A B23-interacting sequence as a tool to visualize protein interactions in a cellular context.
We report the characterization of a nucleolar localization sequence (NoLS) that targets the green fluorescent protein (GFP) into the granular component (GC) of nucleoli. This NoLS interacts in vitro specifically and directly with the major nucleolar protein B23 and more precisely with the region of B23 including the two acidic stretches. The affinity of NoLS for B23 is stronger than that of the HIV-1 Rev protein in vitro. Moreover, B23-NoLS interaction also occurs in vivo. Indeed, (1) NoLS confers on the GFP the behavior of B23 throughout the cell cycle, (2) the GFP-NoLS fusion and B23 remain colocalized after drug treatments, (3) a selective delocalization of B23 from nucleoli to nucleoplasm induces a concomitent delocalization of the GFP-NoLS fusion, and (4) the fusion of NoLS to fibrillarin makes it possible to colocalize fibrillarin and B23. Interestingly, by fusing NoLS to fibrillarin, both fibrillarin and the fibrillarin partner Nop56 are mislocalized in the GC of nucleoli. Similarly, by fusing the NoLS to MafG, part of the nuclear transcription factor NF-E2 composed of both MafG and p45 NF-E2, NF-E2 is redirected from the nucleoplasm to the nucleoli. Thus, we propose that the NoLS may be used as a tool to visualize and prove protein interactions in a cellular context.
Lechertier
Tanguy
T
Institut Jacques Monod, UMR 7592 CNRS/Universités Paris 6 et 7, 2 Place Jussieu, 75251 Paris Cedex 05, France.
Sirri
Valentina
V
Hernandez-Verdun
Danièle
D
Roussel
Pascal
P
D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D002466:Cell Nucleolus / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D007211:Indoles; C000715570:Npm1 protein, mouse; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000090243:Nucleophosmin; D049452:Green Fluorescent Proteins; C007293:DAPI; D005982:Glutathione Transferase