.1 antigen is lost from the basal body region coincident with the loss of the MTCS. The BN5.1 antigen, composed of approximately equal amounts of two subunits of 46 kDa and 51 kDa, both of which carry the BN5.1 epitope, has been named BN46/51. BN46/51 in the basal body region comigrates with the nucleolar antigen by two-dimensional gel electrophoresis. Approximately 75% of the nucleolar BN46/51 is solubilized by extracton with 0.4 M NaCl. However, the antigen associated with the basal body region is resistant to extraction, even with 2 M NaCl. Solubilized BN46/51 exists as a heterogeneous multimer that elutes on gel filtration with a peak at 400 to 500 kDa and sediments on sucrose gradients at 5.5 S. The multimers consist of only the 46 kDa and the 51 kDa subunits in equal amounts as judged by glutaraldehyde cross-linking and by chromatography on BN5.1 affinity columns. Nucleolar BN46/51 is associated with the dense fibrillar and granular components of the nucleolus. However, it does not resemble any previously described nucleolar protein. Neither BN5.1, nor three other mAbs that recognize additional epitopes on both the 46 kDa and 51 kDa subunits of BN46/51, binds to nucleoli from Saccharomyces cerevisiae or mammalian cells. BN5.1 does not bind to the nucleoli of Dictyostelium discoideum or Euglena gracilis. Thus BN46/51 is an unusual and perhaps unique nucleolar component whose presence in the basal body region presents a challenge to our understanding of the cytoskeleton.