Principles of protein targeting to the nucleolus

Abstract

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.

APOBEC3H Subcellular Localization Determinants Define Zipcode for Targeting HIV-1 for Restriction

“…2A). While no strict consensus motif has been defined for nucleolar targeting, numerous proteins have been characterized as having targeting sequences comprised of stretches of basic amino acids (76,77). To determine if these residues have a role in nucleolar targeting, we changed each basic residue within loop 1 to glutamate and assessed the effect on localization.…”

Nucleolar Reorganization Upon Site-Specific Double-Strand Break Induction

“…A recent study indicates that as little as four basic arginine residues in tandem are sufficient to create an NoLS in a protein that already harbors a nuclear localization signal. 49 Even though the localization of NBS1-GFP in the nucleolus might be artefactuous, it remains nevertheless true that it responds to rRNA gene cleavage by accumulation to the NCs. We also show that endogenous NBS1 is recruited to NCs 8 hr after transfection, which underscores the consistency of our results.…”

The Tat inhibitor didehydro‐cortistatin A suppresses SIV replication and reactivation

“…Passive diffusion is the dominant mechanism of Tat nuclear transport (38,39), whereas nucleolus trafficking requires Tat's arginine residues of the basic domain (40). We previously showed that dCA redistributes HIV-1 Tat to the periphery of the nucleolus while remaining nuclear in HeLa-CD4 and astrocyte cell lines (19,26).…”