NAP57, a Mammalian Nucleolar Protein with a Putative Homolog in Yeast and Bacteria

We report the identification and molecular characterization of a novel nucleolar protein of rat liver. As shown by eoimmunoprecipitation this protein is associated with a previously identified nucleolar protein, Noppl40, in an apparently stoichiometric complex and has therefore been termed NAP57 (Noppl40-associated protein of 57 kD). Immunofluorescence and immunogold electron microscopy with NAP57 specific antibodies show colocalization with Nopp140 to the dense fibrillar component of the nucleolus, to coiled bodies, and to the nucleoplasm. Immunogold staining in the nucleoplasm is occasionally seen in the form of curvilinear tracks between the nucleolus and the nuclear envelope, similar to those previously reported for Noppl40. These data suggest that Noppl40 and NAP57 are indeed associated with each other in these nuclear structures. The eDNA deduced primary structure of NAP57 shows a protein of a calculated molecular mass of 52,070 that contains a putative nuclear localization signal near its amino and earboxy terminus and a hydrophobic amino acid repeat motif extending across 84 residues. Like Noppl40, NAP57 lacks any of the known consensus sequences for RNA binding which are characteristic for many nucleolar proteins. Data bank searches revealed that NAP57 is a highly conserved protein. A putative yeast (S. cerevisiae) homolog is 71% identical. Most strikingly, there also appears to be a smaller prokaryotic (E. coli and B. subtilis) homolog that is nearly 50% identical to NAP57. This indicates that NAP57 and its putative homologs might serve a highly conserved function in both proand eukaryotes such as chaperoning of ribosomal proteins and/or of preribosome assembly. F UNCTIONAL ribosomal subunits of E. coli have been reconstituted in vitro from their RNA and protein components some time ago (Traub and Nomura, 1968). Reconstitution was observed at empirically defined windows of ionic strength and temperature (unphysiologically high) and proceeded unassisted by nonribosomal proteins. Little is known how ribosomal subunit assembly occurs in bacteria in vivo. What, for example, is the order in which ribosomal proteins associate with the ribosomal RNAs coand posttranscriptionally? To what extent is ribosomal subunit assembly assisted by nonribosomal proteins? In eukaryotes, ribosomal subunit assembly occurs in the nucleolus. Unlike in bacteria where ribosomal protein and RNA synthesis take place in one compartment, the bacterial cytosol in eukaryotes the two processes occur in two compartments, the cytosol and the nucleolus. For ribosomal subunit assembly, ribosomal proteins therefore need to be Please address all correspondence to Dr. U. Thomas Meier, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Tel.: (718) 430-3294. Fax: (718) 518-7236. transported into the nucleolus (Warner and Soeiro, 1967). The assembled subunits are then transported into the cytosol to function in protein biosynthesis. Little is known either about how ribosomal proteins are transported into the nucleolus or their order of assembly with the ribosomal RNAs coor posttranscriptionally. Not all of the ribosomal proteins may be transported into the nucleolus as indicated by the differences in protein composition between nucleolar and cytoplasmic (pre)ribosomal particles (Kumar and Warner, 1972; Tsurugi et al., 1973; Prestayko et al., 1974; Kumar and Subrarnanian, 1975) and by the in vivo exchange of certain proteins between ribosome associated and soluble proteins (Warner, 1966; Zinker and Warner, 1976). Does, therefore, a distinct cytoplasmic phase of ribosomal subunit assembly yield functionally active cytoplasmic subunits (as opposed to functionally inactive nuclear subunits)? To what extent are nonribosomal proteins involved in assembly and subsequent nucleocytoplasmie transport of ribosomal subunits? A number of nonribosomal nucleolar proteins, largely of unknown function, have now been identified. Some of these proteins could represent structural proteins of a nucleolar skeleton (Franke et al., 1981). Others, such as fibrillarin, are © The Rockefeller University Press, 0021-9525/94/12/1505/10 $2.00 The Journal of Cell Biology, Volume 127, Number 6, Part 1, December 1994 1505-1514 1505 on N ovem er 9, 2017 jcb.rress.org D ow nladed fom involved in processing of ribosomal RNA (Tollervey et al., 1991). These two groups of proteins can be considered resident nucleolar proteins. A third group of nucleolar proteins appears to shuttle between the nucleolus and the cytoplasm (Borer et al., 1989; Meier and Blobel, 1992). It is likely that these proteins function in ribosomal subunit assembly and in nucleocytoplasmic transport. Noppl40, a nucleolar phosphoprotein of 140 kD, is a representative of the shuttling variety of nucleolar proteins (Meier and Blobel, 1992). Immunogold electron microscopy localized Nopp140 primarily to the dense fibrillar component (DFC) ~ of the nucleolus. In addition, Noppl40 could be seen in the nucleoplasm and occasionally was found in curvilinear tracks that extended for microns across the nucleoplasm from the DFC of the nucleolus to nuclear pore complexes (Meier and Blobel, 1992). Similar tracks have now been observed with antibodies to the ribosomal protein S1 (Raska et al., 1992) and to the human immunodeficiency virus type 1 Nefprotein (Murti et al., 1993). These localization data are consistent with Nopp140 shuttling between the nucleolus and the cytoplasm. It is not known, however, why and with whom Nopp140 shuttles. Noppl40 was identified as a protein that binds nuclear localization signals (NLS) in vitro (Meier and Blobel, 1990). If NLS binding were its physiological function (or one of its functions), Nopp140 might serve in protein import and therefore be associated with proteins to be imported. In addition or alternatively, the repetitive acidic, serine-rich motifs of Nopp140 and the basic stretches separating them (Meier and Blobel, 1992) could serve a chaperone function in ribosomal subunit assembly by interacting with regions of opposite charge (ribosomal RNA, ribosomal proteins) displayed by ribosomal subunit assembly intermediates. In this scenario Noppl40 might remain associated with a preribosomal particle that is transported from the nucleolus to the cytoplasm. In an attempt to elucidate the basis of the Noppl40 tracks and the Noppl40 function, we present here the identification and molecular characterization of the Noppl40 associated protein, NAP57. We show that NAP57 coprecipitates and colocalizes with Nopp140 and that it is evolutionary highly conserved. Possible NAP57(/Nopp140) functions are discussed in this context. Materials and Methods