Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state.
The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.
Roussel
P
P
Institut Jacques Monod, Paris, France.
Belenguer
P
P
Amalric
F
F
Hernandez-Verdun
D
D
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009697:Nucleolus Organizer Region / Q000648:ultrastructure*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D016601:RNA-Binding Proteins; D012834:Silver; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured; D000097796:Nucleolin
D034961:Antigens, Nuclear; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D011494:Protein Kinases; D016601:RNA-Binding Proteins; D012834:Silver; D000097796:Nucleolin; C051963:nucleolar organizer region associated proteins