Determination of the functional domains involved in nucleolar targeting of nucleolin

• Published in: Molecular biology of the cell 1993-12, Vol.4 (12), p.1239-1250
• Main Authors: GREANCIER, L, PRATS, H, ZANIBELLATO, C, AMALRIC, F, BUGLER, B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Biological Transport; Cell Line; Cell Nucleolus - metabolism; Chlorocebus aethiops; Contractile proteins; Cytoplasm - metabolism; Fundamental and applied biological sciences. Psychology; Holoproteins; L Cells; Mice; Molecular Sequence Data; Nuclear Proteins - chemistry; Nuclear Proteins - metabolism; Nucleolin; Phosphoproteins - chemistry; Phosphoproteins - metabolism; Protein Engineering; Protein Structure, Tertiary; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Sequence Deletion
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.4.12.1239

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.