Principles of protein targeting to the nucleolus.
The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of rRNA synthesis and assembly of ribosomes. Nucleolar proteins dynamically localize and accumulate in this nuclear compartment relative to the surrounding nucleoplasm. In this study, we have assessed the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside the nucleoli of living cells. The data showed that positively charged peptide entities composed of arginines alone and with an isoelectric point at and above 12.6 are necessary and sufficient for mediating significant nucleolar accumulation. A threshold of 6 arginines is necessary for peptides to accumulate in nucleoli, but already 4 arginines are sufficient when fused within 15 amino acid residues of a nuclear localization signal of a protein. Using a pH sensitive dye, we found that the nucleolar compartment is particularly acidic when compared to the surrounding nucleoplasm and, hence, provides the ideal electrochemical environment to bind poly-arginine containing proteins. In fact, we found that oligo-arginine peptides and GFP fusions bind RNA in vitro. Consistent with RNA being the main binding partner for arginines in the nucleolus, we found that the same principles apply to cells from insects to man, indicating that this mechanism is highly conserved throughout evolution.
a Instituto de Medicina Molecular ; Faculdade de Medicina ; Universidade de Lisboa ; Lisboa , Portugal.
Robert M
RM
Martin
Gohar
G
Ter-Avetisyan
Henry D
HD
Herce
Anne K
AK
Ludwig
Gisela
G
Lättig-Tünnemann
M Cristina
MC
Cardoso
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D019913:Nuclear Localization Signals / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D021381:Protein Transport; D014162:Transfection
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D001120:Arginine
GFP; fluorescence microscopy; nucleolar localization sequence; nucleolus; protein targeting