Protein localization to the nucleolus: a search for targeting domains in nucleolin

Abstract

Nucleolin, a major nucleolar phosphoprotein, is presumed to function in rDNA transcription, rRNA packaging and ribosome assembly. Its primary sequence was highly conserved during evolution and suggests a multi-domain structure. To identify structural elements required for nuclear uptake and nucleolar accumulation of nucleolin, we used site-directed mutagenesis to introduce point- and deletion-mutations into a chicken nucleolin cDNA. Following transient expression in mammalian cells, the intracellular distribution of the corresponding wild-type and mutant proteins was determined by indirect immunofluorescence microscopy. We found that nucleolin contains a functional nuclear localization signal (KRKKEMANKSAPEAKKKK) that conforms exactly to the consensus proposed recently for a bipartite signal (Robbins, J., Dilworth, S.M., Laskey, R.A. and Dingwall, C. (1991) Cell 64, 615–623). Concerning nucleolar localization, we found that the N-terminal 250 amino acids of nucleolin are dispensible, but deletion of either the centrally located RNA-binding motifs (the RNP domain) or the glycine/arginine-rich C terminus (the GR domain) resulted in an exclusively nucleoplasmic distribution. Although both of these latter domains were required for correct subcellular localization of nucleolin, they were not sufficient to target non-nucleolar proteins to the nucleolus. From these results we conclude that nucleolin does not contain a single, linear nucleolar targeting signal. Instead, we propose that the protein uses a bipartite NLS to enter the nucleus and then accumulates within the nucleolus by virtue of binding to other nucleolar components (probably rRNA) via its RNP and GR domains.

Multiple biological responses activated by nuclear protein kinase C

“…Since PKC-␦ seems to localize to the nucleolus, the question arises as to whether or not this isoform possesses a nucleolar targeting signal [Hatanaka, 1990]. However, the issue of nucleolar targeting sequences is even more complex and under debate than that of NLS, also because so far it has been proven difficult to identify sequences targeting proteins to the nucleolus clearly separated from those necessary for nuclear import [Schmidt-Zachmann and Nigg, 1993;Russo et al, 1997]. Recently, Das et al [1998] identified in the Trypanosoma Brucei Nopp44/46 protein a 27 amino acid sequence within the amino terminal region whose deletion abrogated nucleolar but not nuclear localization.…”

Nucleolin Undergoes Partial N- and O-Glycosylations in the Extranuclear Cell Compartment

“…Hence, nucleolin was proposed as a mediator for the extracellular regulation of nuclear events (3). Except for the presence of a nuclear localization sequence (NLS) upstream from the first RBD (11), the structural features that govern the putative shuttling activity of nucleolin in the cell are not well-defined. Post- translational modifications of nucleolin such as phosphorylation with CKII, cdc2, and PKC-ζ kinases, arginine methylation, and autodegradation probably play critical roles in the function and traffic of the protein (12)(13)(14)(15), but no glycosylation was clearly evidenced.…”

Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

“…NCL contains a bipartite nuclear localization signal (NLS), and importins are known to recognize the NLS of NCL to mediate the nuclear import of NCL and its extracellular ligand as a complex [ 45 , 47 ]. Therefore, we examined the role of importins in the nuclear trafficking of the NCL-TS5-p45 complex using an importin inhibitor Ivermectin.…”