Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.

Abstract

To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein N038 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large a-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleoplasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein N038 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino-and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein N038 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture.

p300/CBP-associated Factor Drives DEK into Interchromatin Granule Clusters

“…However, glutathione S-transferase-pull-down experiments, yeast two-hybrid analysis, and native gel electrophoresis demonstrate that DEK can physically associate with itself and exists in several multimeric forms (data not shown). Dimerization would allow mutant forms of DEK to associate with endogenous DEK and "piggyback" to IGCs following TSA treatment (65). This phenomenon therefore masks the identification of the lysine responsible for sub-nuclear movement of DEK using site-directed mutagenesis.…”

Mapping the Functional Domains of Nucleolar Protein B23

“…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 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.…”

“…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.…”

Nucleophosmin and human cancer

“…Under conditions where significant amounts of the monomer were present, NPM was capable of DNA binding, whereas conditions that strongly favored oligomerization caused a nearly complete abolition of this DNA binding activity [32]. Recently, it was reported that the NPM isoforms, NPM1 and NPM1.2, can form oligomers in vivo and in vitro [28,35,36], but the RNA binding activity of NPM1 is impaired by hetero-oligomerization with NPM1.2 [28]. Another study has shown that the reversible change of oligomerization between the hexameric and monomeric forms of NPM is found to occur most frequently in cells in G1 and G1/S phases [37].…”