Abstract Karyopherin-β2 (Kapβ2) is a nuclear-import receptor that recognizes proline-tyrosine nuclear localization signals (PY-NLSs) of diverse cytoplasmic cargo for transport to the nucleus. Kapβ2 cargo include several disease-linked RNA-binding proteins (RBPs) with prion-like domains (PrLDs), such as FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2. These RBPs with PrLDs are linked via pathology and genetics to debilitating degenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and multisystem proteinopathy (MSP). Remarkably, Kapβ2 prevents and reverses aberrant phase transitions of these cargo, which is cytoprotective. However, the molecular determinants of Kapβ2 that enable these activities remain poorly understood, particularly from the standpoint of nuclear-import receptor architecture. Kapβ2 is a superhelical protein comprised of 20 HEAT repeats. Here, we design truncated variants of Kapβ2 and assess their ability to antagonize FUS aggregation and toxicity in yeast and FUS condensation at the pure protein level and in human cells. We find that HEAT repeats 8-20 of Kapβ2 recapitulate all salient features of Kapβ2 activity. By contrast, Kapβ2 truncations lacking even a single cargo-binding HEAT repeat display reduced activity. Thus, we define a minimal Kapβ2 construct for delivery in adeno-associated viruses as a potential therapeutic for ALS/FTD, MSP, and related disorders.

Molecular determinants of Karyopherin-β2 chaperone and disaggregation activity