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.

Keywords: GFP; fluorescence microscopy; nucleolar localization sequence; nucleolus; protein targeting.

Figure 1.

Distribution of poly-(R)and poly-(K)peptides in living cells. Intracellular distribution of poly-R peptides in (A) and poly-K peptides (B) in living C2C12 mouse cells. In each panel, the fluorescence image is on top of the corresponding phase contrast image. Nucleoli are clearly visible as dark round structures within the nuclei in the phase contrast images. The bar diagrams in (C) show the quantification of the poly-K and poly-R peptide mean fluorescence in cytoplasm, nucleoplasm and nucleoli averaged for 10 cells from 2 independent experiments. The nucleoplasmic values were used for normalization. Areas for quantification were defined as described in methods and overlayed with fluorescence images as shown. Intracellular distribution of R10 peptide in living cells of different species as indicated is shown in (D). In (E) living yeast cells were further stained with DRAQ5 for better visualization of the nucleus and a line intensity profile in arbitrary units (a.u.) of both DNA and peptide is shown. The intracellular distribution of (D) and L-R10 peptides in living C2C12 cells and the corresponding quantification of mean fluorescence intensities is shown in (F). Scalebars: 5 µm.

Figure 2.

Intracellular distribution of peptide tagged GFP tracer proteins in living cells. (A) Representative microscopic images of live C2C12 cells transfected with GFP, NLS-GFP and a fusion of NLS-GFP with 8 glycines (8G) as example for the addition of neutral amino acids is shown in the first panel. The second panel shows images of cells with fusions of an increasing number of aspartates as examples for acidic amino acid fusions to NLS-GFP. The GFP fluorescence is depicted in the upper row with the corresponding differential interference contrast (DIC) images below. (B) Microscopic Images of the distribution of the NLS-GFP fused to 4 to 7 additional arginines. The graphs in (C) represent the mean distribution of the GFP constructs depicted in (A) and (B) in cytoplasm and nucleoli in relation to the nucleoplasm for an average of 10 cells from 2 independent experiments. Scalebars: 5 µm.

Figure 3.

Intracellular pH landscape by ratiometric fluorescence microscopy toward the generation of an intracellular pH landscape. The figure in (A) displays fluorescence microscopy images of the pH sensitive dye SNARF-4F in live HeLa cells (color). The green channel shows the emission at 587 and the red channel the emission of the dye at 640 nm during excitation with the same wavelength. The resulting intensity ratio image (gray scale) shows ratiometric differences between the intensities of the 2 fluorescence emission peaks of the dye due to pH variations in subcellular structures. The fluorescence color scale and the ratio scale in gray indicate the relative range from more acidic to more basic pH. Panel (B) illustrates a map of the intracellular pH landscape by measuring the pH in various cellular compartments and substructures like nucleoplasm and nucleolus (modified from⁵³). Scalebars: 5 µm.

Figure 4.

Analyses of RNA binding to poly-(R)peptides and GFP fusions. (A) Total RNA used for in vitro RNA binding assays separated by size showing the absence of genomic DNA contamination, as well as characteristic bands for the 28S and 18S rRNA, which are primarily synthesized and localized in nucleoli. The slot blot in (B) (representative from 2 independent experiments), rows 1 and 2, show a methylene blue stained PDVF membrane in the absence (1) and presence (2) of different amounts of total RNA (0.5 µg and 1 µg) and were used as a loading control. The rest of the blot shows the binding of fluorescently tagged L-R10 (3 and 4) and D-R10 (5 and 6) in the absence (3 and 5) and presence (4 and 6) of total RNA. Slots lacking RNA (rows 5 and 6) do not show binding of L- and D-R10, respectively. In contrast, slots probed with increasing amounts of RNA show increasing L- and D-R10 binding. (C) In vitro RNA pulldown assay using NLS-R7-GFP immobilized to sepharose beads via the GFP binding protein (GBP) (scheme). RNA was stained using propidium iodide (PI) and GFP alone was used as a negative control. RNA binding was measured on a fluorescent plate reader (bar plot) and imaged by confocal microscopy (microscopic images). Plots represent the average plus standard deviation of 2 independent experiments. Scalebar: 100 µm.

Figure 5.

Chart of peptide and protein pI values, intracellular distribution and sequence composition. (A) Display of the pI value of FITC labeled peptides and full-length proteins coupled to different peptides along a continuous pI scale. Representative images illustrating the intracellular and intranuclear distribution are indicated along the scale. (B) Subsets of the pI scale with the values exclusively for the charged amino acid domains in the proteins and peptides tested combined with an illustrative representation of the domains. For the proteins the domain considered starts with the nuclear localization signal (NLS) and continues through the different motifs of charged amino acids (poly-R and poly-D) introduced into the GFP open reading frame. The peptides tested consist exclusively of basic amino acids. The clustering of the charged amino acids is displayed as color bars in the box adjacent to the respective fluorophore FITC or GFP. A legend for the charged amino acids is given above. Representative images for the distribution inside the cells and nuclei are displayed on the right for the respective peptide or protein. Scalebar: 5 µm.