Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Abstract

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high-speed super-resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy-ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs. K E Y W O R D Sintrinsically disordered proteins (IDPs), nuclear pore complex (NPC), nucleocytoplasmic transport, super-resolution Note: Disorder level score was calculated using IUPRED. 58 Approximately 500-1,200 spatial locations were collected in order to obtain reliable 3D spatial transport route for each candidate in our measurements based on computation simulation (Section 4). Abbreviations: Ch/Hy, charged/hydrophobic; CREST, calcium-responsive transactivator; IDP, intrinsically disordered protein; IUPRED, intrinsically unstructured proteins prediction.

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

“…Therefore, importins are capable of binding to both paralogues, NUPR1L and NUPR1, as we had already demonstrated the binding to the latter protein [ 23 ]. These findings are at variance with recent results obtained by fluorescence, where other IDPs were suggested to translocate into the nucleus without the need for the nucleus-cytoplasm transport machinery [ 39 ].…”

Novel SOX10 Mutations in Waardenburg Syndrome: Functional Characterization and Genotype-Phenotype Analysis

“…The present results also suggested that not only the location (first two coding vs. last coding exons) and type (missense vs. truncation/frameshift) of SOX10 mutations, but also the altered levels of protein expression of the SOX10 missense variants and their molecular weight, including the aa composition (ratio of charged/hydrophobic aas) of the mutant proteins, may determine the severity of the expressed phenotype (Junod et al, 2020). These results also suggest that phenotypic severity may be regulated by the differential expression of each mutant protein, influenced by the activation versus escaping NMD pathway and other yet unknown factors.…”

“…However, a mutant protein was also produced, as shown by immunofluorescence study, but the protein was too small (3.1 kD) to be detected by western blot. Despite its lack of a NLS, the p.Ser30 ∗ variant was partly present in the nucleus, probably as a result of passive diffusion of the small (<63 kD) protein through the nuclear pore complex ( Gorlich and Mattaj, 1996 ; Junod et al, 2020 ). In summary, the molecular evidence supports a haploinsufficiency mechanism for this variant, resulting in classic WS4.…”

“…The p.Cys71Hisfs ∗ 62 variant was assumed to enter the nuclear compartment by passive diffusion, but this was not observed. In addition to molecular weight, an aberrant charged/hydrophobic aa ratio is also known to affect the nuclear transport of proteins, which might explain the restriction of this mutant protein to the cytosol ( Junod et al, 2020 ). Surprisingly, the in vitro data demonstrated high protein expression levels of this variant, indicating unactivated NMD machinery, despite the fact that the mutation was located in the first coding exon (exon 3) of the SOX10 gene.…”

Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy

“…Additionally, recently, we found that the nucleocytoplasmic transport of intrinsically disordered proteins (IDPs) was independent of molecular sizes and transport receptors that are well executed for folded proteins. Instead, the number of the charge and hydrophobic amino acid residues of the IDPs dominates their nuclear transport mechanisms [ 66 ]. In detail, the IDPs with a higher concentration of hydrophobic residues diffused through the NPCs with higher successful transport rates.…”