Entry created on 1 July 2019 (Revision 1.0) Annotator: Rita Pancsa; Orsolya Kovács
Basic protein information
Accession P06748
Common name NPM1
Gene NPM1
Organism Homo sapiens
Uniprot name Nucleophosmin
Basic LLPS information
Organelle nucleolus; granular component
Type of experimental evidence
Protein region(s) mediating LLPS
1
-
294
Full protein sequence contributes to LLPS: oligomerization domain, linker with acidic tracts, RRM
Based on the experimental results of the following publication: 26836305
Molecular features viewer
PDB structures
View on PDBe
Extended LLPS information
Functional description
The nucleolus has a layered tripartite organization that consists of the fibrillar center (FC), where the RNA polymerase I (POL1) machinery is active; the dense fibrillar component (DFC) that is enriched in the protein fibrillarin (FIB1); and the granular component (GC) that is enriched in the protein nucleophosmin (NPM1/B23) (PMID:27212236). NPM1 participates in the organization of the liquid-like structure of the granular component of the nucleolus (PMID:25349213) and consequently may actively participate in stress signal integration and transmission, thereby explaining its known roles in ribosome biogenesis, tumor suppression and other processes. Of 132 NPM1-binding proteins, 97% exhibited at least one R-motif, multivalency of acidic tracts within NPM1 and R-motifs within nucleolar substrates mediates liquid-liquid phase separation. Nucleolar localization of NPM1 in vivo requires multi-modal interactions with both R-motif-containing nucleolar proteins and rRNA. Multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, are the structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli in vivo (PMID:26836305). Surfeit locus protein 6 (SURF6/S6N) tunes the composition and material properties of NPM1 droplet scaffolds. Electrostatically-driven interactions between disordered regions of NPM1 and SURF6 drive liquid-liquid phase separation. Co-existing heterotypic (NPM1-SURF6) and homotypic (NPM1-NPM1) scaffolding interactions within NPM1-SURF6 liquid-phase droplets dynamically and seamlessly interconvert in response to variations in molecular crowding and protein concentrations (PMID:30498217).
Literature supporting the LLPS: 26836305, 27212236, 25349213, 29483575, 30498217
Functional class of membraneless organelle: activation/nucleation/signal amplification/bioreactor
Binding partners (at biological protein concentrations)
1) (r)RNA (strictly required) 2) partner preotein(s) with multivalent (at least 2) R-motifs (strictly required)
Type of RNA(s) required/used for the LLPS at biological protein concentrations
wheat germ rRNA
Molecular interaction types contributing to LLPS
discrete oligomerization (PMID:26836305) protein-RNA interaction (PMID:26836305) multivalent domain-motif interactions (PMID:26836305)
Determinants of phase separation and droplet properties
1) protein concentration 2) valency of protein partners
Membrane cluster No
Partner-dependent Yes
RNA-dependent Yes
PTM required No
Domain-motif interactions Yes
Discrete oligomerization Yes
Regulation and disease
Post-translational modifications affecting LLPS
Position Residue PTM Effect Reference Modifying enzyme Notes
Isoforms known to affect LLPS
Isoform Effect Reference
All known isoforms containing sequence changes in the LLPS region(s)
Position type Isoform names from UniProt
Disease mutations affecting LLPS
Mutation dbSNP Disease OMIM Effect Reference Notes
Experimental information
Experimental techniques applied to prove/investigate LLPS
In vivo NPM1 depletion is associated with disruption of nucleolar structure (morphology) (PMID:25349213). In vitro at 150 mM NaCl, NPM1 requires high concentrations of protein (2 μM) and rRNA (100 μg/ml). Phase separation of NPM1 required rRNA and cannot be induced by the addition of heparin or poly-U50. When deleting the N-terminal oligomerization domain to create NPM1ΔN, and deleting the C-terminal RNA binding domain to create NPM1ΔC neither mutant was able to form droplets (particle size and count by microscopy) in vitro. In vitro FIB1 and NPM1 coexist as multiphase droplets, with the NPM1 rich phase tending to partially envelope the FIB1 rich phase (PMID:27212236). In vitro, titration of four R-motif-containing peptides caused phase separation into liquid-like droplets (particle size and count by microscopy) at critical concentrations that varied with R-motif composition, valency and affinity for N130 segment of NPM1. Phase separation was not observed (particle size and count by microscopy) when a truncated NPM1 construct containing only the oligomerization domain OD (residues 1–122) was titrated with the rpL5 peptide, therefore the minimal multivalency requirements for phase separation are the acidic A1 and A2 tracts within NPM1 and at least two complementarily charged R-motifs within a polypeptide binding partner. The molecular basis of this type of NPM1 phase separation is the formation of non-covalent, inter-N130 pentamer interactions via the two R-motifs within the same rpL5 peptide molecule. These rpL5-mediated interactions establish the inter-pentamer spacing within the droplet phase. Full length NPM1 (N294) could phase separate with either rpL5 or wheat germ rRNA. Neither of the OD- or RRM-truncated constructs experienced phase separation in the presence of rRNA (particle size and count by microscopy), confirming that multivalent display of the NBD is required for the co-localization of rRNA with NPM1 within liquid-like droplets. Multi-modal binding to two classes of macromolecules, R-motif-containing nucleolar proteins (binding mode 1) and rRNA (binding mode 2), is likely critical for NPM1-dependent formation of multi-component liquid-like droplets (PMID:26836305).
Experimental observations supporting the liquid material state of the condensate
morphological traits (PMID:26836305, PMID:27212236) rheological traits (PMID:27212236) dynamic movement/reorganization of molecules within the droplet (PMID:27212236)