Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.

The [PSI ؉ ] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the structure of amyloid fibrils formed in vitro from purified recombinant Sup35 1-253 , consisting of the glutamine-and asparagine-rich Nterminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole-dipole couplings among 13 C nuclei in a series of Sup35NM fibril samples, 13 C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular 13 C-13 C distances of Ϸ0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of Ϸ0.5 nm. These results indicate that an in-register parallel ␤-sheet structure underlies the [PSI ؉ ] prion phenomenon. The Sup35p prion domain (Sup35N, residues 1-123) is asparagine-and glutamine-rich, is poor in charged residues, and has five imperfect nine-residue repeats with consensus YQQYN-PQGG. Sequence shuffling shows that the repeats are not necessary for prion generation or propagation and that amino acid content of the prion domain (not the sequence) determines whether a protein can form a prion (13). Certain point mutations in the prion domain can block propagation of [PSI ϩ ] introduced with the wild sequence (14, 15), although the mutant sequence may itself form a prion (16). Thus, propagation of an existing prion is very sequence-specific, as in the species barriers of mammalian prion diseases (reviewed in ref. 17).Amyloid fibrils are filamentous protein aggregates exhibiting ''cross-␤'' x-ray fiber diffraction patterns, indicating the presence of ␤-sheets formed by ␤-strands that are oriented approximately perpendicular to the fiber axis, with interstrand hydrogen bonds approximately parallel to the fiber axis (reviewed in ref. 18). The fact that the prion domains of Ure2p (another yeast prion protein with an N-terminal prion domain rich in asparagine and glutamine) and Sup35p can be shuffled and yet still form prions and amyloid (13,19) suggests that the amyloid on which these prions are based has an in-register parallel ␤-sheet structure (20, 21). A prion amyloid structure based on antiparallel ␤-sheets or ␤-helices would necessarily be stabilized by interactions among specific sets of unlike residues. These interactions would likely be destroyed by shuffling the sequence. In contrast, an in-register parallel ␤-sheet structure can be stabilized by intermolecular hydrophobic interactions (22,23) or polar side chain interactions [e.g., the ''polar zipper'' interactions suggested by Perutz et al. (24)] among like residues. Shuffling the sequence would still allow like residues to align and interact in such a structure. Thus, shuffleability of a prion domain suggests an in-register parallel ␤-sheet structure.The molecular structures of amyloid fibrils, particularly those formed by bona fide proteins, are difficult t...

[URE3] is a prion (infectious protein), a self-propagating amyloid form of Ure2p, a regulator of yeast nitrogen catabolism. We find that overproduction of Btn2p, or its homologue Ypr158 (Cur1p), cures [URE3]. Btn2p is reported to be associated with late endosomes and to affect sorting of several proteins. We find that double deletion of BTN2 and CUR1 stabilizes [URE3] against curing by several agents, produces a remarkable increase in the proportion of strong [URE3] variants arising de novo and an increase in the number of [URE3] prion seeds. Thus, normal levels of Btn2p and Cur1p affect prion generation and propagation. Btn2p-green fluorescent protein (GFP) fusion proteins appear as a single dot located close to the nucleus and the vacuole. During the curing process, those cells having both Ure2p-GFP aggregates and Btn2p-RFP dots display striking colocalization. Btn2p curing requires cell division, and our results suggest that Btn2p is part of a system, reminiscent of the mammalian aggresome, that collects aggregates preventing their efficient distribution to progeny cells.