Valosin-containing protein counteracts ATP-driven dissolution of FUS condensates through its ATPase activity in vitro.

Fused in sarcoma (FUS) forms phase-separated condensates implicated in amyotrophic lateral sclerosis (ALS). Although millimolar ATP concentrations paradoxically dissolve FUS condensates through hydrotropic activity, condensates nevertheless persist in cells, suggesting active regulatory mechanisms. Here, using a reconstituted system, we show that the AAA+ATPase valosin-containing protein (VCP) counteracts ATP-driven dissolution of FUS condensates. VCP preserved both wild-type and ALS-linked P525L condensates under high ATP conditions, and this protection required catalytic ATPase activity rather than stable partitioning into condensates. The effect was abolished by the D2-specific inhibitor ML240. Our findings establish direct biochemical evidence that VCP ATPase activity maintains FUS condensates under high ATP conditions, highlighting ATPase-driven enzymatic control of liquid-liquid phase separation as a potential general principle with implications for neurodegeneration.