RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-β2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-β1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-β2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-β2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

Kapβ2 (also called transportin) recognizes PY nuclear localization signal (NLS), a new class of NLS with a R/H/Kx (2)(3)(4)(5) PY motif. Here we show that Kapβ2 complexes containing hydrophobic and basic PY-NLSs, as classified by the composition of an additional N-terminal motif, converge in structure only at consensus motifs, which explains ligand diversity. On the basis of these data and complementary biochemical analyses, we designed a Kapβ2-specific nuclear import inhibitor, M9M.Ten different import karyopherin-βs (Kapβs, also called importin-βs) 1 mediate trafficking of human proteins into the cell nucleus through recognition of distinct NLSs. Large panels of import substrates are known only for Kapβ1 (importin-β) and Kapβ2 (transportin) 1,2 . The substrate repertoire of each Kapβ and the functional consequences of pathway specificities are some of the main challenges in understanding intracellular signaling and trafficking. In the case of nuclear export, Crm1 inhibitor leptomycin B has been crucial for identifying many Crm1 substrates 3,4 . Such specific inhibitors of nuclear import could be invaluable for proteomic analyses to map extensive nuclear traffic, but none has been found. Two classes of NLS are currently known: short, basic classical NLSs that bind the heterodimer 5), and newly identified PY-NLSs that bind Kapβ2 (ref.2). PY-NLSs are 20-to 30-residue signals with intrinsic structural disorder, overall basic character, C-terminal R/K/Hx 2-5 PY motifs (where x 2-5 is any sequence of 2-5 residues) and N-terminal hydrophobic or basic motifs. These weak but orthogonal characteristics have provided substantial limits in sequence space, enabling the identification of over 100 PY-NLS-containing human proteins 2 . Two subclasses, hPY-NLSs and bPY-NLSs, are defined by their N-terminal motifs: hPY-NLSs contain ϕG/A/Sϕϕ motifs (where ϕ is a hydrophobic residue), whereas bPY-NLSs are enriched with basic residues.We have previously solved the structure of human Kapβ2 bound to the hPY-NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) 2 . Here we have solved the 3.1-Å Fig. 1a,b; Kapβ2 435-780 Cα r.m.s. deviation is 0.9Å). Their NLS termini are structurally diverse, consistent with their apparent lack of sequence conservation 2 . At the N terminus, hnRNP A1 residues 263-266 bind the convex side of Kapβ2 (ref.2), whereas the N terminus of hnRNP M proceeds toward the Kapβ2 arch opening. At the C terminus, hnRNP A1 is disordered beyond Pro288-Tyr289, whereas hnRNP M extends 5 residues beyond its Pro-Tyr motif.Residues 51-64 of hnRNP M and residues 273-289 of hnRNP A1 contact a common Kapβ2 surface, with the highest overlap at their Pro-Tyr motifs (Fig. 1b). R.m.s. deviations for all Pro-Tyr atoms and for arginine guanido group atoms in the R/H/Kx (2-5) PY motifs are 0.9Å and 1.2Å, respectively, upon Kapβ2 superposition. At the N-terminal motifs, hnRNP M residues 51-54 in the basic 50-KEKNIKR-56 motif and hnRNP A1 residues 274-277 in the hydrophobic motif also overlap (main chain r.m.s. deviation ...

Mutations in the proline/tyrosine-nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif, a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (K D for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUSKapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.T he proline/tyrosine-nuclear localization signal (PY-NLS) of RNA-binding protein Fused in Sarcoma (FUS) was first identified in a structure-based bioinformatics approach and shown to bind the import-karyopherin, karyopherinβ2 (Kapβ2) in a Ran-sensitive manner (1). More recently, the signal was shown to direct Kapβ2-mediated nuclear import in cells (2) and to be heavily mutated in ∼5% of familial amyotrophic lateral sclerosis (ALS) (3-5), a progressive and fatal neurodegenerative disorder. ALS mutations in the PY-NLS disrupted nuclear import of FUS, causing its mislocalization and aggregation in the cytoplasm, as evidenced by cytoplasmic FUS inclusions in motor neurons of ALS patients (2, 6-9). The PY-NLS of FUS is also mutated in another neurodegenerative disease, frontotemporal lobar dementia (FTLD), which also is characterized by cytoplasmic mislocalization and aggregation of FUS (10, 11). The pathogenic role of FUS PY-NLS mutants was further confirmed by observations that expression of such proteins in Drosophila, Caenorhabditis elegans, zebrafish, and rats caused neurodegeneration (12-15). Proper FUS localization is important in maintaining neuronal homeostasis, and it is thus important to understand the factors that govern localization of both wild-type and mutant proteins.PY-NLSs are recognized by the import-karyopherin Kapβ2 (also known as Transportin) for transport through the nuclear pore complex into the nucleus (1, 16-18). These 15-to 100-residue-long sequences are large and diverse and cannot be sufficiently described by a traditional consensus sequence but are instead described by a collection of physical rules that include requirements for intrinsic structural disorder, overall basic character, and a set of sequence motifs. PY-NLS motifs consist of an N-terminal hydrophobic or basic motif and a C-terminal RX 2-5 PY motif (Fig. ...

Summary Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them the contributions of centrosomes, chromosomes and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.