PROTEIN LOCALIZATION TO THE NUCLEOLUS - A SEARCH FOR TARGETING DOMAINS IN NUCLEOLIN

Nucleolin, a major nucleolar phosphoprotein, is presumed to function i n rDNA transcription, rRNA packaging and ribosome assembly. Its primar y 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, th e 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 signa l (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 nucleola r localization, we found that the N-terminal 250 amino acids of nucleo lin are dispensible, but deletion of either the centrally located RNA- binding motifs (the RNP domain) or the glycine/arginine-rich C terminu s (the GR domain) resulted in an exclusively nucleoplasmic distributio n. Although both of these latter domains were required for correct sub cellular localization of nucleolin, they were not sufficient to target non-nucleolar proteins to the nucleolus. From these results we conclu de that nucleolin does not contain a single, linear nucleolar targetin g 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.