Mechanism of karyopherin-ß2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed. In unstressed cells, wild type FUS resides predominantly in the nucleus as it is imported by the importin Karyopherin-ß2 (Kapß2), which binds with high affinity to the C-terminal PY-NLS of FUS. Here, we analyze the interactions between two ALS-related variants FUS(P525L) and FUS(R495X) with importins, especially Kapß2, since they are still partially localized to the nucleus despite their defective/missing PY-NLSs. The crystal structure of the Kapß2·FUS(P525L)PY-NLS complex shows the mutant peptide making fewer contacts at the mutation site, explaining decreased affinity for Kapß2. Biochemical analysis revealed that the truncated FUS(R495X) protein, although missing the PY-NLS, can still bind Kapß2 and suppresses LLPS. FUS(R495X) uses its C-terminal tandem arginine-glycine-glycine regions, RGG2 and RGG3, to bind the PY-NLS binding site of Kapß2 for nuclear localization in cells when arginine methylation is inhibited. These findings suggest the importance of the C-terminal RGG regions in nuclear import and LLPS regulation of ALS variants of FUS that carry defective PY-NLSs.

Individual binding pockets of importin-beta for FG-nucleoporins have different binding properties and different sensitivities to RanGTP.

Importin-beta mediates protein transport across the nuclear envelope through the nuclear pore complex (NPC) by interacting with components of the NPC, called nucleoporins, and a small G protein, Ran. Although there is accumulated knowledge on the specific interaction between importin-beta and the Phe-Gly (FG) motif in the nucleoporins as well as the effect of RanGTP on this interaction, the molecular mechanism by which importin-beta shuttles across the nuclear envelope through the NPC is unknown. In this study, we focused on four binding pockets of importin-beta for the FG motifs and characterized the interaction using a single-molecule force-measurement technique with atomic-force microscopy. The results from a series of importin-beta mutants containing amino acid substitutions within the FG-binding pockets demonstrate that the individual FG-binding pockets have different affinities to FG-Nups (Nup62 and Nup153) and different sensitivities to RanGTP; the binding of RanGTP to the amino-terminal domain of importin-beta induces the conformational change of the entire molecule and reduces the affinity of some of the pockets but not others. These heterogeneous characteristics of the multiple FG-binding pockets may play an important role in the behavior of importin-beta within the NPC. Single-molecule force measurement using the entire molecule of an NPC from a Xenopus oocyte also implies that the reduction of the affinity by RanGTP really occurs at the nucleoplasmic side of the entire NPC.

Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore.

Nuclear pore complexes (NPCs) mediate and control the transport of virtually all material between the cytosol and the nucleus. It is, therefore, unsurprising that they have long taken centre stage in physiology. A precise understanding of the NPC structure and function that remain to be thoroughly investigated yet is, thus, of crucial importance. The NPC can mediate transport both actively and passively. It remains to be clarified, however, whether transport of small molecules and macromolecules proceeds through the same route in the NPC. Furthermore, it has been shown that surface hydrophobicity represents a major sorting criterion for the active transport through NPCs. Transport factors like importin beta, which exhibit a rather large surface hydrophobicity, bind to their cargo and are believed to interact with a supposedly hydrophobic meshwork that is assumed to reside in the central channel of the NPC but has not yet been visualised. This interaction is presumed to lead to a partial breakdown of the meshwork, thereby, permitting the transport-cargo complexes to pass through. In this study, by using the nano-imaging approach, atomic force microscopy, we visualised under near-physiological conditions, for the first time, the presence of a hydrophobic meshwork in the NPC central channel. Furthermore, our data lend strong support for the existence of two segregated transport routes in the NPC.

A pathway separate from the central channel through the nuclear pore complex for inorganic ions and small macromolecules.

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.