Membrane-less biomolecular compartmentalization is a core phenomenon involved in many physiological activities that occur ubiquitously in cells. Condensates, such as promyelocytic leukemia (PML) bodies, stress granules, and P-bodies (PBs), have been investigated to understand the process of membrane-less cellular compartmentalization. In budding yeast, PBs dispersed in the cytoplasm of exponentially growing cells rapidly accumulate in response to various stresses such as osmotic stress, glucose deficiency, and heat stress. In addition, cells start to accumulate PBs chronically in post-exponential phases. Specific protein–protein interactions are involved in accelerating PB accumulation in each circumstance, and discovering the regulatory mechanism for each is the key to understanding cellular condensation. Here, we demonstrate that Nst1 of budding yeast Saccharomyces cerevisiae is far more densely associated with PBs in post-exponentially growing phases from the diauxic shift to the stationary phase than during glucose deprivation of exponentially growing cells, while the PB marker Dcp2 exhibits a similar degree of condensation under these conditions. Similar to Edc3, ectopic Nst1 overexpression induces self-condensation and the condensation of other PB components, such as Dcp2 and Dhh1, which exhibit liquid-like properties. Altogether, these results suggest that Nst1 has the intrinsic potential for self-condensation and the condensation of other PB components, specifically in post-exponential phases.

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid–liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1_∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1_∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1_∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components.

SUMMARY: Liquid-liquid phase separation of TDP-43 is implicated in ALS and FTLD, yet how its condensates convert into pathological amyloid in living cells remains poorly defined. Here we identify a polyampholyte motif (PAM) in the linker between RRM2 and the amyloid-forming low-complexity domain as a charge-tunable determinant of fibrillization that operates independently of condensate solidification. In budding yeast expressing GFP-TDP-43ΔNLS, charge reversal of three PAM arginines (R268E/R272E/R275E; 3R-E) converted liquid puncta into ProteoStat-positive fibrils. Unexpectedly, fluorescence recovery after photobleaching revealed that 3R-E condensates were more mobile than wild-type condensates, whereas the least mobile condensates, in both yeast and human U2OS cells, converted least. Across 24 PAM charge variants, the proportion of fibril-bearing cells scaled linearly with net charge independently of residue identity, and charge neutralization delayed rather than abolished conversion. Phosphomimetic substitution of the PAM serines alone induced fibrils, implicating phosphorylation as a potential physiological modulator of PAM charge. HIGHLIGHTS: A polyampholyte motif (PAM) outside the TDP-43 amyloid core governs fibril formation. Fibril-bearing cells scale linearly with PAM net charge, not with residue identity. Conversion precedes loss of exchange; exchange falls only where amyloid has formed. Phosphomimetic substitution of two PAM serines is sufficient for conversion. ETOC BLURB: Choi et al. show that amyloid conversion of TDP-43 condensates in cells tracks the net charge of a short motif outside the amyloid core, not condensate solidification. Phosphomimetic substitution of two serines in the motif is sufficient for conversion, suggesting phosphorylation as a possible physiological route.

SUMMARY: Intracellular condensation is a hallmark of many proteins containing intrinsically disordered regions (IDRs). Decoding the regulatory mechanisms of condensation encoded within the amino acid sequences of condensate-forming proteins is essential for understanding both how intracellular condensates assemble and how they contribute to various disease pathologies. Here, we investigated the budding yeast P-body proteins Nst1 and Edc3, which exhibit pronounced intracellular self-condensation, and identified charged amino acid clusters in the low complexity regions (LCRs) that modulate their phase behavior. Guided by the stickers-and-spacers framework, we hypothesized that clusters of charged residues within IDRs function in promoting intracellular condensation process. Using a sliding-window approach, we designated and dissected the polyelectrolyte (PE) domains of Nst1 and Edc3 and established their essential contributions to condensate coalescence. In Nst1, the polyanionic (PA) subdomain enhances intermolecular attraction and promotes coalescence, whereas the polyampholyte (PAM) subdomain imparts rigidity to the condensate as a distinct modulatory element and functions as a platform in condensation process of other P-body components. PA of Edc3 also regulates coalescence. Extending these findings to the neurodegenerative disease-associated protein TDP-43, we show that its PA regions are critical for coalescence, consistent with the presence of negatively charged amino acid clusters as potent molecular stickers functioning in condensate coalescence in cells. These results highlight charged-residue clusters as important sequence determinants of protein phase behavior and suggest that they represent promising novel regulatory targets for modulating TDP-43 condensation in cells.